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https://openalex.org/W2114019393	https://biblio.ugent.be/publication/5720924/file/8730384	Dutch	null	Bruno Benvindo en Evert Peeters, Scherven van de oorlog. De strijd om de herinnering aan de Tweede Wereldoorlog 1945-2010	Bijdragen en mededelingen betreffende de geschiedenis der Nederlanden	2,014	cc-by	1,195	© 2014 Royal Netherlands Historical Society \| KNHG Creative Commons Attribution 3.0 Unported License URN:NBN:NL:UI:10-1-110054 \| www.bmgn-lchr.nl \| E-ISSN 2211-2898 \| print ISSN 0615-0505 © 2014 Royal Netherlands Historical Society \| KNHG Dit tot ongenoegen van de oud-politieke gevangenen die Breendonk alleen als een patriottistisch monument wilde zien. en homo’s. Bovendien ging een nieuwe generatie ‘de gruwel van Breendonk’ in verband brengen met hedendaagse repressieve regimes. Dit tot ongenoegen van de oud-politieke gevangenen die Breendonk alleen als een patriottistisch monument wilde zien. Net als Breendonk fungeerde ook de Congreskolom in Brussel (het Graf van de onbekende soldaat) als een monument van ‘goed’ vaderlanderschap. Anders dan bij Breendonk werd deze herinnering ingebed in de bestaande patriottistische herinnering aan de Eerste Wereldoorlog. Hier werd gedroomd van de eenheid van het land. Herhaling van fascisme kon in de toekomst alleen worden voorkomen via eendracht, zo klonk het. De uitsluiting van de voormalige communistische verzetslieden, de polariserende Koningskwestie en het oprukkende federalisme zorgde vanaf de jaren zestig echter voor ontgoocheling bij deze ‘ware’ Belgen. Er diende zich geen nieuwe generatie aan die de plek gebruikte voor haar herinnering aan de oorlog en de Congreskolom raakte als gevolg hiervan in de vergetelheid. Hoe anders verging het de IJzertoren in Diksmuide. Deze plek groeide uit als tegenpool van Breendonk en de Congreskolom. Hier werden geen Belgische, maar Vlaams-nationalistische helden geëerd. Na de oorlog had de Belgische regering tevergeefs geprobeerd om van de IJzertoren een gedeeld verleden te maken. De Vlaams- nationalistische herinneringsgemeenschap slaagde er echter in om van de IJzertoren het symbool van de collaboratie te maken. Dit werd de plek waar de naoorlogse ‘repressie’ ter discussie werd gesteld. De auteurs benadrukken niet genoeg dat het Belgische herinneringsparcours op dit punt behoorlijk afwijkt van de rest van West-Europa. Een fenomeen als de IJzertoren en de bijbehorende herinneringsgemeenschap was bijvoorbeeld in het Nederland van na de Tweede wereldoorlog ondenkbaar. Interessant is hoe vanaf de jaren tachtig, onder invloed van de toenemende federalisering, de Vlaamse overheid de IJzertoren gebruikte voor de legitimatie van haar zelfstandig bestaan. Het moest een symbool van alle Vlamingen worden, ontdaan van alle radicale elementen. De greep van de collaboratieherinnering verzwakte volgens auteurs als gevolg hiervan. Hierbij wordt niet verder uitgewijd over de rol van de media of de rol van een nieuwe Vlaamse generatie historici die tegen deze tijd brak met de traditie om de Vlaams-nationalistische collaboratie te vergoelijken. De opkomst vanaf de jaren tachtig van een nieuw racistisch en rechts-radicaal Vlaams-nationalisme had hier alles mee te maken. BMGN - Low Countries Historical Review \| Volume 129-1 (2014) \| review 19 Bruno Benvindo en Evert Peeters, Scherven van de oorlog. De strijd om de herinnering aan de Tweede Wereldoorlog 1945-2010 (Antwerpen: De Bezige Bij, 2011, 260 pp., ISBN 978 90 8542 324 9). De historici Bruno Benvindo en Evert Peeters schreven een mooi boek over de sociale herinnering aan de Tweede Wereldoorlog in België. Of beter gezegd, over de pijnlijke strijd die de verschillende betrokkenen hiervoor sindsdien tot op de dag van vandaag met elkaar voeren. Want die herinnering aan de jaren 1940 -1945 was allesbehalve eenduidig. België zou volgens de socioloog Luc Huyse ‘ziek van zijn jaren veertig’ zijn, of er toch tenminste een groot ‘verdriet’ aan overhouden, zoals Hugo Claus in zijn roman suggereerde (zie Luc Huyse, ‘Waarom België ziek is van zijn jaren veertig’, Bijdragen tot de Eigentijdse Geschiedenis 10 (2002) 185-193; Hugo Claus, Het verdriet van België (Amsterdam 1983)). Scherven van de oorlog vertrekt vanuit de gedachte dat er ‒ net als in andere (West-) Europese landen ‒ in België geen gedeelde herinnering aan de Tweede Wereldoorlog bestaat. In plaats daarvan bestaan er meerdere herinneringsgemeenschappen die de afgelopen decennia onderhandelden over de duiding van dit verleden. De auteurs proberen deze onderhandelingsprocessen in kaart te brengen aan de hand van vier gedenkplekken (lieux de mémoire): het gevangenkamp Fort van Breendonk, de Congreskolom in Brussel, de IJzertoren in Diksmuide en de Dossinkazerne in Mechelen. De strijd om de herinnering wordt al snel duidelijk in de bespreking van het voormalige gevangenkamp Breendonk dat na de oorlog uitgroeide tot het ultieme symbool van de Duitse repressie. De Belgische regering voerde lange tijd geen beleid om de herinnering hieraan levend te houden, maar gaf in plaats daarvan de slachtoffers zelf ruimte om dit vorm te geven. Maar juist door de afwezigheid van een centraal beleid, vlogen de oud-politieke gevangenen elkaar dikwijls in de haren over de invulling van die herinnering, bijvoorbeeld wanneer het ging om de plek van de communistische gevangenen. Desondanks groeide onder invloed van de agency van deze oud-politieke gevangenen Breendonk uit tot een centrum van het Belgisch patriottisme. Dat veranderde toen een sterker wordend mensenrechtendiscours er in de jaren zeventig voor zorgde dat meer groepen slachtoffers aanspraak op de plek maakten, zoals Joden en homo’s. Bovendien ging een nieuwe generatie ‘de gruwel van Breendonk’ in verband brengen met hedendaagse repressieve regimes. De groeiende belangstelling voor de Jodenvervolging die in België eigenlijk pas vanaf de jaren tachtig op gang kwam, zorgde er daarnaast voor dat de collaboratie kritischer tegen het licht werd gehouden. Die aandacht voor de Jodenvervolging wordt in het laatste hoofdstuk over de Dossinkazerne behandeld. Hier zien we hoe de Jodenvervolging in België pas recent uitgroeide tot het symbool bij uitstek van de Tweede Wereldoorlog – nog zo’n punt waarop België internationaal gezien behoorlijk afweek. In de eerste periode na de oorlog werd de Joodse herinnering aan de oorlog ingepast in het dominante discours van heldenmoed en patriottistische strijd. De Mechelse Dossinkazerne, waar vanuit meer dan 25.000 Joden en Roma werden gedeporteerd paste als symbool niet in dit discours en kreeg na de oorlog direct een andere bestemming. Eerst diende het als militaire kazerne en later werden er luxe appartementen ingebouwd. In de jaren negentig begon de Vlaamse overheid zich met de Dossinkazerne te bemoeien en wilde van deze ‘vergeten’ plek een belangrijk gedenkteken maken. Ook hier lag het nieuw racistisch en rechts- radicaal Vlaams-nationalisme aan de basis van de herontdekking van de plek. Via Holocausteducatie zou de Vlaamse overheid de burger op deze plek alert moeten maken voor de gevolgen van (politiek) extremisme. In de behandeling van de plekken laten de auteurs overtuigend zien dat meerdere partijen over de jaren heen met elkaar in de clinch raakte over de duiding van het verleden. De Belgische overheid speelde hier lange tijd nagenoeg geen rol bij. Pas op het moment dat de Belgische staat steeds verder verbrokkelde, begon de Vlaamse overheid de plekken te gebruiken ter legitimering van haar bestaan. Een van de vragen die achterblijft na lezing van het boek is welke scherven van de oorlog er in Wallonië bestaan. Kende de Waalse Beweging dan geen herinneringsgemeenschap met bijbehorende plekken? Dit komt in het boek niet ter sprake, wat er misschien op wijst dat dit een beduidend minder belangrijke plek in de oorlogsherinnering achter liet. Tot slot moet worden opgemerkt dat dit boek werd geschreven in opdracht van het federale Studie- en Documentatiecentrum voor Oorlog en Hedendaagse Maatschappij (SOMA). De gefragmenteerde herinnering aan de oorlog wordt door de Franstalige Benvindo en Nederlandstalige Peeters vanuit het bredere Belgisch perspectief bestudeerd. Dat geeft dit boek een grote meerwaarde. Helen Grevers, NIOD Instituut voor Oorlogs-, Holocaust- en Genocidestudies
https://openalex.org/W2934747589	https://hal.archives-ouvertes.fr/hal-02323824/document	English	null	Recurrent changes in cichlid dentition linked to climate‐driven lake‐level fluctuations	Ecosphere	2,019	cc-by	11,977	Recurrent changes in cichlid dentition linked to climate-driven lake-level fluctuations Jorunn Dieleman, Bert Van Bocxlaer, Wanja Dorothy Nyingi, Anna Lyaruu, Dirk Verschuren To cite this version: Jorunn Dieleman, Bert Van Bocxlaer, Wanja Dorothy Nyingi, Anna Lyaruu, Dirk Verschuren. Recur- rent changes in cichlid dentition linked to climate-driven lake-level fluctuations. Ecosphere, 2019, 10 (4), pp.e02664. 10.1002/ecs2.2664. hal-02323824 JORUNN DIELEMAN ,1 BERT VAN BOCXLAER,1,2 WANJA DOROTHY NYINGI,3 ANNA LYARUU,4 AND DIRK VERSCHUREN 1, JORUNN DIELEMAN ,1 BERT VAN BOCXLAER,1,2 WANJA DOROTHY NYINGI,3 ANNA LYARUU,4 AND DIRK VERSCHUREN 1, 1Limnology Unit, Department of Biology, Ghent University, K. L. Ledeganckstraat 35, Ghent B-9000 Belgium 2UMR 8198 – Evolution, Ecology, Paleontology, CNRS, University of Lille, Lille F-59000 France 3Ichthyology Section, Zoology Department, National Museums of Kenya, P.O. Box 40658-00100, Nairobi, Kenya 4Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam 1090 GE The Netherlands Citation: Dieleman, J., B. Van Bocxlaer, W. D. Nyingi, A. Lyaruu, and D. Verschuren. 2019. Recurrent changes in cichlid dentition linked to climate-driven lake-level ﬂuctuations. Ecosphere 10(4):e02664. 10.1002/ecs2.2664 Abstract. Climate-induced habitat change has often been invoked as an important driver of speciation and evolutionary radiation in cichlid ﬁshes, yet studies linking morphological change directly to long-term envi- ronmental ﬂuctuations are scarce. Here, we track changes through time in the oral dentition of Oreochromis hunteri, the endemic and only indigenous ﬁsh species inhabiting the East African crater Lake Chala (Kenya/ Tanzania), in relation to climate-driven lake-level ﬂuctuations during the last 25,000 yr. Fossil teeth were recovered from six periods representing alternating high and low lake-level stands associated with late- Pleistocene and Holocene trends in rainfall and drought. To construct a reference framework for morpho- logical variation in fossil assemblages, we ﬁrst analyzed the variability in oral tooth morphology within the modern-day ﬁsh population. These analyses established that, like other cichlids, O. hunteri gradually replace their bicuspid/tricuspid oral dentition with more unicuspid teeth as they grow. In the fossil assemblages, we found systematic and recurrent changes in the relative abundance of these oral tooth types, with a higher proportion of unicuspids during low-stands. Moreover, O. hunteri living during low-stands systematically developed unicuspid dentition at a smaller body size, compared with conspeciﬁcs living during high-stands. Considering that low-stand conditions created a sizable area of oxygenated soft-bottom habitat that is lacking during high-stands, we propose that the associated change in oral dentition reﬂects the ﬁshes’ exploitation of this new habitat either for feeding or for reproduction. The recurrent nature of the observed shifts provides evidence for the ability of O. hunteri to systematically adapt to local habitat change, and strongly suggests that morphological change in oral dentition promoted its long-term population persistence in an aquatic ecosystem presenting the dual challenge of being both sensitive to climate change (creating constantly shifting selective pressures) and isolated from surrounding populations (limiting gene ﬂow). JORUNN DIELEMAN ,1 BERT VAN BOCXLAER,1,2 WANJA DOROTHY NYINGI,3 ANNA LYARUU,4 AND DIRK VERSCHUREN 1, In Lake Chala, ancient climate- driven lake-level ﬂuctuations did not directly promote speciation in O. hunteri, but the population bottlenecks they may have caused potentially contributed to its divergence from sister species Oreochromis jipe. Key words: adaptation; Africa; Cichlidae; climate change; ﬁsh fossils; Lake Chala; morphometrics; oral dentition; phenotypic plasticity; population persistence; selection pressure; tooth morphology. Received 17 January 2019; accepted 22 January 2019; ﬁnal version received 3 March 2019. Corresponding Editor: Debra P. C. Peters. Copyright: © 2019 The Authors. This is an open access article under the terms of the Creative Commons License, which permits use, distribution and reproduction in any medium, provided the original work is pro Copyright: © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. E-mail: dirk.verschuren@ugent.be E-mail: dirk.verschuren@ugent.be E-mail: dirk.verschuren@ugent.be HAL Id: hal-02323824 https://hal.science/hal-02323824v1 Submitted on 16 Aug 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. ❖www.esajournals.org April 2019 ❖Volume 10(4) ❖Article e02664 INTRODUCTION driving force of speciation (Thompson et al. 2018). Especially when environmental change gives rise to new, unexploited habitats (i.e., when it creates ecological opportunity), rapid The ability of species to adapt their ecology and behavior to changing environments is a ❖www.esajournals.org April 2019 ❖Volume 10(4) ❖Article e02664 1 DIELEMAN ET AL. DIELEMAN ET AL. et al. 2014), but cichlids are plagued by a scarcity of continuous fossil deposits, the typically disar- ticulated nature of their remains, and difﬁculty to assign individual fossils to speciﬁc lineages. These problems have hampered detailed study of long-term patterns of morphological change in a coupled ecological–evolutionary context (Mur- ray 2001). Cichlid teeth and scales preserved in the sediments of existing African lakes hold the best hope for a continuous record (Cohen 2012). Although such records are now tentatively being explored (Reinthal et al. 2011, Muschick et al. 2018), diverse methodological and confounding factors have thus far prevented detailed study of environment–phenotype relations in individual cichlid species lineages (Dieleman et al. 2015). adaptation provides ample opportunities for populations to diverge into separate species (Sch- luter 2000). Such ecological speciation is promi- nent in the cichlid ﬁshes (Cichlidae) of the African Great Lakes, where it has rapidly pro- duced highly diverse species ﬂocks (Fryer and Iles 1972). In these lakes, lake-level ﬂuctuations induced by Quaternary climate change have caused phases of extreme contraction (Lake Malawi), fragmentation (Lake Tanganyika), or even complete desiccation (Lake Victoria; reviewed by Salzburger et al. 2014). Alternat- ingly creating and eliminating certain types of freshwater habitat, these ﬂuctuations have been invoked as an important driver of evolutionary radiation in African cichlids (Rossiter 1995, Dan- ley et al. 2012, Ivory et al. 2016). p g ( ) One African lake of particular interest in this context is Lake Chala, an isolated crater lake on the border between Kenya and Tanzania near Mt. Kilimanjaro (Fig. 1b, c). This small (4.2 km2) but ~90-m deep lake with permanently stratiﬁed lower water column (Buckles et al. 2014, Wolff et al. 2014) has a ﬁnely laminated sediment record indicating long-term continuity of lacus- trine conditions (Verschuren et al. 2009, Moer- naut et al. 2010), and it contains abundant fossil teeth, bones, and scales of cichlid ﬁshes through- out the last 25,000 yr (Fig. 1a). INTRODUCTION Currently, Lake Chala harbors the endemic Oreochromis hunteri (Chala tilapia) as only indigenous ﬁsh species, but following a series of introductions in the 1980s, this species now co-occurs with three non- native cichlids, namely another but less well- deﬁned Oreochromis species originally attributed to O. korogwe; the tilapiine Coptodon rendalli; and a much smaller haplochromine species of the Astatotilapia bloyeti complex (Dadzie et al. 1988, Seegers et al. 2003, Dieleman et al. 2019, Moser et al. 2018). y y ) Intrinsic biological factors also inﬂuence whether cichlids radiate, as colonization of new lakes and habitats does not automatically result in diversiﬁcation and radiation (Wagner et al. 2012). One group of cichlids whose members rarely radiate is the paraphyletic species group formerly referred to as tilapia (Brawand et al. 2014). Compared to the often stenotopic and haplochromine cichlids, the tilapiine cichlids, including the genus Oreochromis, display a less specialized but highly versatile morphology, making them more tolerant to habitat change and less prone to extinction (Ribbink 1990). One trait that has enabled cichlids in general to cope with new environments is the large ﬂexibility of their trophic apparatus, which consists of both oral and pharyngeal jaws (Muschick et al. 2011, van Rijssel et al. 2015). Oreochromines are gener- ally herbivorous mouth-brooders and hence have a trophic morphology reﬂecting trade-offs between multiple functions (Trewavas 1983). Nevertheless, subtle differences in jaw and tooth morphology still permit them to specialize in cer- tain ecological niches, especially during periods of food scarcity. Lake Chala is the focus of a multifaceted research program aiming to reconstruct the long- term environmental and climate history of equa- torial East Africa. These studies revealed that the lake experienced pronounced ﬂuctuations during the last 25,000 yr (Moernaut et al. 2010; Fig. 1a), creating a succession of high-stand and low- stand conditions sufﬁciently contrasting to have had an impact on the diversity and nature of aquatic habitats available to O. hunteri (Fig. 1d). Assuming that the indigenous tilapiine would over time have adapted to these habitat changes Morphological variation in dentition among Oreochromis populations has been directly linked to differences in habitat (Ndiwa et al. 2016), but evidence of morphological shifts within a single population in response to local habitat change is rare, especially over the substantial periods typi- cally required for speciation. Study of fossils has already proven informative in this respect for other teleost ﬁshes (Purnell et al. ❖www.esajournals.org April 2019 ❖Volume 10(4) ❖Article e02664 INTRODUCTION The light gray shading highlights the area of gently sloping lake bottom at the base of the steep underwater rock face nearshore that was likely at least seasonally oxygenated during the ancient low-stands and hence provided additional habitat for Oreochromis hunteri at those times. (d) Schematic representation of lacustrine habitat available to O. hunteri during high-stand (left) and low-stand (right) episodes, the former situation also characterizing Lake Chala today. The water column is divided into the approximate Fig. 1. (a) Abundance (no. per gram dry mass) of fossil cichlid teeth and of other fossil ﬁsh remains (bones, scales) in the sediment record of Lake Chala, in relation to the reconstructed sequence of past lake-level changes during the last 25,000 yr (Moernaut et al. 2010). The horizontal gray bars delineate the six time intervals studied here, representing four ancient high-stand episodes and two ancient low-stand episodes. (b) Map of equatorial East Africa with location of the principal great lakes Victoria, Tanganyika, and Malawi, and with Lake Chala indicated as a star. (c) Bathymetry of Lake Chala as recorded in March 2003 with indication of the coring site (star), depth contours at 10-meter intervals (except the deepest contour at 94 m), and the crater–rim catchment boundary (dashed line). The dark gray shading shows the approximate extent of lake bottom area that remained anoxic throughout the past 25,000 yr. The light gray shading highlights the area of gently sloping lake bottom at the base of the steep underwater rock face nearshore that was likely at least seasonally oxygenated during the ancient low-stands and hence provided additional habitat for Oreochromis hunteri at those times. (d) Schematic representation of lacustrine habitat available to O. hunteri during high-stand (left) and low-stand (right) episodes, the former situation also characterizing Lake Chala today. The water column is divided into the approximate Fig. 1. (a) Abundance (no. per gram dry mass) of fossil cichlid teeth and of other fossil ﬁsh remains (bones, scales) in the sediment record of Lake Chala, in relation to the reconstructed sequence of past lake-level changes during the last 25,000 yr (Moernaut et al. 2010). The horizontal gray bars delineate the six time intervals studied here, representing four ancient high-stand episodes and two ancient low-stand episodes. (b) Map of equatorial East Africa with location of the principal great lakes Victoria, Tanganyika, and Malawi, and with Lake Chala indicated as a star. INTRODUCTION 2007, Bellwood ❖www.esajournals.org April 2019 ❖Volume 10(4) ❖Article e02664 2 DIELEMAN ET AL. Fig. 1. (a) Abundance (no. per gram dry mass) of fossil cichlid teeth and of other fossil ﬁsh remains (bones, scales) in the sediment record of Lake Chala, in relation to the reconstructed sequence of past lake-level changes during the last 25,000 yr (Moernaut et al. 2010). The horizontal gray bars delineate the six time intervals studied here, representing four ancient high-stand episodes and two ancient low-stand episodes. (b) Map of equatorial East Africa with location of the principal great lakes Victoria, Tanganyika, and Malawi, and with Lake Chala indicated as a star. (c) Bathymetry of Lake Chala as recorded in March 2003 with indication of the coring site (star), depth contours at 10-meter intervals (except the deepest contour at 94 m), and the crater–rim catchment boundary (dashed line). The dark gray shading shows the approximate extent of lake bottom area that remained anoxic throughout the past 25,000 yr. The light gray shading highlights the area of gently sloping lake bottom at the base of the steep underwater rock face nearshore that was likely at least seasonally oxygenated during the ancient low-stands and hence provided additional habitat for Oreochromis hunteri at those times. (d) Schematic i f l i h bi il bl O h t i d i hi h d (l f ) d l d ( i h ) i d Fig. 1. (a) Abundance (no. per gram dry mass) of fossil cichlid teeth and of other fossil ﬁsh remains (bones, scales) in the sediment record of Lake Chala, in relation to the reconstructed sequence of past lake-level changes during the last 25,000 yr (Moernaut et al. 2010). The horizontal gray bars delineate the six time intervals studied here, representing four ancient high-stand episodes and two ancient low-stand episodes. (b) Map of equatorial East Africa with location of the principal great lakes Victoria, Tanganyika, and Malawi, and with Lake Chala indicated as a star. (c) Bathymetry of Lake Chala as recorded in March 2003 with indication of the coring site (star), depth contours at 10-meter intervals (except the deepest contour at 94 m), and the crater–rim catchment boundary (dashed line). The dark gray shading shows the approximate extent of lake bottom area that remained anoxic throughout the past 25,000 yr. ❖www.esajournals.org April 2019 ❖Volume 10(4) ❖Article e02664 INTRODUCTION (c) Bathymetry of Lake Chala as recorded in March 2003 with indication of the coring site (star), depth contours at 10-meter intervals (except the deepest contour at 94 m), and the crater–rim catchment boundary (dashed line). The dark gray shading shows the approximate extent of lake bottom area that remained anoxic throughout the past 25,000 yr. The light gray shading highlights the area of gently sloping lake bottom at the base of the steep underwater rock face nearshore that was likely at least seasonally oxygenated during the ancient low-stands and hence provided additional habitat for Oreochromis hunteri at those times. (d) Schematic representation of lacustrine habitat available to O. hunteri during high-stand (left) and low-stand (right) episodes, the former situation also characterizing Lake Chala today. The water column is divided into the approximate April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 3 DIELEMAN ET AL. depth zones with year-round oxygenation (0–20 m depth, light blue), seasonal oxygenation (20–45 m depth, light-to-dark blue gradient), and permanent anoxia (>45 m depth, dark blue). During high-stand episodes, O. hunteri is restricted to rocky benthic habitat nearshore and the limnetic habitat offshore; food resources associ- ated with soft-bottom benthic habitat are accessible only during low-stand episodes. Fish and substrate drawings by Tim Goethals, combined with a seismic–reﬂection proﬁle (cf. Moernaut et al. 2010) showing a representative cross section of Lake Chala bathymetry. to ensure optimal exploitation of available food and reproductive resources, we hypothesized that this process would have induced morpho- logical changes in the oral dentition of O. hunteri, which might be traceable through time by mor- phometric analysis of fossil teeth preserved in the sediment record. Lake Chala offers a uniquely controlled setting for such a study, because of its isolation from other freshwater sys- tems, its continuous sedimentary archive con- taining abundant ﬁsh fossils, its well-studied environmental past, and the known recent tim- ing of the arrival of other ﬁsh species. However, despite being the type species of its genus, cur- rently only very general morphological and eco- logical descriptions of O. hunteri are available (Trewavas 1983). Reliable interpretation of its fos- sil record hence necessitated the construction of a quantitative reference frame for observed varia- tion in oral tooth morphology. Thus, we ﬁrst quantify the intraspeciﬁc variability in oral tooth morphology among O. INTRODUCTION hunteri in the modern- day population, followed by analyzing variabil- ity in the morphology of fossil oral teeth depos- ited during six periods with contrasting habitat conditions over the last 25,000 yr. Finally, we assess whether variation in tooth shape observed through time can be linked to independently documented environmental changes, and whet- her the observed change can be attributed to the sorting of genotypes under natural selection or to phenotypic plasticity. dissolved oxygen throughout the water column indicates that during the long dry season of July– September the well-oxygenated zone extends to a depth of ~45 m (Wolff et al. 2011, Buckles et al. 2014). For most of the year, only the uppermost 20–30 m is oxygenated, thereby restricting ﬁsh habitat to the steep-sloping rocky crater walls and the open-water (limnetic) zone. The rocks are covered by epilithic algae and interspersed with small sandy patches, providing a relatively continuous food source throughout the year. By contrast, limnetic productivity is largely restricted to a phytoplankton bloom in the dry season (Buckles et al. 2014, van Bree et al. 2018); during the rest of the year, low phytoplankton productivity implies poor open-water feeding conditions. The depositional center of Lake Chala has been continuously anoxic throughout the last 25,000 yr (Verschuren et al. 2009), allowing for excellent preservation of organic fossils (Meyer et al. 2018). However, the major lake-level decli- nes (up to ~40 m, Moernaut et al. 2010; Fig. 1a) which occurred during prolonged periods of regionally drier climate (Verschuren et al. 2009) must have created at least seasonally oxygenated conditions in soft-sediment areas of lake bottom at the base of the underwater rock face, which are currently too deep to be supplied with oxy- gen (Fig. 1d). Considering the low primary pro- ductivity of Lake Chala overall, availability of this additional benthic habitat during ancient low-stands is likely to have affected the ecology of O. hunteri. April 2019 ❖Volume 10(4) ❖Article e02664 Study system To study the extant population of O. hunteri, we purchased 88 specimens from ﬁshermen, fresh-caught at monthly intervals between Jan- uary 2014 and September 2015. Fish were pho- tographed in lateral view, and their standard length (SL; distance in cm between the tip of the snout and the posterior end of the last vertebra) The water level of modern-day Lake Chala is high: Steep, rocky shorelines all around quickly drop down to ~55–60 m depth, from where soft sediments slope more gently toward a maximum depth of ~90 m in the middle of the lake (Moer- naut et al. 2010; Fig. 1d). Monthly monitoring of April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 4 DIELEMAN ET AL. DIELEMAN ET AL. was measured to generate a body size–frequency distribution of the population (Fig. 2c). The heads of 12 specimens with SL values covering the full range of non-juvenile body sizes (here ≥16 cm, based on ﬁsherman’s catches) were fro- zen and transported to Belgium. Obtaining insight into variation of tooth morphology across the largest possible range of body sizes is important because individual cichlid ﬁsh develop multiple sets of teeth during their lives (polyphyodonty; Fryer and Iles 1972), all of which may end up in the fossil record. Upon arri- val in the laboratory, the ﬁsh were preserved in 10% formaldehyde for later tooth extraction. For this purpose, the oral jaws (left dentary, DTL; left premaxilla, PML) were dissected, cleared, and stained using a protocol adapted from Taylor and Van Dyke (1985); for details, see Dieleman et al. (2015). Oreochromis species typically pos- sess many oral teeth (between ~350 and ~950 in O. hunteri), implanted in the jaws in multiple rows (Trewavas 1983). O. hunteri generally has bicuspid teeth in the front row (FR; the outer ser- ies in ofﬁcial terminology; Barel et al. 1975) and tricuspid teeth in the second row (SR) and inner row (IR) of the jaw, together the so-called inner series. This large morphological disparity among the oral teeth of a single ﬁsh implies that establishing differences in average tooth mor- phology between specimens or populations (either in space or in time) requires measuring large numbers of teeth (Dieleman et al. 2015). For morphometric analysis of the modern-day population, we extracted 60 teeth from each specimen. This number was determined by itera- tively reducing the sample included in morpho- metric analysis of two test specimens (Dieleman et al. Fossil study material Extraction of teeth was done by stirring sediments for 30 min in H2O, after which the solution was sieved into two size fractions (150– 250 lm and >250 lm) that were both searched for fossils under a binocular microscope. Only oral teeth were retrieved, representing 27% of all fossil teeth encountered (Table 1). Pharyngeal teeth were counted but not analyzed further. The collected teeth were photographed at 609 mag- niﬁcation, clipped, re-photographed, and treated following the same procedure as adopted for modern-day teeth. Photographs of the strongly asymmetric bicuspid teeth were mirrored for direct comparisons, but the quasi-symmetric tri- cuspid and unicuspid teeth were not. Fossil teeth were grouped in .tps ﬁles per time interval. y In 2003 and 2005, the CHALLACEA project recovered a 20.82 m long composite sequence of ﬁnely laminated lacustrine muds from the center of Lake Chala, deposited continuously during the past 25,000 yr (Verschuren et al. 2009). For this study, we ﬁrst assessed the temporal distri- bution of ﬁsh fossils (teeth, scales, and bones) over this period by determining their abundance in 1026 contiguous 2-cm intervals throughout the core sequence, starting at 30 cm depth. For each interval, 2 g of untreated wet mud was rinsed on a 250-lm mesh sieve, and the retained residue was scanned under a binocular microscope at 25–409 magniﬁcation. These counts were visual- ized in R v.3.3.2 (R Development Core Team 2016) using the package rioja v.0.9.15 (Juggins 2017). Next, we used the lake-level reconstruc- tion based on seismic stratigraphy (Moernaut et al. 2010) to select six time intervals of between ~500 and ~1500 yr duration, from alternating lake high-stands and low-stands and which pro- mised sufﬁcient fossil teeth for comprehensive morphometric analyses (Fig. 1a). Given the con- siderable volume of sediment required for extracting sufﬁcient fossil teeth, the exact posi- tion and duration of selected intervals also depended on availability of adequate core mate- rial (Table 1). Therefore, samples for this study were not extracted from the already partly depleted master core sequence, but from overlap- ping duplicate core sections. The continuous lamination of Lake Chala sediments permits unambiguous cross-correlation of these duplicate sections to the master sequence. Study system 2015) to ﬁnd the lowest number that did not affect the total combined level of morpholog- ical variation represented in the dataset. These 60 teeth were comprised of 10, 5, and 15 teeth extracted randomly from respectively the FR, SR, and IR of both the DTL and the PML of each ana- lyzed specimen. y p Teeth were photographed at 609 magniﬁca- tion under a binocular microscope. The curvature of longer teeth hampered standardized orienta- tion of tooth crowns. Therefore, the crowns were clipped off and photographed again in a stan- dardized manner. To optimize digitization, the contrast of photographs was enhanced using Photoshop CS6, and PML teeth were mirrored for direct comparison with DTL teeth. The edited photographs were bundled per jaw bone and per specimen into .tps ﬁles using tpsUtil version 1.58 (Rohlf 2010a). 2 1 3 2 1 3 L W b 0 5 10 15 20 15 20 25 30 Standard Length (cm) a c Fig. 2. (a) Linear dimensions and morphometric reference points (landmark and semi-landmark positions) col- lected from representative bicuspid and tricuspid fossil teeth. (b) Images of the three main types of fossil teeth, namely unicuspid, bicuspid, and tricuspid. (c) Size–frequency distribution (white bars) of the standard length of the 88 studied Oreochromis hunteri specimens. Gray bars highlight the size distribution of the 12 specimens that were selected for morphometric analysis of oral tooth morphology in the modern-day population. 2 1 3 2 1 3 L W b 0 5 10 15 20 15 20 25 30 Standard Length (cm) a c a Fig. 2. (a) Linear dimensions and morphometric reference points (landmark and semi-landmark positions) col- lected from representative bicuspid and tricuspid fossil teeth. (b) Images of the three main types of fossil teeth, namely unicuspid, bicuspid, and tricuspid. (c) Size–frequency distribution (white bars) of the standard length of the 88 studied Oreochromis hunteri specimens. Gray bars highlight the size distribution of the 12 specimens that were selected for morphometric analysis of oral tooth morphology in the modern-day population. ❖www.esajournals.org April 2019 ❖Volume 10(4) ❖Article e02664 5 DIELEMAN ET AL. DIELEMAN ET AL. DIELEMAN ET AL. ❖www.esajournals.org April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 6 April 2019 ❖Volume 10(4) ❖Article e02664 Analysis of tooth size and shape 2015, 2019), a gen- eral comparison of the total morphospace occu- pied by the collections of fossil and modern-day teeth, and their respective overall average mor- phology, seemed appropriate. Subsequently, we statistically compared the morphospace occupa- tion of fossil teeth from the two low-stand and four high-stand episodes, ﬁrst by pooling tooth assemblages per type of lake phase and then pair- wise between each of the six fossil assemblages, with Bonferroni correction. Assumptions for mul- tivariate parametric tests, that is, multivariate normality and homogeneity of the variance–co- variance, were not met; consequently, we used permutation tests as implemented in the package Hotelling v.1.0.4 (Curran 2017). Using the inter- cusp angle method, teeth were then assigned to one of the three major tooth types, allowing exam- ination of the relative abundances of these tooth types in the six fossil assemblages. The signiﬁ- cance of differences in tooth-type abundances between low-stands and high-stands was tested by comparing the observed abundances with those obtained for 10,000 randomly sampled data- sets of the same sample size. the least-square correlations with Spearman’s rank tests. Finally, we compared the median EW of fossil teeth, per tooth type, from high-stands and low-stands using the two-sample Wilcoxon tests. Non-parametric tests were used through- out, due to violation of the assumptions for para- metric tests. the least-square correlations with Spearman’s rank tests. Finally, we compared the median EW of fossil teeth, per tooth type, from high-stands and low-stands using the two-sample Wilcoxon tests. Non-parametric tests were used through- out, due to violation of the assumptions for para- metric tests. The shape of tooth crowns was quantiﬁed with semi-landmark analysis including three land- marks (LMs) and two curves containing 20 semi- landmarks each, anchored to the LM (Dieleman et al. 2015; Fig. 2a). Tooth shape was digitized in tpsDig2 v.2.17 (Rohlf 2010b) and imported in IMP6 (Sheets 2008) for Procrustes superimposi- tion and perpendicular projection of semi-land- marks. Teeth for which one or more landmarks could not be assigned unambiguously were excluded, resulting in a dataset of 671 modern- day teeth and 886 fossil teeth, which was further analyzed in R. These morphometric data were examined with non-metric multidimensional scaling (NMDS) to reconstruct patterns of mor- phospace occupation (i.e., the distribution of morphological trait variation in ordination space) in two dimensions, using functions in the pack- ages vegan v.2.4.3 (Oksanen et al. 2017) and MASS v.7.3.45 (Venables and Ripley 2002). Analysis of tooth size and shape y p Data on tooth size and shape were obtained following methods described in Dieleman et al. (2015). Tooth length and narrowest width in micrometer were measured using ImageJ v. 1.49 m (Rasband 1997; Fig. 2a). Measurements were log-transformed (base 10) and further ana- lyzed in R. The neck of fossil teeth was often bro- ken, preventing measurement of total length, although their tops were usually sufﬁciently intact for inclusion in the morphometric dataset. Therefore, we examined to what extent enamel- oid (or more precisely, acrodin) width (EW, the Euclidean distance between landmarks 1 and 3 in Fig. 2a) can serve as a proxy for tooth length (TL) and body size (SL). Speciﬁcally, we corre- lated these measures from modern-day speci- mens for each tooth type separately and tested Table 1. Overview of the six fossil teeth assemblages (indicated with Roman numerals I–VI) extracted from sedi- ment samples that represent alternating lake high-stand (H) and low-stand (L) periods during the last 25,000 yr, with indication of their composite core depth, sample wet weight, and the numbers of oral teeth that were respectively recovered and included in the morphometric dataset. Table 1. Overview of the six fossil teeth assemblages (indicated with Roman numerals I–VI) extracted from sedi- ment samples that represent alternating lake high-stand (H) and low-stand (L) periods during the last 25,000 yr, with indication of their composite core depth, sample wet weight, and the numbers of oral teeth that were respectively recovered and included in the morphometric dataset. Assemblage Core depth (cm) Approximate age range (yrs BP) Lake level Weight (g) No. of oral teeth retrieved No. of oral teeth in dataset Top Bottom I 66.5 123 500–1000 H 645 148 104 II 239 275 2500–3000 H 383 199 162 III 600.5 640 7500–8000 L 491 544 463 IV 787 830.5 10,500–11,000 H 399 86 79 V 1328.5 1501 17,500–19,000 L 1860 77 63 VI 1861.5 1959 22,000–23,500 H 1224 26 15 Total 5002 1080 886 ❖www.esajournals.org 6 April 2019 ❖Volume 10(4) ❖Article e02664 DIELEMAN ET AL. Considering the unavoidable difference in sam- pling methods, our collections of modern-day and fossil teeth cannot be considered fully equivalent, precluding detailed quantitative comparisons. However, as both collections derive from the same species (Dieleman et al. April 2019 ❖Volume 10(4) ❖Article e02664 Analysis of tooth size and shape The resulting stress value (8.9) is low according to the criteria of Kruskal (1964) and Clarke (1993), indi- cating no risk for false interpretations. g p As mentioned, tooth-shape variation within the modern-day O. hunteri population was ana- lyzed separately to develop a comparative framework for morphological variation in the fossil teeth. Although the oral teeth of cichlids are generally described as unicuspid, bicuspid, or tricuspid (Trewavas 1983; Fig. 2b), the assign- ment of individual teeth to one of these tooth types is often somewhat ambiguous. Hence, we examined the robustness of our results under two quantitative classiﬁcation methods, one based on the angles between cusps and one based on the clustering of tooth shape in mor- phospace using a Gaussian mixture model. As the two methods yielded highly similar results (Appendix S1: Figs. S1, S2), we here only present results based on inter-cusp angles. In this method, the number of cusps is determined by counting the number of crown incisions with an angle <135°. Tricuspids possess two such inci- sions, bicuspids one, and unicuspids none. Modern-day reference framework y f f The SL of the 88 O. hunteri specimens collected in Lake Chala ranged from 16 to 30 cm, with 81% of all specimens measuring between 23 and 29 cm (Fig. 2c). Modern-day teeth become both longer and wider as body size increases, and because the increase in width is proportionally larger, they become more robust (Fig. 3). Enameloid width of all three tooth types correlates strongly with tooth length (unicuspids, r = 0.85, P < 0.001; bicuspids, r = 0.82, P < 0.001; tricuspids, r = 0.90, P < 0.001; Appendix S1: Fig. S3a) and with body size (uni- cuspids, r = 0.55, P < 0.001; bicuspids, r = 0.71, P < 0.001; tricuspids, r = 0.74, P < 0.001; Appen- dix S1: Fig. S3b). Therefore, we can use EW in fossils as a proxy of tooth length, and the EW dis- tributions of fossil ﬁsh assemblages as an esti- mate of median body size. Inferring the mechanism of morphological change Inferring the mechanism of morphological change Observed differences in tooth morphology between low-stand and high-stand episodes may be caused by genotype sorting driven by natural selection or by phenotypic plasticity. To differen- tiate between these potential drivers, we exam- ined the variation in morphospace occupation among fossil assemblages, based on the expec- tation that selection intensity would be different between low-stands and high-stands, and hence produce a systematic difference in the amount of morphological variation. Alternatively, if obse- rved morphological change is mainly driven by phenotypic plasticity, we expected to ﬁnd similar levels of morphological variation during low- stands and high-stands. For this analysis, we pooled all specimens in a high-stand and a low- stand group, and compared the morphospace occupation between both lake states with the betadisper function of vegan. The multivariate April 2019 ❖Volume 10(4) ❖Article e02664 7 ❖www.esajournals.org DIELEMAN ET AL. DIELEMAN ET AL. gradient of symbol color reﬂecting body size of the source specimens. Non-metric multidimen- sional scaling axis 1 discriminates between uni- or bicuspids (negative values) and tricuspids (positive values; Fig. 4b), whereas NMDS axis 2 reﬂects the distinction between unicuspids (neg- ative values) and bi- or tricuspids (positive val- ues; Fig. 4c). Tooth-shape changes related to growth are evident along both NMDS axes: The predominantly bi- and tricuspid teeth of small specimens (green symbols) are gradually replaced by unicuspid teeth in larger specimens (red symbols). The same morphospace with teeth classiﬁed by tooth type based on inter- cusp angles is visualized in Fig. 5a. Despite some overlap, teeth assigned to each of the three tooth types mostly occupy distinct loca- tions in morphospace. The FR consists predomi- nantly of bicuspid teeth, whereas the SR and IR consist mostly of tricuspid teeth (Fig. 5b), especially in small individuals. As ﬁsh grow, their dentition becomes increasingly unicuspid (Fig. 5c). homogeneity of variances between both was then tested with an ANOVA on the Euclidean dis- tances of group members to the group centroid after principal coordinate analysis. April 2019 ❖Volume 10(4) ❖Article e02664 Fossil teeth assemblages −0.2 −0.1 0.0 0.1 −0.2 −0.1 0.0 0.1 0.2 NMDS1 NMDS2 15 20 25 30 cm a c b SL b Fig. 4. (a) Morphospace occupation plot of the shape variation in oral tooth crowns of modern-day Oreochromis hunteri as reconstructed by non-metric multidimensional scaling (NMDS), with symbol colors reﬂecting the body size (SL, standard length) of the ﬁsh from which each tooth was sampled. (b, c) Tooth crown shape as repre- sented by semi-landmark positions, showing the outlines which correspond with the highest (black) and lowest (gray) values along respectively NMDS 1 (b) and NMDS 2 (c). −0.2 −0.1 0.0 0.1 NMDS2 −0.2 −0.1 0.0 0.1 0.2 NMDS1 a 0.00 0.25 0.50 0.75 1.00 Proportion Inner Front Second b 0.00 0.25 0.50 0.75 1.00 Proportion C142 C119 C129 C003 C124 C126 C131 C019 C122 C023 C018 C151 c Unicuspid Bicuspid Tricuspid Fig. 5. (a) Morphospace occupation plot of the shape variation in oral tooth crowns of modern-day Oreochromis hunteri as reconstructed by non-metric multidimensional scaling (NMDS), with symbols color-coded according to tooth type. (b) Average proportion of uni-, bi-, and tricuspid teeth in the front, second, and inner teeth rows of the oral jaw of 12 specimens. (c) Proportions of the three tooth types in each of 12 specimens, ordered by increas- ing body size from C142 (standard length [SL] = 16 cm) to C151 (SL = 30 cm). 0.2 0.00 0.25 0.50 0.75 1.00 Proportion Inner Front Second b 0.00 0.25 0.50 0.75 1.00 Proportion C142 C119 C129 C003 C124 C126 C131 C019 C122 C023 C018 C151 c Unicuspid Bicuspid Tricuspid −0.2 −0.1 0.0 0.1 NMDS2 −0.2 −0.1 0.0 0.1 0.2 NMDS1 a Fig. 5. (a) Morphospace occupation plot of the shape variation in oral tooth crowns of modern-day Oreochromis hunteri as reconstructed by non-metric multidimensional scaling (NMDS), with symbols color-coded according to tooth type. (b) Average proportion of uni-, bi-, and tricuspid teeth in the front, second, and inner teeth rows of the oral jaw of 12 specimens. (c) Proportions of the three tooth types in each of 12 specimens, ordered by increas- ing body size from C142 (standard length [SL] = 16 cm) to C151 (SL = 30 cm). O. hunteri (Fig. 6b). When the fossil assemblages are grouped according to lake level, a signiﬁcant difference is observed between the average shape of oral teeth from O. Fossil teeth assemblages g Enameloid width distributions show that the recovered fossil oral teeth (n = 886) are signiﬁ- cantly smaller, on average, than our reference collection of modern-day O. hunteri teeth (Fig. 6a; W = 114,790, P < 0.001). Grouping fossil teeth assemblages according to lake level reveals that teeth deposited during a high-stand episode (subtotal n = 360) are signiﬁcantly smaller, on average, than those deposited during a low- stand (subtotal n = 526; W = 77, 434, P < 0.001). However, in all three tooth types separately the individual assemblages display a marked decline in median tooth size from older (VI) to younger (I) age since deposition (Appendix S1: Fig. S4), indicating that the former result may be biased by the abundant material from assemblage III (Table 1). Also, although most samples are skewed toward larger teeth, this skewness is enhanced in older samples (Appendix S1: Fig. S4), suggesting that post-depositional corro- sion results in a disproportionate loss of smaller teeth over long time periods. The morphospace occupation of all 671 mod- ern-day teeth is plotted in Fig. 4a, with a Tooth length (µm) Tooth width (µm) 15 20 25 30 cm 400 300 200 100 1000 2000 3000 SL Fig. 3. Relationship between the length and width of oral teeth in 12 modern-day Oreochromis hunteri specimens as a function of body size (SL, standard length), indicated with a gradient of symbol colors, with linear regression lines reﬂecting variation in this relationship among specimens. The morphospace occupation of fossil teeth, as represented by six fossil assemblages covering the past 25,000 yr, overlaps almost completely with that of the modern-day population of April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 8 DIELEMAN ET AL. −0.2 −0.1 0.0 0.1 −0.2 −0.1 0.0 0.1 0.2 NMDS1 NMDS2 15 20 25 30 cm a c b SL Fig. 4. (a) Morphospace occupation plot of the shape variation in oral tooth crowns of modern-day Oreochromis hunteri as reconstructed by non-metric multidimensional scaling (NMDS), with symbol colors reﬂecting the body size (SL, standard length) of the ﬁsh from which each tooth was sampled. (b, c) Tooth crown shape as repre- sented by semi-landmark positions, showing the outlines which correspond with the highest (black) and lowest (gray) values along respectively NMDS 1 (b) and NMDS 2 (c). April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org Fossil teeth assemblages 7a), of which the most recent (from episode I) are between ~500 and ~1000 yr old (Table 1). Multivariate permutation tests yield signiﬁcant differences in ﬁve out of eight Bonferroni-cor- rected pairwise comparisons of the individual high- and low-stand assemblages (Table 2). Clas- sifying fossil teeth based on inter-cusp angles reveals a somewhat larger overlap between tooth types in fossils (Fig. 7b) than in modern-day teeth (Fig. 5a). Consistent with our observations in NMDS morphospace (Fig. 7a), the proportion of unicuspid teeth is found to be markedly higher during the two ancient low-stand epi- sodes than during high-stand episodes (Fig. 7c), with the average increase in unicuspid teeth dur- ing low-stands (~10%) being signiﬁcantly larger (P < 0.01) than expected by chance. Finally, the variance in morphospace occupation of fossil teeth from ancient high-stands is signiﬁcantly shape explained by the two NMDS axes is identi- cal to that in modern-day teeth (Fig. 4b, c), on average more negative NMDS2 values similarly indicate a greater proportion of unicuspid teeth in ancient O. hunteri which lived during low- stands. As might be expected, the average tooth morphology of modern-day O. hunteri, which experience high-stand conditions (Fig. 1a), plot- ted closest to the centroids of fossil teeth assem- blages deposited during past high-stands (Fig. 7a), of which the most recent (from episode I) are between ~500 and ~1000 yr old (Table 1). Multivariate permutation tests yield signiﬁcant differences in ﬁve out of eight Bonferroni-cor- rected pairwise comparisons of the individual high- and low-stand assemblages (Table 2). Clas- sifying fossil teeth based on inter-cusp angles reveals a somewhat larger overlap between tooth types in fossils (Fig. 7b) than in modern-day teeth (Fig. 5a). Consistent with our observations in NMDS morphospace (Fig. 7a), the proportion of unicuspid teeth is found to be markedly higher during the two ancient low-stand epi- sodes than during high-stand episodes (Fig. 7c), with the average increase in unicuspid teeth dur- ing low-stands (~10%) being signiﬁcantly larger (P < 0.01) than expected by chance. Finally, the variance in morphospace occupation of fossil teeth from ancient high-stands is signiﬁcantly larger than that of those from low-stands (F = 5.7742, DF = 1; 888, P = 0.016; Fig. 8), indi- cating that overall morphological variation is reduced during low-stands. Fossil teeth assemblages hunteri living during high-stands and those living during low-stands (T2 = 85.25, P < 0.001). Moreover, when comparing the two low-stand and four high-stand assem- blages separately, this shift in tooth morphology is shown to be recurrent through time (Fig. 7a). The difference is mainly situated along NMDS2, with on average more negative values for “low- stand” teeth. Since the variation in fossil tooth April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 9 DIELEMAN ET AL. 0 30 60 90 100 200 300 400 Enameloid width (µm) 100 200 300 400 −0.2 −0.1 0.0 0.1 0.2 −0.2 −0.1 0.0 0.1 0.2 NMDS1 NMDS2 a b Modern Fossil Fossil Modern Number of teeth Fig. 6. (a) Enameloid width (EW) distributions of the 671 oral teeth of modern-day Oreochromis hunteri (ochre) and of 886 fossil teeth (green) from the sediment record of Lake Chala. Inset boxplots show the respective median EW values (thick vertical line), interquartile ranges (box width), and outliers (black dots). (b) Morphospace occu- pation plot comparing the overall shape variation in oral tooth crowns of modern-day O. hunteri (ochre circles) and fossil teeth (green triangles) as reconstructed by non-metric multidimensional scaling (NMDS). −0.2 −0.1 0.0 0.1 0.2 −0.2 −0.1 0.0 0.1 0.2 NMDS1 NMDS2 b 0 30 60 90 100 200 300 400 Enameloid width (µm) 100 200 300 400 a Modern Fossil F Number of teeth Fossil Modern Fig. 6. (a) Enameloid width (EW) distributions of the 671 oral teeth of modern-day Oreochromis hunteri (ochre) and of 886 fossil teeth (green) from the sediment record of Lake Chala. Inset boxplots show the respective median EW values (thick vertical line), interquartile ranges (box width), and outliers (black dots). (b) Morphospace occu- pation plot comparing the overall shape variation in oral tooth crowns of modern-day O. hunteri (ochre circles) and fossil teeth (green triangles) as reconstructed by non-metric multidimensional scaling (NMDS). shape explained by the two NMDS axes is identi- cal to that in modern-day teeth (Fig. 4b, c), on average more negative NMDS2 values similarly indicate a greater proportion of unicuspid teeth in ancient O. hunteri which lived during low- stands. As might be expected, the average tooth morphology of modern-day O. hunteri, which experience high-stand conditions (Fig. 1a), plot- ted closest to the centroids of fossil teeth assem- blages deposited during past high-stands (Fig. April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org The oral dentition of modern-day Oreochromis hunteri Our data show that during their lifetime, mod- ern-day O. hunteri in Lake Chala undergo signiﬁ- cant changes in oral dentition. First, the teeth become gradually wider and more robust with increasing body size (Fig. 3). Secondly, larger ﬁshes develop a greater proportion of unicuspid teeth (Figs. 4, 5). Speciﬁcally, small ﬁshes have bicuspid FR teeth followed by tricuspid SR and IR teeth, whereas very large ﬁshes possess an almost entirely unicuspid oral dentition. Individ- uals of intermediate body size have an interme- diate dentition: They largely retain the bicuspid/ tricuspid arrangement, but the major/middle tooth cusp becomes more dominant relative to the side cusp(s). The change toward a predomi- nantly unicuspid dentition in very large speci- mens is thus gradual rather than abrupt (Fig. 4). g p g This observed change to unicuspid FR teeth and, eventually, an (almost) entirely unicuspid oral dentition is known to occur in large April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 10 DIELEMAN ET AL. DIELEMAN ET AL. −0.2 −0.1 0.0 0.1 0.2 NMDS2 −0.2 −0.1 0.0 0.1 0.2 NMDS1 −0.2 −0.1 0.0 0.1 0.2 NMDS2 −0.2 −0.1 0.0 0.1 0.2 NMDS1 0.00 0.25 0.50 0.75 1.00 Proportion V I II VI IV III a b c V I III IV II VI Unicuspid Tricuspid Bicuspid Fig. 7. (a) Morphospace occupation plot of shape variation in oral tooth crowns among the six f sil teeth assemblages from Lake Chala, as reco structed by non-metric multidimensional scali (NMDS) and with separate colors for each of the fo ancient high-stand episodes (green triangles) and t ancient low-stand episodes (yellow and och individuals of several other Oreochromis species, mainly in sexually mature males (Trewavas 1983). Most cichlids display dietary shifts as they grow (Njiru et al. 2008), and these are often accompanied by changes in different aspects of their trophic morphology (Streelman et al. 2007). This study is, to our knowledge, the ﬁrst to docu- ment this ontogenetic change in cichlid oral den- tition with geometric morphometrics, and it reveals that the changes occur gradually, at least in O. hunteri. In other cichlid taxa, a unicuspid oral dentition is often linked to feeding at a higher trophic level (Fryer and Iles 1972), but ore- ochromines are rarely entirely piscivorous or insectivorous. Most Oreochromis species, includ- ing modern-day O. hunteri in Lake Chala, are herbivorous (Trewavas 1983). The oral dentition of modern-day Oreochromis hunteri We therefore con- cur with Trewavas (1983) that the shift to unicus- pid dentition in O. hunteri is likely associated instead with sexual maturation and enhanced territorial behavior linked to breeding. −0.2 −0.1 0.0 0.1 0.2 NMDS2 −0.2 −0.1 0.0 0.1 0.2 NMDS1 a V I III IV II VI −0.2 −0.1 0.0 0.1 0.2 NMDS2 −0.2 −0.1 0.0 0.1 0.2 NMDS1 b Unicuspid Tricuspid Bicuspid Trends in fossil teeth abundance and preservation p The presence of ﬁsh remains throughout the studied sediment record (Fig. 1a) demonstrates that ﬁsh have been continuously present in Lake Chala over at least the past 25,000 yr. Lack of a systematic contrast in the total abundance of ﬁsh fossils between high-stand and low-stand epi- sodes further suggests that lake-level ﬂuctuations had a negligible inﬂuence on post-mortem trans- port of ﬁsh remains to the mid-lake coring site. Nevertheless, fossil abundances vary consider- ably over the last 25,000 yr, showing a distinct increase in abundance ~10,500 yr ago, that is, shortly after the Pleistocene–Holocene transition. 0.00 0.25 0.50 0.75 1.00 Proportion V I II VI IV III c triangles). Also indicated are their respective centroid scores (ﬁlled stars), in relation to the centroid score of tooth-shape variation in the modern-day Oreochromis hunteri population (red-ﬁlled star). (b) The same mor- phospace occupation plot of all fossil teeth as in (a) but with colors representing the three principal tooth types. (c) Proportions of the three tooth types in each of the six fossil assemblages from alternating high- stand (I, II, IV, and VI) and low-stand (III and V) epi- sodes. Low-stand assemblages (gray bars) display a signiﬁcantly larger proportion of unicuspid teeth. (Fig. 7. continued) triangles). Also indicated are their respective centroid scores (ﬁlled stars), in relation to the centroid score of tooth-shape variation in the modern-day Oreochromis hunteri population (red-ﬁlled star). (b) The same mor- phospace occupation plot of all fossil teeth as in (a) but with colors representing the three principal tooth types. (c) Proportions of the three tooth types in each of the six fossil assemblages from alternating high- stand (I, II, IV, and VI) and low-stand (III and V) epi- sodes. Low-stand assemblages (gray bars) display a signiﬁcantly larger proportion of unicuspid teeth. (Fig. 7. continued) Fig. 7. (a) Morphospace occupation plot of the shape variation in oral tooth crowns among the six fos- sil teeth assemblages from Lake Chala, as recon- structed by non-metric multidimensional scaling (NMDS) and with separate colors for each of the four ancient high-stand episodes (green triangles) and two ancient low-stand episodes (yellow and ochre Fig. 7. Trends in fossil teeth abundance and preservation (a) Morphospace occupation plot of the shape variation in oral tooth crowns among the six fos- sil teeth assemblages from Lake Chala, as recon- structed by non-metric multidimensional scaling (NMDS) and with separate colors for each of the four ancient high-stand episodes (green triangles) and two ancient low-stand episodes (yellow and ochre April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 11 DIELEMAN ET AL. last 25,000 yr was found in genetic analyses of modern-day O. hunteri (Dieleman et al. 2019); however, the sample size of that study may have been too small to pick up such a signature. In principle, the observed trend in fossil abundance down-core could also partly be due to tapho- nomic effects. Chemical corrosion by organic acids in sediments makes teeth progressively more brittle over time, which may not only decrease the overall abundance of teeth in older samples, but also progressively reduce the pro- portion of smaller teeth, as they are likely to cor- rode faster. However, the relatively modest changes in mean tooth size with age across the six assemblages (Appendix S1: Fig. S4) compared to the abrupt, order-of-magnitude increase in the abundance of ﬁsh fossils ~10,500 BP (Fig. 1a) indicate that the latter almost certainly reﬂects a true increase in the O. hunteri population at that time. Table 2. Bonferroni-corrected P-values of permutation tests for each pairwise comparison of oral tooth mor- phology between the six fossil tooth assemblages. High High Low High Low I II III IV V High II 0.74 Low III <0.01* <0.01* High IV 1.00 1.00 0.03* Low V 0.02* <0.01* 1.00 0.17 High VI 1.00 1.00 1.00 1.00 1.00 Notes: Direct pairwise comparison between the morphol- ogy of fossil and modern-day tooth assemblages is not appro- priate, because the two datasets are not fully equivalent (see Methods and Discussion). * Signiﬁcance at the 5% level. Notes: Direct pairwise comparison between the morphol- ogy of fossil and modern-day tooth assemblages is not appro- priate, because the two datasets are not fully equivalent (see Methods and Discussion). ) * Signiﬁcance at the 5% level. Our data on EW indicate that fossil teeth are somewhat smaller, on average, than those of modern-day ﬁshes (Fig. 6a). ❖www.esajournals.org Trends in fossil teeth abundance and preservation The main reason for this difference is probably that the smallest speci- mens in our modern-day survey are 16 cm, due to the prescribed large mesh size of ﬁshing nets and the minimum size of ﬁsh deemed suitable for selling, whereas teeth of smaller ﬁsh also con- tribute to the fossil record. On the other hand, these smaller teeth may be more susceptible to corrosion after long-term burial, and/or our 150- lm mesh sieve may not have retained all of those present in the sediment. Also, cichlids are poly- phyodont (Fryer and Iles 1972), and each tooth has an average life span of around 100 d (Tuisku and Hildebrand 1994). Assuming that the life span of O. hunteri is similar to that of other Ore- ochromis species (up to ~10 yr; Froese and Pauly 2017), each large specimen contributes up to three dozen sets of teeth to the sediment record during its life, further increasing the proportion of small teeth. Thus, the smaller median size of fossil teeth conforms to expectation. In the end, whereas the minimum tooth size in our modern-day reference collection is mainly controlled by the mesh size of ﬁsh nets, that in fossil assemblages is controlled by the sieves used for sediment processing. Mod- ern-day and fossil assemblages are hence not exactly equivalent as to tooth size; however, their morphospace occupation is nevertheless highly similar (Figs. 6b, 7a). Fig. 8. Principal coordinate analysis plot showing that overall morphological variation is signiﬁcantly larger among fossil teeth deposited during ancient high-stand episodes (black circles; n = 363) than among fossil teeth deposited during low-stand epi- sodes (red triangles; n = 527), as measured by the Euclidean distances of individual teeth to their respec- tive group centroid (solid black and red circles). Along this same transition, a marked increase in the organic carbon content of the sediments is observed (Blaauw et al. 2011), presumably reﬂecting increased aquatic primary production (Meyer et al. 2018) associated with regional cli- mate warming around that time (Loomis et al. 2017). This enhanced productivity may in turn have increased the lake’s carrying capacity to sustain a larger ﬁsh population. No evidence of a pronounced demographic expansion within the April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 12 DIELEMAN ET AL. DIELEMAN ET AL. Trends in fossil teeth abundance and preservation Temporal trends in oral tooth shape dentition could have been advantageous to prey on the invertebrate macrobenthos that likely developed during low-stands in the then oxy- genated and accessible soft-bottom sediments at the foot of the steep underwater rock face (Fig. 1d). However, insectivorous oreochromines are rare in all of the diverse aquatic habitats they occupy, so the probability that O. hunteri would have acquired that habit when presented the opportunity may be rather low. Alternatively, an earlier shift to unicuspid dentition might indicate that low-stand ﬁshes reached sexual maturity at a smaller body size (i.e., earlier age) than those living during high-stands. Population-level intraspeciﬁc variability in body size and age at maturity is common in Oreochromis species, and strongly linked to environmental variation (Duponchelle and Panﬁli 1998). p p The near-complete congruence between fossil and modern-day oral teeth in morphospace (Fig. 6b) conﬁrms pre-1980s historical accounts (G€unther 1889, Dadzie et al. 1988) and genetic evidence (Dieleman et al. 2019) that O. hunteri has been the only ﬁsh species inhabiting Lake Chala before the recent introductions, and this for at least the last 25,000 yr. The overall variabil- ity in tooth shape in the living population has thus been remarkably stable over time, implying that our examination of modern-day O. hunteri provides a robust framework for interpretation of the fossil assemblages representing ancient O. hunteri populations. The similarity of mod- ern-day teeth with those of ancient O. hunteri liv- ing under comparable high-stand conditions as recently as ~500–1000 yr ago (Fig. 7a) also implies that any change in oral tooth morphol- ogy that may have occurred since the 1980s, due to several decades of co-habitation and, hence, possible interference competition with the intro- duced ﬁsh species (character displacement; Brown and Wilson 1956), is modest compared with the mean difference in tooth morphology between ancient high-stand and low-stand populations. ( p ) Besides marked trends in the proportion of uni- cuspid teeth potentially reﬂecting changes in life- history traits, we also documented signiﬁcantly smaller overall variation in the oral tooth mor- phology of ﬁshes living during low-stand periods compared to high-stand periods (Fig. 8). April 2019 ❖Volume 10(4) ❖Article e02664 Trends in fossil teeth abundance and preservation This adaptive response increased its chance of long-term popu- lation persistence in an aquatic ecosystem pre- senting the dual challenge of being both sensitive to climate change (producing constantly shifting selective pressures) and hydrographically iso- lated from neighboring populations (limiting gene ﬂow). Indeed, this endemic ﬁsh population survived at least four major climate-driven envi- ronmental upheavals (potential population bot- tlenecks) within the currently documented part of lake history. In a broader context, the results of this study support the often invoked but rarely documented hypothesis that past climate-driven lake-level ﬂuctuations have stimulated evolu- tionary radiation in the cichlid communities of East Africa’s large rift lakes. populations of Oreochromis jipe (at least 25,000 yr but possibly >200,000 yr; Dieleman et al. 2019) eventually led to the evolution of endemic O. hunteri. Thus, even if a sequence of ancient lake-level ﬂuctuations did not directly promote speciation in O. hunteri, they may still have been instrumental in creating genetic divergence from its more widespread sister species O. jipe. Modiﬁcation of the dental apparatus in response to climate-driven habitat change has frequently been hypothesized to be an important step in cichlid radiation (Kocher 2004, Danley et al. 2012, Ivory et al. 2016). However, evidence supporting this hypothesis is thus far mainly derived from phenotype–environment correla- tions that do not necessarily imply causality. In this study, we exploited the unique setting of Lake Chala’s sediment record and a reference framework of tooth morphology in modern-day O. hunteri to link morphological trends in fossil teeth assemblages directly to the independently reconstructed evidence for long-term habitat change. Speciﬁcally, our analyses revealed sys- tematic and recurrent changes in O. hunteri tooth morphology in response to a sequence of ancient lake-level ﬂuctuations. Our data further suggest that selection played an important role in the observed morphological change, notwithstand- ing the substantial level of phenotypic plasticity that has been documented for the dental appara- tus of oreochromine and other cichlids (Ribbink 1990, van Rijssel et al. 2015, Ndiwa et al. 2016). Currently, we cannot exclude the possibility that phenotypic plasticity played a role in providing the initial variation on which selection subsequently acted. In fact, the long-term persis- tence of O. hunteri in Lake Chala, and other oreochromines inhabiting ﬂuctuating aquatic ecosystems (Ribbink 1990), may result from plas- ticity-driven adaptation, that is, the ability to plastically modify morphological features and subsequently reﬁne them through selection. Trends in fossil teeth abundance and preservation This ﬁnding suggests that natural selection either reduced the amount of standing variation in the oral dentition of low-stand populations, for exam- ple, by increased competition or a reduction of total population size, or alternatively that diversi- fying selection (as opposed to simply relaxing selection) may have occurred during high-stands. Our present data and earlier genetic analysis (Dieleman et al. 2019) indicate that such selection did not cause population divergence in O. hunteri, contrasting with the rapid phenotypic and geno- mic differentiation and diversiﬁcation being reported in haplochromine cichlids (Malinsky et al. 2015, Moser et al. 2018). Apparently, the recurrent morphological changes documented here have enabled the endemic population of O. hunteri to cope with successive events of local environmental change without promoting specia- tion. In this and possibly other oreochromine cich- lids, long-term population separation and the genetic drift associated with any population bot- tlenecks during this time may be more probable mechanisms for the formation of new species than adaptation to ecologically contrasting habitats. The operation of these mechanisms is exempliﬁed in Lake Chala, where long-term isolation of the local ﬁsh population from neighboring Focusing on the fossil tooth assemblages, our data revealed signiﬁcant and recurrent changes in the morphospace occupation of oral teeth from ﬁsh populations living at different times in the past (Fig. 7), synchronous with the indepen- dently documented lake-level changes (Moer- naut et al. 2010; Fig. 1a). Speciﬁcally, during past episodes of low lake level, an average increase of 10% in the relative abundance of unicuspid teeth is observed (Fig. 7c). The differences in oral dentition between past high-stand and low-stand populations of O. hunteri in Lake Chala are over- all similar to the ontogenetic changes in oral dentition which occur in the modern-day popu- lation. However, averaged per tooth type the mean size of teeth from low-stand assemblages is not systematically larger than that of teeth from high-stand assemblages (Appendix S1: Fig. S4), arguing against a larger mean body size of low-stand O. hunteri populations. Rather, our data suggest that during low-stand episodes O. hunteri shifted toward a unicuspid dentition earlier in life, likely triggered by a change in available habitat. For example, the unicuspid April 2019 ❖Volume 10(4) ❖Article e02664 ❖www.esajournals.org 13 DIELEMAN ET AL. DIELEMAN ET AL. local habitat change, and to repeatedly do so over very long periods of time. ACKNOWLEDGMENTS This study was carried out under Memorandum of Understanding A14/TT/0923 between Ghent University and the National Museums of Kenya (NMK), and insti- tutional afﬁliation of Dirk Verschuren with NMK. We thank Caxton Oluseno and the ﬁshermen of Lake Chala for ﬁeld assistance, and Maarten Van Daele for assis- tance in cross-correlating duplicate core sections to the dated master core sequence. Walter Salzburger, Jos Snoeks, George Turner, Ann Huysseune, Dries Bonte, and Dominique Adriaens provided many insightful comments which improved this paper. This work was ﬁnanced by Ghent University Research Council through Collaborative Research Activity “Deep- CHALLA” (2013–2018; Jorunn Dieleman and Dirk Ver- schuren) and by ANR-JCJC-EVOLINK (Bert Van Bocxlaer). The lake-sediment cores used in this study were collected under permit 13/001/11C of the Kenyan Ministry of Education, Science and Technology, in the framework of the European Science Foundation’s Euro- CLIMATE project CHALLACEA (2005–2008). Trends in fossil teeth abundance and preservation Such adaptation may play an important role in specia- tion (Thompson et al. 2018) and in evolutionary radiation (West-Eberhard 2005, Pfennig et al. 2010, Muschick et al. 2011, Laland et al. 2014); however, in Oreochromis species it is perhaps pri- marily a powerful mechanism to cope with recurrent environmental ﬂuctuation. Our data, certainly, provide evidence for the ability of O. hunteri to rapidly and systematically adapt to LITERATURE CITED Barel, C. D. N., F. Witte, and M. J. P. Van Oijen. 1975. The shape of the skeletal elements in the head of a generalized Haplochromis species: H. elegans Tre- wavas 1933 (Pisces, Cichlidae). Netherlands Jour- nal of Zoology 26:163–265. Bellwood, D. R., A. S. Hoey, O. Bellwood, and C. H. R. Goatley. 2014. Evolution of long-toothed ﬁshes and the changing nature of ﬁsh-benthos interactions on coral reefs. 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Science 333:743–747. q Wagner, C. E., L. J. Harmon, and O. Seehausen. 2012. Ecological opportunity and sexual selection together predict adaptive radiation. Nature 487: 366–369. Wolff, C., I. Kristen-Jenny, G. Schettler, B. Plessen, H. Meyer, P. Dulski, R. Naumann, A. Brauer, D. Ver- schuren, and G. H. Haug. 2014. Modern seasonality in Lake Challa (Kenya/Tanzania) and its sedimen- tary documentation in recent lake sediments. Lim- nology and Oceanography 59:1621–1636. West-Eberhard, M. J. 2005. Developmental plasticity and the origin of species differences. Proceedings of the National Academy of Sciences USA 102: 6543–6549. April 2019 ❖Volume 10(4) ❖Article e02664 SUPPORTING INFORMATION SUPPORTING INFORMATION Additional Supporting Information may be found online at: http://onlinelibrary.wiley.com/doi/10.1002/ecs2. 2664/full nal Supporting Information may be found online at: http://onlinelibrary.wiley.com/doi/10.1002/ecs2 ❖www.esajournals.org April 2019 ❖Volume 10(4) ❖Article e02664 17
https://openalex.org/W2519651977	https://www.ssoar.info/ssoar/bitstream/document/50327/1/ssoar-politicsgovernance-2016-3-bang_et_al-The_Paris_Agreement_Short-Term_and.pdf	English	null	The Paris Agreement: Short-Term and Long-Term Effectiveness	Politics and governance	2,016	cc-by	8,724	www.ssoar.info Issue This article is part of the issue “Climate Governance and the Paris Agreement”, edited by Jon Hovi and Tora Skodvin (University of Oslo, Norway). © 2016 by the authors; licensee Cogitatio (Lisbon, Portugal). This article is licensed under a Creative Commons Attribu- tion 4.0 International License (CC BY). term effectiveness we mean the extent to which Paris may be expected to contribute to reducing global emissions in its first five-year period. By long-term ef- fectiveness we mean the extent to which it is likely to reduce global emissions in a time perspective longer than five years. The Paris Agreement: Short-Term and Long-Term Effectiveness Guri Bang 1, Jon Hovi 2,* and Tora Skodvin 2 Guri Bang 1, Jon Hovi 2,* and Tora Skodvin 2 1 Center for International Climate and Environmental Research, 0349 Oslo, Norway; E-Mail: guri.bang@cicero.oslo.no 2 Department of Political Science, University of Oslo, 0317 Oslo, Norway; E-Mails: jon.hovi@stv.uio.no (J.H.), tora.skodvin@stv.uio.no (T.S.) * Corresponding author Submitted: 10 April 2016 \| Accepted: 9 July 2016 \| Published: 8 September 2016 Keywords l h Keywords climate change mitigation; climate cooperation; effectiveness, international leadership; Paris Agreemen Abstract The 2015 Paris Agreement was widely greeted with enthusiasm. We assess the short-term and long-term potential effectiveness of Paris. Concerning short-term effectiveness, we contend that while Paris scores high on participation, and reasonably high on the depth of the parties’ commitments (ambition), its Achilles’ heel will likely be compliance. Concerning long-term effectiveness, we argue that Paris does little to restructure states’ incentives so as to avoid free riding. At worst, it might end up as a failure, much like Kyoto did. On the other hand, domestic and international norms could continue to develop in a direction that makes it more and more difficult for individuals, firms, and states alike to ignore the plea to limit and reduce their carbon footprints. Technological progress that gradually reduces abatement costs, combined with leadership by major emitters such as the United States, might further strengthen climate cooperation and enhance other countries’ willingness to follow through. However, deep political polarization continues to represent a significant barrier to U.S. leadership on climate change. www.ssoar.info The Paris Agreement: Short-Term and Long-Term Effectiveness Bang, Guri; Hovi, Jon; Skodvin, Tora Veröffentlichungsversion / Published Version Zeitschriftenartikel / journal article Empfohlene Zitierung / Suggested Citation: Bang, G., Hovi, J., & Skodvin, T. (2016). The Paris Agreement: Short-Term and Long-Term Effectiveness. Politics and Governance, 4(3), 209-218. https://doi.org/10.17645/pag.v4i3.640 Empfohlene Zitierung / Suggested Citation: Bang, G., Hovi, J., & Skodvin, T. (2016). The Paris Agreement: Short-Term and Long-Term Effectiveness. Politics and Governance, 4(3), 209-218. https://doi.org/10.17645/pag.v4i3.640 Empfohlene Zitierung / Suggested Citation: Bang, G., Hovi, J., & Skodvin, T. (2016). The Paris Agreement: Short-Term and Long-Term Effectiveness. Politics and Governance, 4(3), 209-218. https://doi.org/10.17645/pag.v4i3.640 Terms of use: This document is made available under a CC BY Licence (Attribution). For more Information see: https://creativecommons.org/licenses/by/4.0 Nutzungsbedingungen: Dieser Text wird unter einer CC BY Lizenz (Namensnennung) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by/4.0/deed.de Politics and Governance (ISSN: 2183-2463) 2016, Volume 4, Issue 3, Pages 209-218 doi: 10.17645/pag.v4i3.640 Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 2. Criteria for Evaluation To be effective, a climate agreement must satisfy three requirements (Barrett, 2003). First, it must attract broad participation to ensure that most (or preferably all) of the world’s anthropogenic greenhouse gas (GHG) emissions are covered by the agreement. Second, to make a real difference for curbing climate change the agreement must reflect high ambitions, in the sense that it must commit the participating countries to deep emis- sions reductions. Finally, the agreement must achieve high compliance rates, that is, the participating countries must actually meet their emissions reduction targets. An agreement can be effective in the short and/or in the long term. Short-term effectiveness concerns the extent to which the agreement is effective in its own lifetime (here defined as the first 5-year period). Long- term effectiveness concerns the extent to which the agreement triggers future actions that enhance effec- tiveness in a longer time perspective (beyond the first 5-year period). The Paris Agreement includes both individual and collective goals. Each party reports a nationally deter- mined contribution (NDC), which constitutes that party’s individual goal. Two key collective goals in the Paris Agreement are, first, to keep the rise in global mean temperatures well below 2 °C and “pursue efforts” to limit warming to 1.5 °C and, second, to achieve worldwide car- bon neutrality sometime between 2050 and 2100. We emphasize that these three conditions for ef- fectiveness apply to all climate agreements, regardless of their type. In particular, they apply regardless of whether the participating countries’ commitments to curbing emissions are determined top down or bottom up. They also apply regardless of the extent to which the participating countries are held responsible for their ac- tions internationally or only at the national level. y An assessment of the agreement’s effectiveness thus needs to take into account (1) whether the parties comply with their individual goals, and (2) whether the aggregate effect of a successful implementation of the individual goals enables the parties to reach the agreement’s collective goals. In terms of Barrett’s three requirements, the latter point concerns whether the parties’ (self-determined2) emissions reduction targets qualify as deep commitments. If the aggregate effect of reaching the individual goals is inadequate to reach the collective goals of the agreement, we further need to consider whether the agreement includes mechanisms that may trigger deeper (more ambitious) commit- ments over time. 2. Criteria for Evaluation The long-term effectiveness of the Paris Agreement thus depends on its ability to preserve ambitious commitments over time—and further deep- en them if necessary—while preserving broad partici- pation and high compliance levels. Importantly, satisfying one or two requirements is not enough. In the words of Simmons (1998, p. 78), “while compliance may be necessary for effectiveness, there is no reason to consider it sufficient”. The same can be said about broad participation and deep com- mitments: Whereas each condition is necessary, all three must be fulfilled to ensure effectiveness. Conversely, any climate agreement that fails to sat- isfy at least one of the requirements will experience free riding in one or more forms. Consider the Kyoto Protocol. Kyoto 1 seems to have achieved perfect compliance levels; indeed, all Annex B countries met or even exceeded their emissions reduction or limitation targets in the first commitment period.1 Nevertheless, when Kyoto 1 expired in 2012 global emissions were approximately 50% higher than they were in 1990 (Kyoto’s baseline year). The reason is that Kyoto 1’s ef- fectiveness was hampered by at least four forms of free riding (Hovi, Skodvin, & Aakre, 2013). First, a few countries—most notably the United States—failed to ratify. Second, one country—Canada—essentially ig- nored its emissions reductions commitment until it withdrew from the agreement in 2011. Third, some economies in transition participated with extremely shallow targets that they could easily meet or exceed even without implementing any measures specifically intended to curb emissions (the hot air problem). Final- ly, the developing countries ratified without any bind- ing target for emissions reductions or limitations. 1. Introduction While the expectations for the 2015 Paris climate change meeting were modest, the outcome—the Paris Agreement—was widely greeted with enthusiasm (e.g., Brun, 2016; Dimitrov, 2016). A bottom-up agreement, Paris requires countries to submit nationally deter- mined contributions (NDCs) to reducing global emis- sions. It also includes a set of mechanisms for ratchet- ing up these NDCs over time. Partly because the NDCs are not legally binding, Paris does not include any en- forcement measures. Importantly, therefore, the inten- tion is that once key countries make first steps towards cutting their emissions, others will follow suit. Section 2 clarifies the criteria we use to assess the Paris Agreement’s effectiveness. Sections 3 and 4 con- sider the Agreement’s short-term and long-term effec- tiveness, respectively. We argue that its short-term ef- fectiveness will likely be limited. Moreover, its long- term effectiveness remains uncertain. At worst, the Agreement might prove to be a failure. At best, it might cause a race to the top that eventually makes it very ef- fective in the long term. The outcome will likely de- pend on, among other things, whether major emitters In this paper, we assess the short-term and long- term effectiveness of the Paris Agreement. By short- 209 Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 prove able and willing to take the lead. In section 5 we discuss the prospects for such leadership by one major emitter that has a particularly important role as a first mover—the United States. Finally, section 6 concludes. We do not dispute that even an agreement that falls somewhat short of fulfilling all three requirements can make a difference, by causing global emissions to become lower than they would have been without the agreement. However, no agreement that falls signifi- cantly short of fulfilling one or more requirements can even come close to solving the climate change problem. 1 http://phys.org/news/2016-06-kyoto-analysis-compliance.html 2 In contrast to the Kyoto Protocol, where the parties’ individu- al goals were negotiated (a “top-down approach”), the parties’ individual goals in the Paris Agreement are self-determined (a “bottom-up” approach). The climate change problem is a collaboration prob- lem; indeed, it may well be the most challenging col- laboration problem ever (Barrett, 2003). The Kyoto Protocol was ratified by nearly all coun- tries. However, after the United States denounced the agreement, Kyoto imposed an emissions reduction or limitation target on only 37 ratifying countries (36 after Canada’s withdrawal in 2011). Moreover, these 37 countries were responsible for less than 20% of global emissions. In this sense, participation in Kyoto was ra- ther limited. The second factor concerns the depth of the par- ties’ commitments.4 A shallow commitment does not require any costly behavioural change. In contrast, im- plementing a deep commitment might entail very sub- stantial costs (Downs, Rocke, & Barsoom, 1996). Nearly all economic activity entails GHG emissions (Barrett, 2003). Thus, deep commitments in a climate agreement will be very costly to implement. The deeper the com- mitments, the greater the costs involved, and the great- er the risk of noncompliance (other things being equal). In contrast, a major strength of the Paris Agree- ment is its broad participation. In Paris, 195 countries consented to the Agreement’s adoption and by 1 March 2016, 165 countries—including major emitters such as China and the United States—had submitted their NDCs under the Agreement.3 It is thus fair to say that participation is far broader in the Paris Agreement than it was in the Kyoto Protocol. The third factor has to do with the presence or ab- sence of enforcement measures. In general, the im- plementation of a climate agreement may be influ- enced through at least four main types of compliance mechanisms: (1) pressure from domestic stakeholders (including through domestic courts), (2) informal en- forcement by other countries, (3) facilitation by inter- national institutions, and (4) enforcement by interna- tional institutions.5 While Kyoto relied on all four types, it seems that Paris will only rely on the first three. In Kyoto, Annex I countries committed to reducing emissions about 5% below 1990 levels in the first commitment period. Many of these countries have made considerably deeper commitments under the Paris Agreement. For example, the European Union has com- mitted to reducing emissions 40% below 1990 levels by 2030. Moreover, the non-Annex I countries have now joined the Annex I countries in making commitments. 3 NDCs submissions are available at: http://www4.unfccc.int/ submissions/indc/Submission%20Pages/submissions.aspx 3. Short-Term Effectiveness In this section, we consider the short-term effective- ness of the Paris Agreement. We do so by assessing how this Agreement fares regarding participation, the depth of participating countries’ commitments, and the prospects for achieving high compliance levels. Be- cause we expect compliance to be the Achilles’ heel of the Paris Agreement, we place more emphasis on the last requirement than on the first two. 2 In contrast to the Kyoto Protocol, where the parties’ individu- al goals were negotiated (a “top-down approach”), the parties’ individual goals in the Paris Agreement are self-determined (a “bottom-up” approach). 1 http://phys.org/news/2016-06-kyoto-analysis-compliance.html Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 210 4 This means that commitment depth influences the Agree- ment’s effectiveness both directly (a positive effect) and indi- rectly (via reduced compliance, a negative effect). The net ef- fect therefore depends on the size of these direct and indirect effects. 4 This means that commitment depth influences the Agree- ment’s effectiveness both directly (a positive effect) and indi- rectly (via reduced compliance, a negative effect). The net ef- fect therefore depends on the size of these direct and indirect effects. 5 For example, the 1989 Montreal Protocol on Substances that Deplete the Ozone Layer, which is widely regarded as a very successful treaty, includes a possibility of enforcement in the form of trade restrictions against nonparticipating and non- compliant countries (e.g., see Aakre, Helland, & Hovi, 2014). While these enforcement measures have never been used, an- ecdotal evidence suggests that their existence have been im- portant for boosting participation as well as compliance (Brack, 2003, p. 220). 6 S htt // / li / li i /i t ti l/ ti The Paris Agreement requires each country to re- port to other countries— and to the public—its pro- gress in implementing its NDC. It also includes an ambi- tion to “track progress towards the long-term goal through a robust transparency and accountability sys- tem”.6 However, Paris provides no material conse- quences to be implemented against a country that fails to fulfil its NDC. Work is currently underway to develop a compliance system; however, Paris specifically states that any compliance measures should be “expert-based and facilitative in nature and function in a manner that is transparent, non-adversarial and non-punitive”. Some countries’ NDCs are formulated in a way that makes it challenging to determine whether and, if so, to what extent their fulfilment requires emissions re- ductions beyond business as usual. Consequently, it is not easy to determine the average depth of the com- mitments made under the Paris Agreement. However, observers seem to think that the current NDCs, if fully implemented, will curb global warming to somewhere between 2.7 °C and 3.0 °C above preindustrial levels, down from the 3.6 °C expected to result with the poli- cies already in place before (and not including) Paris (Kinver, 2015). Thus, a substantial gap remains between the Agreement’s collective aims and the sum of the parties’ individual NDCs. It is well known that compliance with international environmental agreements is generally good, despite that few such agreements include potent enforcement measures (Chayes & Chayes, 1995). However, it is far from clear that these findings are relevant for deep climate agreements, where the costs of compliance are An important question is whether the current NDCs will be fully implemented. Under Paris, NDCs are not legally binding. Hence, one may well question whether it is meaningful to speak of noncompliance if a country fails to fulfil its NDC. However, for lack of better terms, we will nevertheless use the terms “compliance” and “noncompliance”. Compliance with an international agreement de- pends on many factors, of which we will here briefly consider three. The first is whether the agreement concerned aims to solve a coordination problem or a collaboration problem (Stein, 1990). While countries participating in an agreement of the former type do not face any incentive for noncompliance, countries participating in an agreement of the latter type do (see, for example, Keohane & Oppenheimer, 2016). A way to circumvent the need for enforcement could be to design a climate agreement in such a way as to transform the climate change collaboration prob- lem into a coordination problem. Some agreements that introduce a technology standard in the presence of network externalities might serve as an example (see Barrett, 2003). However, the Paris agreement makes little (if any) attempt at pursuing this strategy. Proponents of the management school provide a forceful argument that the design of international en- forcement measures is ‘a waste of time’, because states are (according to this school) largely motivated by international norms rather than by self-interest (Chayes & Chayes, 1995).7 They also argue that simply doing the best one can to reach compliance is efficient, because constantly looking for opportunities to act as a free rider would consume considerable resources. In summary, the Paris Agreement scores high on participation and scores reasonably high on depth (alt- hough assessing such depth is challenging). Thus, con- cerning short-term effectiveness, the main issue is to what extent the countries participating in Paris will ac- tually fulfil their NDCs. Whereas some countries (such as the EU countries) may be expected to be compliant, it remains an open question whether a number of oth- er countries will be. However, even with some non- compliance, Paris could end up as a reasonably suc- cessful agreement. Lack of enforcement measures may induce some countries to submit deeper NDCs than they would have done otherwise (Victor, 2011). The irony is that the deeper the commitments, the larger the need for enforcement and the less likely that such enforcement will be politically feasible (Aakre & Hovi, 2010). However, at least in principle, an agreement with deep commitments and only moderate compli- ance levels might fare better than one with only shal- low commitments but full compliance. Concerning climate change, however, the record for commitments without international enforcement is not impressive. While participants in the 1988 Toronto conference collectively aimed to reduce global CO2 emissions 20% below 1988 levels by 2005, global emis- sions actually increased more than 30% between 1988 and 2005. As noted by Barrett (2008, p. 240), the prob- lem with such global targets is that “everyone is re- sponsible for meeting them—meaning, of course, that no one is responsible for meeting them”. 5 For example, the 1989 Montreal Protocol on Substances that Deplete the Ozone Layer, which is widely regarded as a very successful treaty, includes a possibility of enforcement in the form of trade restrictions against nonparticipating and non- compliant countries (e.g., see Aakre, Helland, & Hovi, 2014). While these enforcement measures have never been used, an- ecdotal evidence suggests that their existence have been im- portant for boosting participation as well as compliance (Brack, 2003, p. 220). 6 See http://ec.europa.eu/clima/policies/international/negotia tions/paris/index_en.htm Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 211 likely to be much larger than in other international en- vironmental agreements. reduction targets is not good either. After the Toronto conference, Austria, Denmark, Italy, and Luxembourg stated that they would meet the Toronto target individ- ually by reducing their own emissions 20% below 1988 levels by 2005. None of them did. Other individual tar- gets have also been missed (Barrett, 2008).8 Although much has changed since the 1980s, it is far from clear that we can take it for granted that all countries will fulfil their NDCs under the Paris Agreement. Because NDCs are not legally binding, enforcement through domestic legal action is also unlikely. Moreover, judged by Canada’s experience after its withdrawal from Kyoto, we should not expect much informal enforce- ment by other members if a country fails to deliver on its NDC or even withdraws from the Paris agreement. Thus, what remains is the possibility of naming and shaming by domestic and international politicians and pressure groups. The risk of such naming and shaming did not deter the United States from denouncing the Kyoto Protocol in 2001. Nor did it deter Canada from withdrawing 10 years later. It therefore seems perti- nent to question whether the anticipation of informal enforcement will suffice to induce all (or even most) of the 195 parties to the Paris Agreement to implement their NDCs. Technically, failure to do so may not result in noncompliance; it may equally well end in the coun- tries concerned pulling out of the agreement (similar to Canada’s withdrawal from Kyoto). Assuming that enforcement matters, the countries most likely to comply with Paris are the EU members. The reason is that the EU controls a number of en- forcement measures of its own that might be used to spur its members to fulfil their targets. 8 For a complete list of declared targets or commitments un- dertaken by OECD countries at this time, see Paterson and Grubb (1992, p. 301). 7 Following Elster (1989, pp. 98-99), we define a norm as an imperative that is not outcome-oriented. A norm is social if it is shared by several actors and partly sustained by other actors’ approval or disapproval. Barrett’s words also apply to the Paris Agreement’s collective goal of stabilizing the global mean temperature at no more than 2 °C above preindustrial levels, while pursu- ing efforts to limit warming to 1.5 °C. Indeed, the Paris Agreement’s collective goal also suffers from the prob- lem that the global mean temperature partly depends on natural variations; hence, it is even less under the control of the parties than global emissions are. obligations such as the ones mentioned above, it is largely silent regarding the substantive content of the parties’ NDCs. For instance, the Agreement includes a general aim “to reach global peaking of [GHGs] as soon as possible” with “rapid reductions thereafter” (Article 4.1), instructs parties that their NDCs will reflect their “highest possible ambition” (Article 4.3), and states that developed country parties “should continue taking the lead by undertaking economy-wide absolute emis- sion reduction targets” (Article 4.4). However, the par- ties’ commitments will continue to be self-determined and may thus, in aggregate, fall well short of reaching the collective 2 °C long-term goal. If so, the Paris Agreement may, even with full compliance and even in the long term, prove unable to close the gap COP21 expressed its concern about. In its adoption of the Paris Agreement, COP21 em- phasized its “serious concern” for the “urgent need to address the significant gap between the aggregate ef- fect of Parties’ mitigation pledges…and aggregate emission pathways consistent with holding the in- crease in the global average temperature to well below 2 °C above pre-industrial levels and pursuing efforts to limit the temperature increase to 1.5 °C above pre- industrial levels” (Decision 1/CP.21, preamble). In the short term, the Paris Agreement does little to close this gap. A key question, therefore, is whether the Paris Agreement includes mechanisms that contribute to closing the gap over time. The main mechanism to ensure the Agreement’s long-term effectiveness is detailed in Article 4, which states that each party “shall prepare, communicate and maintain successive nationally determined contribu- tions [NDCs] that it intends to achieve” (Article 4.2). These successive NDCs “will represent a progression beyond the Party’s then current [NDCs] and reflect its highest possible ambition” (Article 4.3, emphasis add- ed). Each party is invited to communicate its NDC, at the latest, when it submits its instrument of ratifica- tion, acceptance, approval or accession. Parties are then requested to communicate a new NDC by 2020 and every five years thereafter (Article 4.9; Decision 1/CP.21, paragraphs 21-24). Second, and perhaps more importantly, the Agree- ment’s lack of an enforcement mechanism might gen- erate uncertainty concerning some parties’ willingness to honour their commitments. A “compliance mecha- nism” is established to “facilitate implementation and promote compliance with the provisions of this Agreement” (Article 15.1). But this mechanism will al- most certainly include no incentives to actively dis- courage and sanction noncompliance. On the contrary, the Paris Agreement expressly states that the compli- ance mechanism “shall be…non-adversarial and non- punitive” (Article 15.2). Thus, parties might be tempted to communicate over-ambitious NDCs—for instance, targets they do not know if they are able to implement, are unlikely to implement, or even do not intend to implement—with impunity. An NDC thus represents lit- tle more than a party’s good intentions. If there are reasons to doubt a party’s sincerity, other parties’ will- ingness to implement ambitious emissions reductions might be weakened accordingly. The lack of an en- forcement mechanism, therefore, could limit the Agreement’s long-term effectiveness, because it means that even if all parties intend to implement ambitious emissions reductions, they might nevertheless be re- luctant to do so simply because they lack confidence in other parties’ willingness to honour their commit- ments. Thus, even if we might see an initial upward cy- cle of progressively rigorous NDCs, the long-term effec- tiveness of the Agreement could be modest. This mechanism is supported by the Agreement’s reporting, transparency, and monitoring provisions. First, a party is obliged to “regularly provide a national GHG inventory and the information necessary to track progress in implementing and achieving its NDC” (Bo- dansky & O’Connor, 2016, p. 10), (Article 13.7a, b). Second, parties are required to submit their NDCs to the secretariat at least 9–12 months in advance of the relevant COP/MOP meeting “with a view to facilitate the clarity, transparency and understanding of these contributions”. The NDCs are then summarized in a synthesis report prepared by the secretariat (Decision 1/CP.21, paragraph 25; Article 4.9). Third, a monitoring function of sorts is established in the inclusion of provi- sions for a “global stocktake”, which implies that the COP “shall periodically take stock of the implementa- tion of this Agreement to assess the collective progress towards achieving the purpose of this Agreement and its long-term goals” (Article 14.1). Such global stocktak- ing shall take place every five years beginning in 2023 (i.e., two years before the parties’ NDCs are up for their first revision). The lack of an enforcement mechanism is arguably the Agreement’s main weakness. 4. Long-Term Effectiveness The long-term effectiveness of the Paris agreement depends on (1) whether a successful implementation of the parties’ individual goals (the NDCs) enables the parties to reach their long-term goals of the agree- ment, and (2) whether the agreement includes mecha- nisms to further deepen commitments over time, while also ensuring high compliance rates and (continued) broad participation. A better strategy might be to aim for individual tar- gets, and the Paris Agreement specifies individual tar- gets in the form of NDCs. However, the record concern- ing compliance with individual non-binding emissions 7 Following Elster (1989, pp. 98-99), we define a norm as an imperative that is not outcome-oriented. A norm is social if it is shared by several actors and partly sustained by other actors’ approval or disapproval. Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 212 Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 5. The United States—A Pivotal Actor in the Paris Agreement The United States is a crucial player in the Paris Agree- ment, not only because it is the world’s second largest emitter of GHGs but also because it has a special role to play in terms of triggering action from other coun- tries. As described above, the Agreement relies inher- ently on vagueness in its specification of commitments, compliance requirements and the ratcheting-up mech- anism (see also Keohane & Oppenheimer, 2016). While such vagueness was necessary diplomacy for forging the Agreement itself and attract broad participation, it locks success to reliance on key countries to go first and set the stage for a snowball effect (or race to the top). As argued by Underdal (1994, pp. 179-180), “the more complex the negotiation setting (that is, the larg- er the number of actors and the number and ‘intricacy’ of issues), the more likely that some actors will emerge as leaders and others as followers…, and the more crit- ical leadership becomes as a determinant of success”. While the Paris Agreement includes provisions in- tended to increase its effectiveness over time, no in- centives are included to support these provisions and to ensure their intended effect. The long-term effec- tiveness of the Paris Agreement thus entirely depends on other factors. One such factor might be the reputational damage that could be associated with nonparticipation and noncompliance. Bodansky and O’Connor suggest that “states risk greater costs to their reputation and to their relations with other states if they violate a treaty commitment than a political commitment, making non- compliance less attractive” (2016, p. 16). But he also suggests that ‘the inclusion of transparency and ac- countability mechanisms in the Paris agreement could accomplish the same result. By making it more likely that poor performance will be detected and criticized, these mechanisms will raise the reputational costs of failing to achieve one’s NDC’ (2016, pp. 16-17). Yet, as we have already seen, the risk of reputational costs does not seem to have significantly influenced the United States’ decision not to ratify Kyoto 1, Canada’s decision to withdraw from it, or the decision of coun- tries such as Belarus, Japan, New Zealand, Russia, and Ukraine not to sign on to Kyoto 2. Arguably, the United States must play an important role in such leadership for at least two reasons. by accelerating investments in “green energy” and de- celerating investments in fossil energy, it could have significant implications for future emissions reductions. While experts see it as “likely” that the Paris Agree- ment “will accelerate investments in technologies like renewable energy and electric vehicles”, the Interna- tional Energy Agency forecasts that “fossil fuels will still account for about 75 percent of energy demand in 2030, with coal hitting a plateau, oil growing slightly and gas surging” (Campbell, 2015). The interlinkage between participation, the depth of commitments, and enforcement implies that even if parties were to supplement the Paris Agreement with an enforcement mechanism in the future (there are no indications at present that they will), that might ad- versely affect the incentive to remain a party and/or the incentive to submit an ambitious NDC. First, with an enforcement mechanism in place, parties that fail to implement their NDCs might withdraw from the Agreement rather than remain a party and suffer the costs of punitive sanctions for noncompliance. Article 28 states that “at any time after three years from the date on which this Agreement has entered into force for a Party, that Party may withdraw from the Agreement” (28.1), and that “any such withdrawal shall take effect upon expiry of one year from the date of receipt by the Depositary of the notification of withdrawal” (28.2). Finally, as noted, the long-term effectiveness of the Paris Agreement mostly depends on the extent to which parties trust each other to implement their commitments. In particular, the Paris Agreement’s long-term effectiveness will likely be significantly influ- enced by the response of large global emitters. In the next section, we consider the likelihood that the United States will act as a leader under the Paris Agreement. Second, with an enforcement mechanism in place, parties might feel inclined to submit less ambitious NDCs, both to be certain of their ability to meet the targets (and hence avoid punitive sanctions) and to en- able them subsequently to submit progressively more rigorous NDCs at low cost. In these scenarios, there- fore, rather than seeing the positive snowball effect everyone hopes for, we might see that the Agreement will have decreasing participation and an increasing gap between the aggregate effect of parties’ mitigation pledges and the required aggregate emission pathway to reach the 2 °C goal. Yet, this feature might also be the cause of the Agreement’s most hailed achievement: participation by a broad scope of countries (including developed countries as well as de- veloping countries and countries with emerging econ- omies) that have set specified emissions reduction or control targets. Indeed, parties may have agreed to join the Agreement with emissions control targets pre- cisely because there is no risk associated with such be- haviour. While noncompliance might entail reputation- al damage, it will not entail immediate material costs. So the Agreement does indeed include mechanisms intended to enhance its long-term effectiveness. How- ever, it remains uncertain whether these mechanisms suffice to ensure the positive snowball effect (or race to the top) many participants and observers hope for. This uncertainty is due to two key features of the Agreement. First, while the Agreement establishes procedural 213 Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 5. The United States—A Pivotal Actor in the Paris Agreement First, as the largest economy in the world and the second larg- est carbon emitter the United States is often pointed to as a key actor because of its major historical responsi- bilities for the climate change problem (e.g. World Re- sources Institute, 2016). What happens with U.S. cli- mate policy has an effect for the entire world. Second, in the history of international climate negotiations the United States has often played the role as crucial lag- gard, blaming lack of participation by all major emitters and flawed treaty design for its own non-participation— for instance in the Kyoto Protocol. Experience thus in- dicates that for any comprehensive international cli- mate agreement to work, it is vital that the world’s most powerful country shows interest in participation and compliance with its pledges. If a pivotal actor like Another factor concerns the market responses to the Agreement. If the Paris Agreement generates new investment patterns in key global markets, for instance 214 Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 gress to pass climate legislation. Several bills were de- bated, and in June 2009 Congressmen Waxman and Markey’s American Clean Energy and Security Act nar- rowly passed a vote in the House yet later died in the Senate (Bang & Skodvin, 2014). Voting on this contro- versial bill largely followed party lines, reflecting deep and bitter polarization between Republicans and Dem- ocrats (Skocpol, 2013). No climate bill has been debat- ed in the U.S. Congress since, and with Republican ma- jorities in both the House and the Senate after the 2012 elections, climate legislation was no longer on the congressional agenda. In his second term, therefore, President Obama decided to use executive powers to circumvent the congressional gridlock on climate poli- cy. Acting on the U.S. Supreme Court’s ruling from 2009, which identified carbon emissions as a pollutant causing risks to the health and welfare of citizens, Obama ordered the Environmental Protection Agency (EPA) to develop regulations under the Clean Air Act to cut CO2 emissions in the power sector by 32% by 2030 (Bang, 2015). The EPA worked with stakeholders and state-level regulators over two years (2013–2015) to set up regulations—the Clean Power Plan—that en- gage states to design individual plans for cutting car- bon emissions from power plants. 5. The United States—A Pivotal Actor in the Paris Agreement States are assigned individual emissions reduction targets, and are encour- aged to find policy solutions adapted to local circum- stances to minimize negative effects on industry and consumers (EPA, 2015). the United States should fail to implement its commit- ments, it will likely negatively affect other parties’ in- centives to adopt and implement ambitious NDCs. In the following pages, we analyse the role that the United States took at COP 21 and in the months pre- ceding the conference to secure the adoption of a bot- tom-up agreement. We argue that the United States took several crucial steps to influence the outcome. We also analyse the domestic politics underlying the U.S. position at COP 21, to enable an evaluation of the prospects for the country to deliver on its Paris pledg- es. The dynamic between domestic politics and inter- national negotiation positions is important in order to understand the scope for acceptance of international commitments in all countries. In the U.S. case this dy- namic is influenced by the separation of powers in the political system that gives the president great free- doms in international negotiations, but allows the Sen- ate decisive powers in issues of treaty ratification and funding of new policy programs. Hence, the interaction between the executive and legislative branches of gov- ernment defines the scope for U.S. pledges at Paris, as well as the prospects to fulfil them. It helps explain why the United States can come to play an important role for the success of the Paris Agreement if it engages in a role as first mover (in a race to the top), but can also explain why in a difficult collaboration problem like climate change the United States may have difficulties in taking on such leadership. In Paris, U.S. diplomacy and personal engagement by President Barack Obama and Secretary of State John Kerry helped craft compromises that were necessary for the adoption of the Agreement. Importantly, Obama engaged with China’s President Xi through cru- cial bilateral contact in the months before the Paris meeting, paving the way for support of the Agreement by both countries (Goldenberg, 2014; Henderson, 2015). 5. The United States—A Pivotal Actor in the Paris Agreement At the Paris meeting, President Obama said: “I’ve come here personally, as the leader of the world’s largest economy and the second-largest emitter, to say that the United States of America not only recognizes our role in creating this problem, we embrace our re- sponsibility to do something about it” (White House, 2015). The Obama administration acknowledged that the United States can play a key role in inspiring and convincing other countries to address their GHG emis- sions, and committed to reducing U.S. GHG emissions 26 to 28% below 2005 levels by 2025, premised on numerous domestic policy measures that have been or are to be implemented (Bang & Schreurs, 2016). Ambi- tious domestic investments in clean energy, energy ef- ficiency programs, and new federal regulations limiting carbon emissions from power plants are among the climate policies initiated by the Obama administration. The Clean Power Plan (CPP) is extremely controver- sial among policymakers at both the federal and the state levels. The controversy centres on whether the Clean Air Act gives the president and the EPA the au- thority to introduce wide-ranging regulations for CO2 emissions without involving the U.S. Congress. Oppo- nents at the federal and state levels have sought to put up barriers. Republican leaders in Congress have vowed to cancel the CPP at the first opportunity. Sen- ate Majority Leader Mitch McConnell (Republican from Kentucky) encouraged states not to start developing plans, arguing that they would be wasting resources since the CPP will likely be removed either through congressional action by the Republican majority or through the courts (Cama, 2016). Several votes in Con- gress in 2013–2015 tried to remove the EPA’s authority on the issue; however, those bills that passed were ve- toed by the president. In October 2015, a coalition of 26 states—many of which depend heavily on coal for power generation—brought litigation against the EPA, arguing that the CPP represented a “power grab” by the federal government over state-level electricity sys- tems that would be excessively burdensome for the states’ economies (Bang & Schreurs, 2016). A signifi- cant blow to the Obama administration’s climate policy came in February 2016 when a 5-4 decision in the Su- preme Court stayed implementation of the CPP until judicial review of its legality. The Court’s decision illus- The Obama administration has fought hard for changes in domestic climate policy. Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 6. Conclusion To build an effective climate agreement and to strengthen it over time, states might rely on two main types of factors—norms and incentives. An ideal agreement would ensure that both norms and incen- tives push the parties to make serious efforts to reduce emissions and to gradually reinforce those efforts. To build an effective climate agreement and to strengthen it over time, states might rely on two main types of factors—norms and incentives. An ideal agreement would ensure that both norms and incen- tives push the parties to make serious efforts to reduce emissions and to gradually reinforce those efforts. The Paris Agreement currently relies disproportion- ately on norms, while doing little to restructure states’ incentives so as to deter free riding. Norms and incen- tives thus pull in opposite directions, meaning that the outcome will depend on the force of each factor. Be- cause virtually all economic activity entails emissions of GHGs, the incentive to free ride is much stronger in the context of climate change than in the context of other international environmental cooperation. Unsurprising- ly, therefore, the historical record of climate change cooperation suggests that the force of incentives has thus far outweighed that of norms. Judged by this rec- ord, the Paris Agreement may well suffer a fate similar to Kyoto’s. Kyoto, too, aimed for a series of 5-year pe- riods with new and more ambitious commitments in every period. Yet already by the end of the first period, this architecture was clearly not viable. g g ( ) These different views regarding the need for cli- mate policy action convey the level of willingness to recognize the Paris Agreement as a priority for the United States. While President Obama and a clear ma- jority in the Democratic Party are fully committed, Re- publican leaders reacted very differently to the Agree- ment’s adoption. The immediate reaction of leading Republicans after COP 21 indicated a looming fight over the commitment to the Paris Agreement. Senate Majority Leader Mitch McConnell said, “Obama is mak- ing promises he can't keep” and should remember that the Agreement “is subject to being shredded” after the 2016 election. With reference to the presidential elec- tion, McConnell said the Agreement could be reversed if the Republicans win the White House (Freking, 2015). Republicans argued that the deal is simply politically binding, not judicially binding, and hence barely worth any serious attention. 5. The United States—A Pivotal Actor in the Paris Agreement At the outset of his first term, President Obama pushed for the U.S. Con- 215 Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 United States from any further involvement with the “non-binding” Paris Agreement. The Democratic Party Convention in 2016 supported both a carbon tax, con- tinuation of Obama’s climate policy programs, and ful- filment of pledges made in Paris. Presidential candi- dates Donald Trump and Hillary Clinton mirror their party’s opposing views, hence representing starkly dif- ferent ways forward for U.S. climate policy. trates the fragility of the Obama administration’s cli- mate policy and the significant role of judicial review when executive power is used to impose policy in a controversial field. If the Supreme Court decides to hear the case, its ruling might influence the future of the CPP as well as the U.S. ability to fulfil its commit- ment in the Paris Agreement. Many U.S. states have put on hold any effort to de- velop state implementation plans for adhering to the CPP, while other states are pursuing climate action re- gardless of the uncertainty surrounding the plan’s fu- ture (C2ES, 2016). Clearly, deep polarization in U.S. cli- mate policy affects the United States’ ability to live up to the promises it made in Paris. Without a firm do- mestic policy strategy, like the CPP or some other fed- eral climate policy, investors and business owners will have weak incentives to make long-term business deci- sions that include a pathway to a low carbon economy. Moreover, the U.S. NDC under the Paris Agreement will be less credible. U.S. political parties greatly disagree on the importance of climate policy action. Most Dem- ocrats accept that climate change is a serious problem that requires political action to reduce emissions. Most Republicans, on the other hand, are not committed to addressing the climate change problem, because they do not believe in the science or because they think it is premature to risk the potential economic hardship that climate action might bring (Leiserowitz et al., 2016). In sum, deep political polarization over climate change prevents the United States from sending a clear signal to other countries that it is ready to address car- bon emissions seriously and to lead the international process envisioned by the Paris Agreement. 5. The United States—A Pivotal Actor in the Paris Agreement Potentially, the 2016 presidential election could upset Obama’s climate leadership and put the United States back in a position where no credible federal climate policy initia- tives exist. For the time being, therefore, the potential and willingness for the United States to lead is unclear. Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 Acknowledgments Center for Climate and Energy Solutions (C2ES). (2016). Map: State action on the clean power plan. Retrieved from http://www.c2es.org/us-states-regions/policy- maps/state-action-clean-power-plan We thank the editorial team at Politics and Governance and three anonymous reviewers for helpful comments. Chayes, A., & Chayes, A. H. (1995). The new sovereignty. Cambridge, MA: Harvard University Press. The authors declare no conflict of interests. The authors declare no conflict of interests. The authors declare no conflict of interests. Brack, D. (2003). Monitoring the Montreal protocol. In T. Findlay (Ed.), Verification yearbook 2003 (pp. 209- 226). London: VERTIC. United States prove able and willing to take the lead, it might further strengthen cooperative norms and limit other countries’ costs of compliance. Such develop- ments might ultimately pave the way for a transfor- mation from a logic of consequences to a logic of appro- priateness in the field of climate change (Mitchell, 2015). Brun, A. (2016). Conference diplomacy: The making of the Paris Agreement. Politics and Governance, 4(3), 115-123. Cama, T. (2016). McConnell tells states to stop planning for EPA climate rule. The Hill. Retrieved from http://thehill.com/policy/energy-environment/2737 74-mcconnell-tells-states-to-stop-planning-for-epa- climate-rule So far, however, deep political polarization has rep- resented a significant barrier to U.S. leadership on cli- mate change. Thus, while the Paris Agreement could become the start of a race to the top that sets the world on a path towards solving the climate change problem, it might also end as a flop, much like the Kyo- to Protocol did. The latter outcome is particularly plau- sible if the United States and other major emitters prove unable or unwilling to lead. Campbell, M. (2015, December 14). Big oil, make way for big solar. The winners and losers in Paris. Bloomberg. Retrieved from http://www.bloomberg.com/news/ articles/2015-12-14/big-oil-make-way-for-big-solar- the-winners-and-losers-in-paris 6. Conclusion Congressman Ed Whitfield (Re- publican from Kentucky) described the Paris Agree- ment as merely a “signal” of the Obama administra- tion’s preferences rather than a treaty. He said, “While some may claim the resulting deal is a grand triumph, the bottom line is that this was a nonbinding political document that does not impose any new obligation on the United States”. He added that Obama “misled the international community in Paris” (Chemnik, 2016). On a more optimistic note, norms can change. For example, the Paris Agreement shows that today’s in- terpretation of the common-but-differentiated- responsibilities norm differs from that of the 1990s and 2000s. Consequently, the cards are now stacked somewhat less in favour of incentives than they were then. Domestic and international norms may well con- tinue to develop such that it becomes increasingly dif- ficult for individuals, firms, and states to ignore pleas to limit and reduce their carbon footprints. In addition, technological progress may gradually lessen abatement costs. Such developments would further favour norms over incentives. Finally, if major emitters such as the The Republican Party Convention in 2016 adopted a political platform that explicitly rejects any form of federal carbon price, and pledged to disengage the Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 216 Conflict of Interests Chemnik, J. (2016). Will Republicans seek to undermine Paris agreement? The politics surrounding the land- mark global climate deal are murky in this election year. Scientific American, January 6. Retrieved from http://www.scientificamerican.com/article/will- republicans-seek-to-undermine-paris-agreement References Aakre, S, Helland, L., & Hovi, J. (2014). When does in- formal enforcement work? Journal of Conflict Resolu- tion. doi:10.1177/0022002714560349 Dimitrov, R. (2016). The Paris agreement on climate change: Behind closed doors. Global Environmental Politics. doi:10.1162/GLEP_a_00361 Aakre, S., & Hovi, J. (2010). Emission trading: Participa- tion enforcement determines the need for compli- ance enforcement. European Union Politics, 11(3), 427-445. Downs, G. W., Rocke, D. M., & Barsoom, P. N. (1996). Is the good news about compliance good news about cooperation? International Organization, 50(3), 379- 406. Bang, G. (2015). The United States: Obama’s push for climate policy change. In G. Bang, A. Underdal, & S. Andresen (Eds.), The domestic politics of global cli- mate change. Key actors in international climate co- operation. Cheltenham: Edward Elgar Publishing. Elster, J. (1989). The cement of society. A study of social order. Cambridge: Cambridge University Press. Environmental Protection Agency (EPA). (2015). Clean power plan. Retrieved from http://www2.epa.gov/ cleanpowerplan Bang, G., & Skodvin T. (2014). U.S. climate policy and the shale gas revolution. In T. Cherry, J. Hovi, & D. McEvoy (Eds.), Toward a new climate agreement: Conflict, resolution and governance. London: Routledge. Freking, K. (2015). Republican lawmakers vocal in oppo- sition to climate deal. The Huffington Post. Retrieved from http://www.huffingtonpost.com/entry/republi can-lawmakers-vocal-in-opposition-to-climate- Bang, G., & Schreurs, M. A. (2016). The United States: The challenge of global climate leadership in a politi- cally divided state. In R. K. Wurzel, J. M. Connelly, & D. Liefferink (Eds.), Still taking a lead? The European Union in international climate change politics. Lon- don: Routledge. Goldenberg, S. (2014). Secret talks and a private letter: How the US–China climate deal was done. The Guardian. Retrieved from http://www.theguardian. com/environment/2014/nov/12/how-us-china-clima te-deal-was-done-secret-talks-personal-letter Barrett, S. (2003). Environment and statecraft. Oxford: Oxford University Press. Henderson, G. (2015, September 15). Chinese and U.S. cities, states and provinces announce climate targets and extensive cooperation. China FAQs: The Network for Climate and Energy Information. Retrieved from http://www.chinafaqs.org/blog-posts/chinese-and- us-cities-states-and-provinces-announce-climate- targets-and-extensive-coopera Barrett, S. (2008). Climate treaties and the imperative of enforcement. Oxford Review of Economic Policy 24(2), 239-258. Bodansky, D., & O’Connor, S. D. (2016). The legal charac- ter of the Paris Agreement. Manuscript submitted for publication. Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 217 Simmons, B. (1998). Compliance with international agreements. Annual Review of Political Science, 1(1), 75-93. Hovi, J., Skodvin, T. & Aakre, S. (2013). Can climate change negotiations succeed? Politics and Govern- ance, 1(2), 138-150. References Keohane, R. O., & Oppenheimer, M. (2016). Paris: Be- yond the climate dead end through pledge and re- view? Politics and Governance, 4(3), 142-151. Skocpol, T. (2013, February 14). Naming the problem: What it will take to counter extremism and engage Americans in the fight against global warming. Paper presented at the Symposium “The Politics of Ameri- ca’s Fight Against Global Warming, Harvard Universi- ty, Cambridge, MA. Kinver, M. (2015, December 14). COP21: What does the Paris climate agreement mean for me? BBC. Re- trieved from http://www.bbc.com/news/science- environment-35092127 Stein, A. A. (1990). Why nations cooperate. Ithaca: Cor- nell University Press. Leiserowitz, A., Maibach, E., Roser-Renouf, C., Feinberg, G., & Rosenthal, S. (2016). Politics and global warm- ing. New Haven, CT: Yale Program on Climate Change Communication. Underdal, A. (1994). Leadership theory. In W. Zartman (Ed.), Rediscovering the arts of management. San Francisco, CA: Jossey-Bass Publishers. Victor, D. (2011). Global warming gridlock. Cambridge, MA: Cambridge University Press. Mitchell, R. B. (2015). The problem structure of climate change and the need for a discursive transition. Re- trieved from https://clas-pages.uncc.edu/inss/annual -meetings/2015-meeting/conference-presentations/ bend-or/the-problem-structure-of-climate-change- and-the-need-for-a-discursive-transition White House. (2015). Remarks by President Obama at the first session of COP21. Retrieved from https:// www.whitehouse.gov/the-press-office/2015/11/30/ remarks-president-obama-first-session-cop21 World Resources Institute. (2016). The United States. Re- trieved from http://www.wri.org/our-work/topics/ united-states Paterson, M., & Grubb, M. (1992). The international poli- tics of climate change. International Affairs, 68(2), 293-310. Politics and Governance, 2016, Volume 4, Issue 3, Pages 209-218 About the Authors Guri Bang is Research Director at the Center for International Climate and Environmental Policy, Oslo (CICERO). Her research focuses on comparative energy and climate policy; low-carbon energy transi- tions; international climate politics, and U.S. climate and energy politics. She is leading research pro- jects on domestic climate policy development in the U.S., EU, China, India, Brazil, Japan, and Russia for the Norwegian Center for Strategic Challenges in International Climate Policy (CICEP) and the Re- search Council of Norway. Jon Hovi is professor of political science at the University of Oslo. His research focuses on the design of international agreements, particularly the aspects pertaining to participation, compliance, and en- forcement. Other research interests include the effectiveness of economic sanctions as well as stra- tegic aspects of international and domestic armed conflict. Tora Skodvin is professor of political science at the University of Oslo. Her main areas of interest in- clude international negotiation with a particular focus on the international negotiations of climate change, the role of non-state actors in international climate policies and the domestic–international interface of U.S. and EU climate and energy policies. 218
https://openalex.org/W2899014495	http://iapress.org/index.php/soic/article/download/20181209/383	English	null	Nonlinear Approximation in the Large Deviations Principle	Statistics, optimization & information computing	2,018	cc-by	4,254	AMS 2010 subject classiﬁcations. 60G25, 60G35 DOI: 10.19139/soic.v6i4.317 DOI: 10.19139/soic.v6i4.317 STATISTICS, OPTIMIZATION AND INFORMATION COMPUTING Stat., Optim. Inf. Comput., Vol. 6, December 2018, pp 600–608. Published online in International Academic Press (www.IAPress.org) STATISTICS, OPTIMIZATION AND INFORMATION COMPUTING Stat., Optim. Inf. Comput., Vol. 6, December 2018, pp 600–608. Published online in International Academic Press (www.IAPress.org) STATISTICS, OPTIMIZATION AND INFORMATION COMPUTING Stat., Optim. Inf. Comput., Vol. 6, December 2018, pp 600–608. Published online in International Academic Press (www.IAPress.org) Keywords Markov random processes and their approximations Keywords Markov random processes and their approximations ∗Correspondence to: Oksana Yarova (Email: Moklyachuk@gmail.com). Department of Theoretical and Applied Statistics, Ivan Franko National University of Lviv,1, University str. Lviv 79000 Ukraine Nonlinear Approximation in the Large Deviations Principle O. A. Yarova ∗, Ya. I. Yeleyko Department of Theoretical and Applied Statistics, Ivan Franko National University of Lviv, Ukraine Abstract The Markov random processes and their approximations are considered. The main object of study is the exponential generator of random processes with independent increments, which are the solution of the problems of large deviations. These processes satisfy the conditions that make it possible to consider the Poisson and L´evy approximation. Generators of random processes are normalized by nonlinear parameters. Found explicit form of normalization parameter estimation. ISSN 2310-5070 (online) ISSN 2311-004X (print) Copyright c⃝2018 International Academic Press and in the L´evy approximation (g3(ε))−1g1(ε)f2(ε) →1, ε →0, (g3(ε))−1g1(ε)f2(ε) →1, ε →0, where g1(ε), g2(ε), g3(ε), f1(ε), f2(ε) are normalization function, such that g2(ε) = o(g1(ε)), g3(ε) = o(g2(ε)), f2(ε) = o(f1(ε)), g1(ε), g2(ε), g3(ε), f1(ε), f2(ε) →0, ε →0. where g1(ε), g2(ε), g3(ε), f1(ε), f2(ε) are normalization function, such that g2(ε) = o(g1(ε)), g3(ε) = o(g2(ε)), f2(ε) = o(f1(ε)), g1(ε), g2(ε), g3(ε), f1(ε), f2(ε) →0, ε →0. 1. Introduction The problem of large deviations was originated as a method of solving statistical problems associated with the estimation of probability of rare events. The ﬁrst work in this direction was the article by Cram´er [1], but ultimately the method was developed in the article by Chernoff [2]. Publications [3, 4] are also related to this problem. The purpose of solving the problem of large deviations is ﬁnding of action functional I(x) = I(x0) + ∫∞ 0 L(x(s), x′(s))ds, where x(s) is a Markov process, function L(x, u) deﬁned by the exponential generator where x(s) is a Markov process, function L(x, u) deﬁned by the exponential generator L(x, u) = sup p∈R {pu −H(v, p)}, where p := φ′(u), H(v, φ′(u)) := HΓφ(u). In the writing [5] in the scheme of Poisson approximation processes with independent increments without diffusion component were considered. Between jumps there were Markov processes with linear normalizing factor. where p := φ′(u), H(v, φ′(u)) := HΓφ(u). In the writing [5] in the scheme of Poisson approximation processes with independent increments without diffusion component were considered. Between jumps there were Markov processes with linear normalizing factor. These processes are deﬁned by the generator These processes are deﬁned by the generator Γδ εφ(u) = ε−1 ∫ R ( φ(u + εv) −φ(u) ) Γδ(dv). in the scale of time t/ε. in the scale of time t/ε. in the scale of time t/ε. In the articles [7]–[9] the generator of Markov process and their evolutions were considered in the scale of time t/g1(ε) and t/g2(ε) in the Poisson and L´evi approximation. ∗Correspondence to: Oksana Yarova (Email: Moklyachuk@gmail.com). Department of Theoretical and Applied Statistics, Ivan Franko National University of Lviv,1, University str. Lviv 79000 Ukraine ISSN 2310-5070 (online) ISSN 2311-004X (print) Copyright c⃝2018 International Academic Press 601 O. A. YAROVA AND YA. I. YELEYKO The purpose of this work is to ﬁnd functions that normalize the generator of random process with independent increments in the scale of time t/g2(ε) in the Large Deviations Principle. These processes are deﬁned by the generator These processes are deﬁned by the generator Γεφ(u) = (g2(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv). Besides this, in the Poisson approximation the next condition take places (g2(ε))−1g1(ε)f1(ε) →1, ε →0, Stat., Optim. Inf. Comput. Vol. 6, December 2018 2. Poisson approximation We consider Markov processes with locally independent increments ηε(·) with trajectories in DR[0, ∞): ηε = g1(ε) η ( t g2(ε) ) , t ≥0. In this normalization g1(ε), g2(ε) →0 as ε →0. These processes are deﬁned by the generator In this normalization g1(ε), g2(ε) →0 as ε →0. These processes are deﬁned by the generator In this normalization g1(ε), g2(ε) →0 as ε →0. These processes are deﬁned by the generator Γεφ(u) = (g2(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv), where φ(u) is a twice differentiable function on R which tends to 0 at inﬁnity and with sup-norm, φ(u) ∈C2 0(R). The kernel of intensity Γε belongs to the class C3(R). This kernel satisﬁes the condition Γε(0) = 0. where φ(u) is a twice differentiable function on R which tends to 0 at inﬁnity and with sup-norm, φ(u) ∈C2 0(R). The kernel of intensity Γε belongs to the class C3(R). This kernel satisﬁes the condition Γε(0) = 0. We consider the problem of large deviations in the scheme of Poisson approximation in the case where the following conditions are satisﬁed: g (P1) Approximation of the mean values: (P1) Approximation of the mean values: bε = ∫ R v Γε(dv) = f1(ε) (b + θε b) and and cε = ∫ R v2 Γε(dv) = f1(ε) (c + θε c) , where b, c < ∞, \|θε b\| →0, \|θε c\| →0, f1(ε) →0, ε →0. (P2) The Poisson approximation condition for the intensity kernel (P2) The Poisson approximation condition for the intensity kernel Γε q = ∫ R q(v) Γε(dv) = f1(ε) ( Γq + θε q ) for all q(·) from C3(R) for all q(·) from C3(R) for all q(·) from C3(R) The kernel Γq has the following representation: ( ) ( ) The kernel Γq has the following representation: Γq = ∫ R q(v) Γ0(dv). Stat., Optim. Inf. Comput. Vol. 6, December 2018 602 Lemma 1 Lemma 1 Lemma 1 The exponential generator in the scheme of Poisson approximation has the following asymptotical representation Hε Γφ(u) = HΓφ(u) + θε Γφ, Hε Γφ(u) = HΓφ(u) + θε Γφ, provided (g2(ε))−1g1(ε)f1(ε) →1, where φ(u) is from C3 0(R) and \|θε Γφ\| →0, as g1(ε), f1(ε) →0, as ε →0, provided (g2(ε))−1g1(ε)f1(ε) →1, where φ(u) is from C3 0(R) and \|θε Γφ\| →0, as g1(ε), f1(ε) HΓφ(u) = bφ′(u) + ∫ R ( evφ′(u) −1 −vφ′(u) ) Γ0(dv). Proof NONLINEAR APPROXIMATION IN THE LARGE DEVIATIONS PRINCIPLE NONLINEAR APPROXIMATION IN THE LARGE DEVIATIONS PRINCIPLE Items θε b, θε c, θε q satisﬁed the conditions \|θε\| →0, f1(ε) →0 as ε →0. (P3) The limiting generator is without diffuse component c = ∫ R v2 Γ0(dv) = const. (P4) Square-integrability condition (P4) Square-integrability condition (P4) Square-integrability condition lim c→∞ ∫ \|v\|>c v2 Γε(dv) = 0. lim c→∞ ∫ \|v\|>c v2 Γε(dv) = 0. (P5) The exponential boundedness (P5) The exponential boundedness ∫ R ep\|v\| Γq(dv) < ∞. ( ) p ∫ R ep\|v\| Γq(dv) < ∞. ∫ R ep\|v\| Γq(dv) < ∞. In the scheme of Poisson approximation the solution of the problem of large deviations for these processes are deﬁned by nonlinear exponential generator: Hε Γφ(u) = e−φ(u) g1(ε) g1(ε)Γεe φ(u) g1(ε) . Hε Γφ(u) = e−φ(u) g1(ε) g1(ε)Γεe φ(u) g1(ε) . Stat., Optim. Inf. Comput. Vol. 6, December 2018 Proof f The generator of Markov processes has the next form Γεφ(u) = (g2(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv), so, for the exponential generator so, for the exponential generator Hε Γφ(u) = (g2(ε))−1g1(ε) ∫ R ( e∆εφ(u) −1 ) Γε(dv), where ∆εφ(u) = (g1(ε))−1( φ(u + g1(ε)v) −φ(u) ) . We can write the generator in the form where ∆εφ(u) = (g1(ε))−1( φ(u + g1(ε)v) −φ(u) ) . We can write the generator in the form where ∆εφ(u) = (g1(ε))−1( φ(u + g1(ε)v) −φ(u) ) . We can write the generator in the form Hε Γφ(u) = (g2(ε))−1g1(ε) ∫ R ( e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2) Γε(dv) + (g2(ε))−1g1(ε) ∫ R ( ∆εφ(u) + 1 2 ( ∆εφ(u) )2) Γε(dv). where the function e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 is from C3(R), and since e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 v2 →0 as v →0. In addition, this function is continuous and bounded, because φ(u) is from C2 0(R). So, from conditions P1 and P2 we obtain: In addition, this function is continuous and bounded, because φ(u) is from C2 0(R). So, from conditions P1 and P2 we obtain: Stat., Optim. Inf. Comput. Vol. 6, December 2018 Stat., Optim. Inf. Comput. Vol. 6, December 2018 603 O. A. YAROVA AND YA. I. YELEYKO Hε Γφ(u) = (g2(ε))−1g1(ε)f1(ε) ∫ R ( e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 ) Γ0(dv)+ (g2(ε))−1g1(ε) ∫ R ( ∆εφ(u) −vφ(u) −g1(ε)v2 2 φ′′(u) ) Γε(dv)+ (g2(ε))−1g1(ε)f1(ε)bφ′(u) + 1 2(g2(ε))−1(g1(ε))2f1(ε)cφ′′(u)+ (g2(ε))−1g1(ε) ∫ R (1 2 ( ∆εφ(u) )2 −v2 2 ( φ′(u) )2 ) Γε(dv)+ (g2(ε))−1g1(ε)f1(ε)1 2c ( φ′(u) )2. (g2(ε))−1g1(ε)f1(ε)1 2c ( φ′(u) )2. Applying the Taylor’s formula for φ(u) and making use of the condition P2: Hε Γφ(u) = (g2(ε))−1g1(ε)f1(ε) ∫ R ( evφ′(u) −1 −vφ′(u) −v2 2 ( φ′(u) )2 ) Γ0(dv)+ (g2(ε))−1g1(ε)f1(ε) ∫ R ( evφ′(u)g1(ε)v2 2 φ′′(˜u) −g1(ε)v2 2 φ′′(˜u) − g1(ε)v4 8 ( φ′′(˜u) )2 ) Γ0(dv)+ (g2(ε))−1g1(ε)f1(ε) ∫ R g1(ε)v3 3! φ′′′(˜u) Γ0(dv)+ (g2(ε))−1g1(ε)f1(ε)bφ′(u) + 1 2g1(g2(ε))−1(g1(ε))2f1(ε)cφ′′(˜u)+ (g2(ε))−1g1(ε)f1(ε) ∫ R g1(ε)v4 4 ( φ′′(˜u) )2 Γ0(dv)+ (g2(ε))−1g1(ε)f1(ε)c1 2 ( φ′(u) )2. Stat., Optim. Inf. Comput. Vol. 6, December 2018 Proof From condition P3 and the condition (g2(ε))−1g1(ε)f1(ε) →1 we obtain Hε Γφ(u) = HΓφ(u) + θε Γφ, Hε Γφ(u) = (g2(ε))−1g1(ε)f1(ε) ∫ R ( evφ′(u) −1 −vφ′(u) −v2 2 ( φ′(u) )2 ) Γ0(dv)+ (g2(ε))−1g1(ε)f1(ε) ∫ R ( evφ′(u)g1(ε)v2 2 φ′′(˜u) −g1(ε)v2 2 φ′′(˜u) − g1(ε)v4 8 ( φ′′(˜u) )2 ) Γ0(dv)+ Hε Γφ(u) = (g2(ε))−1g1(ε)f1(ε) ∫ R ( evφ′(u) −1 −vφ′(u) −v2 2 ( φ′(u) )2 ) Γ0(dv)+ (g2(ε))−1g1(ε)f1(ε) ∫ R ( evφ′(u)g1(ε)v2 2 φ′′(˜u) −g1(ε)v2 2 φ′′(˜u) − g1(ε)v4 8 ( φ′′(˜u) )2 ) Γ0(dv)+ (g2(ε))−1g1(ε)f1(ε) ∫ R g1(ε)v3 3! φ′′′(˜u) Γ0(dv)+ (g2(ε))−1g1(ε)f1(ε)bφ′(u) + 1 2g1(g2(ε))−1(g1(ε))2f1(ε)cφ′′(˜u)+ (g2(ε))−1g1(ε)f1(ε) ∫ g1(ε)v4 4 ( φ′′(˜u) )2 Γ0(dv)+ where θε Γφ →0, as g1(ε), f1(ε) →0. Lemma is proved. where θε Γφ →0, as g1(ε), f1(ε) →0. Lemma is proved. From this Lemma we have that the following Theorem holds true. From this Lemma we have that the following Theorem holds true. From this Lemma we have that the following Theorem holds true. Theorem 1 Theorem 1 The solution for the problem of large deviations for the process ηε = g1(ε) ( t g2(ε) ) , t ≥0, Γεφ(u) = (g2(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv) ηε = g1(ε) ( t g2(ε) ) , t ≥0, Γεφ(u) = (g2(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv) Γεφ(u) = (g2(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv) Stat., Optim. Inf. Comput. Vol. 6, December 2018 Stat., Optim. Inf. Comput. Vol. 6, December 2018 NONLINEAR APPROXIMATION IN THE LARGE DEVIATIONS PRINCIPLE 604 under conditions (P1-P5) and under conditions (P1-P5) and under conditions (P1-P5) and (g2(ε))−1g1(ε)f1(ε) →1, ε →0, is determined by the limit generator HΓ of the form HΓφ(u) = bφ′(u) + ∫ R ( evφ′(u) −1 −vφ′(u) ) Γ0(dv). Remark 1 Condition (g2(ε))−1g1(ε)f1(ε) →1 can be satisﬁed not only for trivial functions g2(ε) = ε2, g1(ε) = ε, f1(ε) = ε, as in the article [6], but for more complex functions, such as g2(ε) = ε2, g1(ε) = sin ε, f1(ε) = cos ε. Remark 1 Condition (g2(ε))−1g1(ε)f1(ε) →1 can be satisﬁed not only for trivial functions g2(ε) = ε2, g1(ε) = ε, f1(ε) = ε, as in the article [6], but for more complex functions, such as g2(ε) = ε2, g1(ε) = sin ε, f1(ε) = cos ε. Stat., Optim. Inf. Comput. Vol. 6, December 2018 3. L´evy approximation Now consider another normalization for the family of Markov processes with trajectories in DR[0, ∞) ηε = g1(ε) η ( t g3(ε) ) , t ≥0. In this normalization g1(ε), g3(ε) →0, as ε →0. In this normalization g1(ε), g3(ε) →0, as ε →0. These processes are deﬁned by generator These processes are deﬁned by generator Γεφ(u) = (g3(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv), where φ(u) is a twice differentiable function on R which tends to 0 at inﬁnity and with sup-norm, φ(u) ∈C2 0(R). The kernel of intensity Γε belongs to the class C3(R). This kernel satisﬁes the condition Γε(0) = 0. where φ(u) is a twice differentiable function on R which tends to 0 at inﬁnity and with sup-norm, φ(u) ∈C2 0(R). The kernel of intensity Γε belongs to the class C3(R). This kernel satisﬁes the condition Γε(0) = 0. y g ( ) The conditions of L´evy approximation are the following: The conditions of Levy approximation are (L1) Approximation of the mean values: y pp g (L1) Approximation of the mean values: y pp (L1) Approximation of the mean values: bε = ∫ R v Γε(dv) = f1(ε)b1 + f2(ε) (b + θε b) and cε = ∫ R v2 Γε(dv) = f2(ε) (c + θε c) bε = ∫ R v Γε(dv) = f1(ε)b1 + f2(ε) (b + θε b) and and cε = ∫ R v2 Γε(dv) = f2(ε) (c + θε c) cε = ∫ R v2 Γε(dv) = f2(ε) (c + θε c) where b < ∞, c < ∞, \|θε b\| →0, \|θε c\| →0, f1(ε), f2(ε) →0. In this normalization f2(ε) = o(f1(ε)). (L2) L´evy approximation condition for the intensity kernel here b < ∞, c < ∞, \|θε b\| →0, \|θε c\| →0, f1(ε), f2(ε) →0. In this normalization f2(ε) = o(f1(ε) re b < ∞, c < ∞, \|θε b\| →0, \|θε c\| →0, f1(ε), f2(ε) →0. In this normalization f2(ε) = o(f1(ε)). where b < ∞, c < ∞, \|θε b\| →0, \|θε c\| →0, f1(ε), f2(ε) →0. In this normalization f2(ε) = o(f1(ε)). (L2) L´evy approximation condition for the intensity kernel where b < ∞, c < ∞, \|θb\| →0, \|θc\| →0, f1(ε), f2(ε) →0. 3. L´evy approximation (L2) L´evy approximation condition for the intensity kernel Γε q = ∫ R q(v) Γε(dv) = f2(ε) ( Γq + θε q ) Γε q = ∫ R q(v) Γε(dv) = f2(ε) ( Γq + θε q ) ( ) This kernel has the following representation: Γq = ∫ R q(v) Γ0(dv). Γq = ∫ R q(v) Γ (dv). Items θε b, θε c, θε q satisﬁed the conditions \|θε\| →0, f1(ε) →0 as ε →0. (L3) Square-integrability condition lim c→∞ ∫ \|v\|>c v2 Γε(dv) = 0. (L5) The exponential boundedness ∫ R ep\|v\| Γq(dv) < ∞. Items θε b, θε c, θε q satisﬁed the conditions \|θε\| →0, f1(ε) →0 as ε →0. (L3) Square-integrability condition lim c→∞ ∫ \|v\|>c v2 Γε(dv) = 0. (L5) The exponential boundedness Items θε b, θε c, θε q satisﬁed the conditions \|θε\| →0, f1(ε) →0 as ε →0. (L3) Square-integrability condition ∫ Items θε b, θε c, θε q satisﬁed the conditions \|θε\| →0, f1(ε) →0 as ε →0. (L3) Square-integrability condition lim ∫ v2 Γε(dv) = 0. Items θε b, θε c, θε q satisﬁed the conditions \|θε\| →0, f1(ε) →0 as ε →0. (L3) Square-integrability condition ∫ lim c→∞ ∫ \|v\|>c v2 Γε(dv) = 0. (L5) The exponential boundedness (L5) The exponential boundedness ∫ R ep\|v\| Γq(dv) < ∞. 605 O. A. YAROVA AND YA. I. YELEYKO Example 3.1 Consider the example of a family of Markov processes αε, for which Example 3.1 Consider the example of a family of Markov processes αε, for which P{αε = f1(ε)α1} = p0 −f2(ε)p1, P{αε = f2(ε)α} = q0, p0 + q0 = 1, P{αε = f1(ε)α1} = p0 −f2(ε)p1, P{αε = f2(ε)α} = q0, p0 + q0 = 1, P{αε = d} = f2(ε)p1. Check the conditions of L´evy approximation. Check the conditions of L´evy approximation. Check the conditions of Levy approximation. Firstly, ﬁnd the ﬁrst and the second moments for the family of Markov processes Firstly, ﬁnd the ﬁrst and the second moments for the family of Markov processes bε = Eαε = p0f1(ε)α1 −f1(ε)f2(ε)α1p1 + q0f2(ε)α + f2(ε)p1d = f1(ε)(p0α1) + f2(ε)(αq0 + p1d) + o(f 2(ε)). bε = Eαε = p0f1(ε)α1 −f1(ε)f2(ε)α1p1 + q0f2(ε)α + f2(ε)p1d = bε = Eαε = p0f1(ε)α1 −f1(ε)f2(ε)α1p1 + q0f2(ε)α + f2(ε)p1d = bε = Eαε = p0f1(ε)α1 −f1(ε)f2(ε)α1p1 + q0f2(ε)α + f2(ε)p1d = f1(ε)(p0α1) + f2(ε)(αq0 + p1d) + o(f 2(ε)). 3. L´evy approximation f1(ε)(p0α1) + f2(ε)(αq0 + p1d) + o(f 2(ε)). f1(ε)(p0α1) + f2(ε)(αq0 + p1d) + o(f 2(ε)). cε = E(αε)2 = f 2 1 (ε)α1(p0 −f2(ε)p1) + f 2 2 (ε)α2q0 + d2f2(ε)p1 = cε = E(αε)2 = f 2 1 (ε)α1(p0 −f2(ε)p1) + f 2 2 (ε)α2q0 + d2f2(ε)p1 = f2(ε)(p1d2) + o(f 2 2 (ε)). f2(ε)(p1d2) + o(f 2 2 (ε)). So, we have the next parameters for condition (L1): b1 = p0α1, b1 = p0α1, b = αq0 + p1d, c = p1d2. b = αq0 + p1d, c = p1d2. Now, ﬁnd the intensity kernel Γε q = q(f1(ε)α1)p0 + q(f2(ε)α)q0 −q(f1(ε)α1)(−f2p1) + q(d)f2(ε)p1 = f2(ε)q(d)p1 Γε q = q(f1(ε)α1)p0 + q(f2(ε)α)q0 −q(f1(ε)α1)(−f2p1) + q(d)f2(ε)p1 = f2(ε)q(d)p1. ese moments satisﬁed the condition of approximation of the mean values. So, Γq = p1d2. us, we have all parameters for process αε in L´evi approximation. These moments satisﬁed the condition of approximation of the mean values. So, Γq = p1d . Thus, we have all parameters for process αε in L´evi approximation. Lemma 2 The exponential generator Hε Γφ(u) = e−φ(u) g1(ε) g1(ε)Γεe φ(u) g1(ε) in the scheme of L´evy approximation has the next asymptotically representation Hε Γφ(u) = (g3(ε))−1 g1(ε)f1(ε)b1φ′(u) + HΓφ(u) + θε Γφ with conditions (g3(ε))−1g1(ε)f2(ε) →1, ε →0, where φ(u) is from C3 0(R) and HΓφ(u) = (b −b0)φ′(u) + 1 2(c −c0) ( φ′(u) )2 + ∫ R ( evφ′(u) −1 ) Γ0(dv), b0 = ∫ R v Γ0(dv), c0 = ∫ R v2 Γ0(dv). The neglected item θε Γφ →0 as g1(ε) →0. Stat., Optim. Inf. Comput. Vol. 6, December 2018 606 NONLINEAR APPROXIMATION IN THE LARGE DEVIATIONS PRINCIPLE Proof Proof Proof φ′′′(˜u) Γ0(dv) + (g3(ε))−1g1(ε)f1(ε)b1φ′(u)+ (g3(ε))−1g1(ε)f2(ε)bφ′(u) + (g3(ε))−1g2 1(ε)f2(ε)1 2cφ′′(˜u)+ (g3(ε))−1g1(ε)f2(ε) ∫ R g1(ε)v4 4 ( φ′′(˜u) )2 Γ0(dv)+ (g3(ε))−1g1(ε)f2(ε)c1 2 ( φ′(u) )2. Using condition (L3) and (g3(ε))−1g1(ε)f2(ε) →1, ﬁnally we obtain Hε Γφ(u) = (g3(ε))−1g1(ε)f1(ε)b1φ′(u) + HΓφ(u) + θε Γφ, where θε Γφ →0 as g1(ε), f1(ε) →0. Lemma is proved g1(ε)v4 8 ( φ′′(˜u) )2 ) Γ0(dv)+ (g3(ε))−1g1(ε)f2(ε) ∫ R g1(ε)v3 3! φ′′′(˜u) Γ0(dv) + (g3(ε))−1g1(ε)f1(ε)b1φ′(u)+ (g3(ε))−1g1(ε)f2(ε)bφ′(u) + (g3(ε))−1g2 1(ε)f2(ε)1 2cφ′′(˜u)+ g1(ε)v4 8 ( φ′′(˜u) )2 ) Γ0(dv)+ (g3(ε))−1g1(ε)f2(ε)bφ′(u) + (g3(ε))−1g2 1(ε)f2(ε)1 2cφ′′(˜u)+ (g3(ε))−1g1(ε)f2(ε) ∫ R g1(ε)v4 4 ( φ′′(˜u) )2 Γ0(dv)+ (g3(ε))−1g1(ε)f2(ε)c1 2 ( φ′(u) )2. (g3(ε))−1g1(ε)f2(ε) →1, ﬁnally we obtain (g3(ε))−1g1(ε)f2(ε) ∫ R g1(ε)v 4 ( φ′′(˜u) )2 Γ0(dv)+ (g3(ε))−1g1(ε)f2(ε)c1 2 ( φ′(u) )2. Using condition (L3) and (g3(ε))−1g1(ε)f2(ε) →1, ﬁnally we obtain Hε Γφ(u) = (g3(ε))−1g1(ε)f1(ε)b1φ′(u) + HΓφ(u) + θε Γφ, where θε Γφ →0 as g1(ε), f1(ε) →0. Lemma is proved Hε Γφ(u) = (g3(ε))−1g1(ε)f1(ε)b1φ′(u) + HΓφ(u) + θε Γφ, From this Lemma we have that the following Theorem holds true. From this Lemma we have that the following Theorem holds true. Theorem 2 The solution for the problem of large deviations for the process Theorem 2 The solution for the problem of large deviations for the process ηε = g1(ε) ( t g3(ε) ) , t ≥0, ηε = g1(ε) ( t g3(ε) ) , t ≥0, Γεφ(u) = (g3(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv) Γεφ(u) = (g3(ε))−1 ∫ R ( φ(u + g1(ε)v) −φ(u) ) Γε(dv) under conditions (L1-L5) and (g3(ε))−1g1(ε)f2(ε) →1, ε →0 under conditions (L1-L5) and (g3(ε))−1g1(ε)f2(ε) →1, ε →0 is determined by the limit generator HΓ of the form is determined by the limit generator HΓ of the form HΓφ(u) = (b −b0)φ′(u) + 1 2(c −c0) ( φ′(u) )2 + ∫ R ( evφ′(u) −1 ) Γ0(dv). Proof oof We can represent the exponential generator in the form f We can represent the exponential generator in the form Hε Γφ(u) = (g3(ε))−1g1(ε) ∫ R ( e∆εφ(u) −1 ) Γε(dv), Hε Γφ(u) = (g3(ε))−1g1(ε) ∫ R ( e∆εφ(u) −1 ) Γε(dv), where ∆εφ(u) = (g1(ε))−1( φ(u + g1(ε)v) −φ(u) ) . Rewrite the generator: Hε Γφ(u) = (g3(ε))−1g1(ε) ∫ R ( e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 ) Γε(dv)+ (g3(ε))−1g1(ε) ∫ R ( e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 ) Γε(dv) (g3(ε))−1g1(ε) ∫ R ( ∆εφ(u) + 1 2 ( ∆εφ(u) )2 ) Γε(dv). Function e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 belongs to C3(R), since e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 v2 →0 as v →0. (g3(ε))−1g1(ε) ∫ R ( ∆εφ(u) + 1 2 ( ∆εφ(u) )2 ) Γε(dv Function e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 belongs to C3(R), since e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 v2 →0 as v → (g3(ε))−1g1(ε) ∫ R ( ∆εφ(u) + 1 2 ( ∆εφ(u) )2 ) Γε(dv). Function e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 belongs to C3(R), since Function e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 belongs to C3(R), since e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 v2 →0 as v →0. In addition, this function is continuous and bounded, since φ(u) from C2 0(R). So, from conditions (L1) and (L2) we obtaine Hε Γφ(u) = (g3(ε))−1g1(ε)f2(ε) ∫ R ( e∆εφ(u) −1 −∆εφ(u) −1 2 ( ∆εφ(u) )2 ) Γ0(dv)+ (g3(ε))−1g1(ε) ∫ R ( ∆εφ(u) −vφ′(u) −g1(ε)v2 2 φ′′(u) ) Γε(dv)+ (g3(ε))−1g1(ε)f1(ε)b1φ′(u) + (g2(ε))−1g1(ε)f2(ε)bφ′(u)+ (g3(ε))−1g1(ε)f1(ε)b1φ′(u) + (g2(ε))−1g1(ε)f2(ε)bφ′(u)+ (g3(ε))−1g1(ε)f1(ε)b1φ′(u) + (g2(ε))−1g1(ε)f2(ε)bφ′(u)+ (g3(ε))−1g2 1(ε)f2(ε)1 2cφ′′(u)+ (g3(ε))−1g1(ε) ∫ R (1 2 ( ∆εφ(u) )2 −v2 2 ( φ′(u) )2 ) Γε(dv)+ (g3(ε))−1g1(ε)f2(ε)1 2c ( φ′(u) )2. Applying the Taylor’s formula for φ(u) and making use the condition L2 we will have Hε Γφ(u) = (g3(ε))−1g1(ε)f2(ε) ∫ R ( evφ′(u) −1 −vφ′(u) −v2 2 ( φ′(u) )2 ) Γ0(dv)+ (g3(ε))−1g1(ε)f2(ε) ∫ R ( evφ′(u)g1(ε)v2 2 φ′′(˜u) −g1(ε)v2 2 φ′′(˜u) − Stat., Optim. Inf. Comput. Vol. 6, December 2018 Stat., Optim. Inf. Comput. Vol. 6, December 2018 Stat., Optim. Inf. Comput. Vol. 6, December 2018 Stat., Optim. Inf. Comput. Vol. 6, December 2018 607 O. A. YAROVA AND YA. I. YELEYKO g1(ε)v4 8 ( φ′′(˜u) )2 ) Γ0(dv)+ (g3(ε))−1g1(ε)f2(ε) ∫ R g1(ε)v3 3! 4. Conclusions. Thus, using normalization parameters as nonlinear functions we can found asymptotic representation for the random process in Poisson and L´evy approximation. This normalization allows to ﬁnd nonlinear exponential generator that is the solution of the Large Deviations Principle.As normalization factors, we can take not only ε, but other nonlinear functions f(ε) and g(ε), that satisﬁed the conditions (g2(ε))−1g1(ε)f1(ε) →1, ε →0, and in the L´evy approximation (g3(ε))−1g1(ε)f2(ε) →1, ε →0. This normalization allows us to consider more processes and their small and large deviations. Stat., Optim. Inf. Comput. Vol. 6, December 2018 Stat., Optim. Inf. Comput. Vol. 6, December 2018 608 Stat., Optim. Inf. Comput. Vol. 6, December 2018 REFERENCES 1. H. Cram´er, Sur un nouveau th´eor`eme-limite de la th´eorie des probabilit´es, Actualit´es Scientiﬁques et Industrielles 736 (1938), 5–23. 2. H. Chernoff, A measure of asymptotic efﬁciency for tests of a hypothesis based on the sum of observations, Ann. Math. Statist. 23 (1952), no. 4, 493–507. 1. H. Cram´er, Sur un nouveau th´eor`eme-limite de la th´eorie des probabilit´es, Actualit´es Scientiﬁques et Industrielles 736 (1938), 5–23. 2. H. Chernoff, A measure of asymptotic efﬁciency for tests of a hypothesis based on the sum of observations, Ann. Math. Statist. 23 (1952), no. 4, 493–507. , p , q ( ), 2. H. Chernoff, A measure of asymptotic efﬁciency for tests of a hypothesis based on the sum of observations, Ann. Math. Statist. 23 (1952), no. 4, 493–507. 2. H. Chernoff, A measure of asymptotic efﬁciency for tests of a hypothesis based on the sum of observations, Ann. Math. Statist. 23 (1952), no. 4, 493–507. ( ), , 3. J. Feng and T. G. Kurtz, Large Deviations for Stochastic Processes, Mathematical Surveys and Monographs, 131, AMS, Providence, RI 2006 3. J. Feng and T. G. Kurtz, Large Deviations for Stochastic Processes, Mathematical Surveys and Monographs RI, 2006. , 4. J. Jacod and A. N. Shiryaev, Limit Theorems for Stochastic Processes, Springer-Verlag, Berlin, 2003. 4. J. Jacod and A. N. Shiryaev, Limit Theorems for Stochas y , f , p g g, , 5. A. Puhalskii, On functional principle of large deviations, New Trends in Probability and Statistics, vol. 1, VSP, Utrecht, 1991, pp. 198– 219. 5. A. Puhalskii, On functional principle of large deviations, New Trends in Probability and Statistics, vol. 1, VS 219. and I. V. Samo˘ılenko, Large deviations problem for processes of random evolution, Teor. Imovir. Mat. Stat. 93 (2016 an) 6. V. S. Korolyuk and I. V. Samo˘ılenko, Large deviations problem for processes of random evolution, Teor. Imovir. 88–95. (Ukrainian) ( ) 7. Ya. I. Yeleyko and O. A. Yarova, The behavior of the generator normalization factor in approximation of random processes, Cybernetics and Systems Analysis. 52 (2016), pp. 147–153. y y ( ), pp 8. O. A. Yarova, About selection a small normalization parameter for generator of random process, Visn. L’viv. Univ., Ser. Mekh.-Mat. 81 (2016), pp. 159–162. 9. Ya. I. Yeleyko and O. A. Yarova, The Problem of Large Deviations for Markov Evolutions in the Scheme of Poisson and L´evy Approximation, Columbia International Publishing. Contemporary Mathematics and Statistics. REFERENCES 4 (2017), pp. 28–40.
https://openalex.org/W2782295271	https://diposit.ub.edu/dspace/bitstream/2445/120906/1/671719.pdf	English	null	Philosophy as attentive gaze	Haser	2,017	cc-by-sa	7,406	PHILOSOPHY AS ATTENTIVE GAZE FILOSOFÍA COMO MIRADA ATENTA JOSEP MARIA ESQUIROL Universidad de Barcelona jmesquirol@ub.edu JOSEP MARIA ESQUIROL Universidad de Barcelona jmesquirol@ub.edu RECIBIDO: 17 DE DICIEMBRE DE 2016 ACEPTADO: 9 DE ENERO DE 2017 HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp.125-144 1 SCOTTI ERIUGENAE, Iohannis: Periphyseon (De divisione naturae), Liber Primus, SHELDON-WILLIAMS (ed.), The Dublin Institute for Advanced Studies, Dublin, 1978. Pag. 125 [481c]. RECIBIDO: 17 DE DICIEMBRE DE 2016 ACEPTADO: 9 DE ENERO DE 2017 Abstract: There is something unusual, even paradoxical, about the way that we look at the world around us: the utter ease of looking contrasts with the difficulty of performing the same act well. If there is light, we just open our eyes and the things around us appear. In contrast, we must pay attention to become aware of certain aspects of reality and, in particular, to perceive things in a different way. Simply seeing, mere visual perception, involves virtually no effort (hence, for example, the success of television and screens in general), but looking with care is harder: directing our gaze and concentrating on something involves an effort and can therefore be tiring. The attentive gaze is more uncommon than we might expect. What is it that attention adds to the gaze to transform it in such a significant way? Why does the effort of directing our attention imply much more than a simple zoom effect? Philosophical gaze and attentive gaze is the same thing. g Keywords: Philosophy, attentive gaze, respect, attention Resumen: La mirada tiene algo de extraño, de paradójico: la total facilidad de mirar contrasta con la dificultad de mirar bien. Si hay luz, con solo abrir los ojos se nos aparecen las cosas que nos rodean, pero en cambio hay que prestar atención, fijarse bien, para darse cuenta de según qué aspectos de la realidad y, sobre todo, para percibir las cosas de otra manera. El solo ver, el mero percibir visual casi no cuesta ningún esfuerzo (de ahí, por ejemplo, el éxito de la televisión y de las pantallas en general), mientras que el mirar bien, eso sí que cuesta: dirigir la mirada y concentrarse en algo supone ya un esfuerzo y acarrea, por tanto, un cansancio. La mirada atenta sea más inusual de lo que en un principio podría pensarse. ¿Qué es lo que la atención añade a la mirada, hasta el HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp.125-144 126 JOSEP MARÍA ESQUIROL JOSEP MARÍA ESQUIROL punto de transformarla tan significativamente? ¿Por qué el esfuerzo de la atención supone mucho más que un simple aumento de la lente? Mirada filosófica y mirada atenta coinciden. punto de transformarla tan significativamente? ¿Por qué el esfuerzo de la atención supone mucho más que un simple aumento de la lente? Mirada filosófica y mirada atenta coinciden. Palabras clave: Filosofía, mirada atenta, respeto, atención 2 MERLEAU-PONTY, Maurice: “Eye and Mind”, in The Primacy of Perception: And Other Essays on Phenomenological Psychology, the Philosophy of Art, History and Politics, EDIE, James (trans.), Northwestern University Press, Evanston, 1964. Pag. 186. 3 ELLUL, Jacques: The Humiliation of the Word, Eerdmans, Michigan, 1985. Pag. 11. The gaze of the attentive gaze Our senses open us to the world. In the West, sight has generally been regarded as the most powerful of the senses. We now know that sight involves light waves reaching our eyes, but historically the active and penetrating nature of the sense was often highlighted by characterising it as the point of origin for seeing, or as a kind of beam. In the 9th century, for example, the great medieval thinker John Scottus Eriugena wrote: ‘For who does not know that the eye is a corporeal part of the head and that it is moist, and that it is that through which the sight pours forth from the brain like rays out of the meninx, that is, membrane. The meninx, however, receives the nature of light from the heart, that is, from the seat of fire.’1 And precisely because of the power of sight, western philosophy, unlike other cultural traditions, has privileged seeing over our other senses. This gives rise to interesting contrasts such as that between Greek philosophy and the Jewish tradition, which has always placed particular emphasis on hearing and the word. In the Jewish tradition, the truth is heard; in the Greek tradition it is seen or intuited. Jewish wisdom depends on listening and obeying; Greek wisdom, on intuition, seeing and clarity. Our eyes, the organ of sight, have come to symbolise wisdom and intelligence. Indeed, they are often described as the ‘windows of the soul’. Merleau-Ponty wrote: ‘The eye accomplishes the HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE 127 prodigious work of opening the soul to what is not soul – the joyous realm of things and their god, the sun.’2 prodigious work of opening the soul to what is not soul – the joyous realm of things and their god, the sun.’2 Given its power, however, sight can become a means of appropriation and control. Certainly, sight is an invitation to action and may provoke action: ‘I see’ immediately translates into ‘I can’. What I look at I can also, ipso facto, act on. At the very least, part of what I see is experienced as ready-to-hand (in Heideggerian terminology). Moreover, for something to become useful, to become pragmata, it must be within our reach, and it is sight that performs the function of bringing things nearer to us. The gaze of the attentive gaze Touch, with its capacity for manipulation, is an ally of sight. So it should not surprise us that it has been said that seeing is the starting point for technology: ‘All techniques are based on visualization and involve visualization.’3 Sight is the sense on which our efficacy depends. Without it, how could we even thread a needle? Are the plans we draft for our construction projects and machines not an emblematic example of what we set down on paper to guarantee good results based on precise measurements and clear perspectives? Without visual images, action is blind and uncertain. Having said this, the time appears ripe for a re-examination of the primacy of sight in our cultural tradition, and possibly even to consider starting or accessing a different tradition. It is doubtful, though, whether such changes are possible, or likely rather to end up involving little more than declarations of intentions or the superficial adoption of other cultural models. In any case, the thesis of this paper is that the shift that really is possible is one concerned HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 128 JOSEP MARÍA ESQUIROL JOSEP MARÍA ESQUIROL primarily with the gaze. Within the confines of a civilisation in which sight retains its privileged status, we can nevertheless examine another dimension of that sense; one that leads us in the direction of wonder rather than control, and towards respect rather than the reduction of what we see to mere resources. Sight is, without doubt, the sense associated with efficacy, but it is also the sense of respect. While up to a point, as Ellul and others have observed, the word has been ‘humiliated’ in the civilisation of sight and images, things clearly could have turned out differently. There is no reason why the word and sight should be at odds when they could just as easily be allies. The attentive gaze is very closely related both to the word and to listening. A respectful attitude always implies a readiness to listen. But we must learn how to look. 4 MERLEAU-PONTY, Maurice: The Visible and the Invisible, LINGIS, Alphonso (trans.), Northwestern University Press, Evanston, 1968. Pag. 4. 5 MERLEAU-PONTY, Maurice: “Eye and Mind”, cit., pag. 166. 6 WITTGENSTEIN, Ludwig: Philosophical Investigations, ANSCOMBE, Elizabeth (trans.), Basil Blackwell, Oxford, 1978. Pag. 211. Learning to look in order to see ‘The world is what we see and… nonetheless, we must learn to see it.’4 Commentators often move directly from the contrast between sight and hearing to that between the image (what is seen) and the word (what is heard). The claim is then made that while images present everything in ‘pre-processed’ form, the word demands a much greater response on our part. With images our role is a passive one; the word requires that we provide greater input. This contrast, however, is a rather simplistic one, mainly because it is almost exclusively screen images that are considered (the so-called ‘civilisation of the image’ would be better described as the ‘civilisation of the screen’). The fact is that we can still see things that do not appear on screens (though this is increasingly LINGIS, HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE 129 uncommon). Looking at non-screen images does require an effort on our part; the world is revealed to us, but not in an automatic fashion. Ultimately, what is revealed depends on us, so we must learn how to look. Only then will we see all that there is to see. ‘What exactly does [the painter] ask of [the mountain]? To unveil the means, visible and not otherwise, by which it makes itself a mountain before our eyes.’5 Virtually the same point can be made in another way. Were it the image that provided meaning, we would need only look. Given that this is clearly not the case, we need to focus on how we can look with care, which means interpreting properly what is revealed to us. On an even more basic level, however, we must first learn to look and also to see. The first thing is to look: if we do not look, neither can we see. Sight is subject to movement. We do not see what we do not look at. ‘In order to see clearly’ – according to Antoine de Saint-Exupery – ‘you often need to change only your line of sight.’ Looking is closely linked to sight, but here we are taking a broader view that encompasses the gaze of the soul, or of the mind. It is possible to look without seeing. As Wittgenstein says in his Philosophical Investigations: ‘“He looked at it without seeing it.” – There is such a thing. WITTGENSTEIN, Ludwig, Culture and Value, WINCH, Peter (trans.), Basil Blackwell, Oxford, 1980. Pag. 63. 8 BACHELARD, Gaston: The Poetics of Reverie. Childhood, Language, and the Cosmos, RUSSELL, Daniel (trans.), Bacon Press, Boston, 1969. Pags. 185-186. Learning to look in order to see But what is the criterion for it? – Well, there is a variety of cases here.’6 One can move one's head, along with the rest of one's body, and apparently even direct the gaze but still see nothing or virtually nothing of what could be seen. How do we learn to look? We learn to look by looking, just as we learn to think by thinking. Exercise is the main teacher. Sight, we can therefore say, does not learn from anything but itself. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 130 JOSEP MARÍA ESQUIROL JOSEP MARÍA ESQUIROL When, for whatever reason, this capacity has not been properly applied or is subject to some kind of distortion, learning to look means looking in a new way, as if seeing things in broad daylight for the first time. Learning to look will also involve pausing to examine what is simple and what is part of our everyday experience. The most penetrating human gaze is that which is capable of detecting the extraordinary character of what is most commonplace. ‘God grant the philosopher insight into what lies in front of everyone’s eyes,’7 wrote Wittgenstein. If the word ‘philosophers’ is used here in a restricted sense, then we would do well to add ‘and not only philosophers’. Looking and being seen: The gaze of the world As Bachelard says: ‘Gentleness of seeing while admiring, pride of being admired, those are human bonds. But they are active, in both directions, in our admiration of the world. The world wishes to see itself; the world lives in an active curiosity with ever open eyes. In uniting mythological dreams (songes), we can say: The Cosmos is an Argus. The Cosmos, a sum of beauties, is an Argus, a sum of ever open eyes. Thus the theorem of the reverie of vision is translated to the cosmic level: everything that shines sees, and there is nothing in the world which shines more than a look.’ 8 That is similar to the words of Antonio Machado, the great spanish poet: ‘The eye you see is not / an eye because you see it; / it is an eye because it sees you.’ The penetration of sight can reach ‘extremes’ when we perceive the world as something that looks at us. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE 131 My gaze makes me the centre of the world by placing me at the point from which I see everything, and everything is thus relative to the point from which I see it. Perhaps we have not given enough thought to the obvious fact that the act of looking makes each of us the centre of the world. I think this inevitable perceived centrality should be counterbalanced. It should not be negated (what price would we pay if we negated the subject?), but does need to be offset: first, by the certainty that other subjects also perceive themselves as central; and second, by the idea that the world we look upon can also, in a manner of speaking, look back at us. This may sound odd and disturbing. It does not negate my centrality, but qualifies it by pointing to a possibility (not a fact). 9 LACAN, Jacques: The Four Fundamental Concepts of Psychoanalysis, SHERIDAN, Alan (trans.), Penguin Books, London, 1994. Pag. 95. Looking and being seen: The gaze of the world The disturbing nature of this hypothesis is reflected in an anecdote recounted by Lacan about an incident that took place when he was a young man: ‘One day, I was on a small boat, with a few people from a family of fishermen in a small port (...) One day, then, as we were waiting for the moment to pull in the nets, an individual known as Petit-Jean (…) pointed out to me something floating on the surface of the waves. It was a small can, a sardine can. It floated there in the sun, a witness to the canning industry, which we, in fact, were supposed to supply. It glittered in the sun and Petit-Jean said to me: “You see that can? Do you see it? Well, it doesn’t see you!” He found this incident highly amusing – I less so. I thought about it. Why did I find it less amusing than he? It’s an interesting question. To begin with, if what petit-Jean said to me, namely, that the can did not see me, had any meaning, it was because in a sense, it was looking at me all the same. It was looking at me at the level of the point of light at which everything that looks at me is situated – and I am not speaking metaphorically.’9 The anecdote suggests something very similar to the experience described by the painter Paul Klee: ‘In a HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 132 JOSEP MARÍA ESQUIROL forest I have felt many times that it was not I who looked at the forest. Some days I have felt that the trees were looking at me...’ In my view, we do not need to revive any form of animism to achieve a richer and more complex view of the world. While it is true that I am at the centre when it comes to my own perception, I am also aware that there are other ‘centres’ of perception, which I call people, and others, such as animals, which have distinct perceptual capacities, and plants, which as living organisms are sensitive to their surroundings. Moreover, the material we describe as ‘inert’ interacts with us. Looking and being seen: The gaze of the world This interaction need not be understood within the framework of the Leibnizian theory of universal perception; it is enough to be open to the implications of quantum theory concerning interaction of elementary particles. In short, my aim in relating these anecdotes (selected from among many that could be used to make the same point) is only to recommend that we look with greater care and put more effort into perception. Paradoxically, when we look with care, we are as likely to become aware of our own relative insignificance as of our centrality. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 10 HUSSERL, Edmund: Ideas: General Introduction to Pure Phenomenology, W. R. BOYCE GIBSON, W.R., (trans.), George Allen/Humanities Press, London/New York. 1969, § 92, pag. 269. 11 HUSSER, Edmund: Logical Investigations, vol. I, FINDLAY, J. N. (trans), Humanity Books, New York, 2000. Pag. 384. 12 MERLEAU-PONTY, Maurice: Phenomenology of Perception, SMITH, Colin (trans.), Routledge, London/New York, 1962. Pag. 27. Paying attention and the spiral of attention Learning to look essentially means learning to pay attention. People often say ‘If you pay attention, you will see that…’ This is the key: paying attention is a condition for seeing or becoming aware of something, as well as a way to accomplish this. What is attention, though? We could characterise it in almost the same way as the gaze. Like the gaze, attention is a kind of ‘spotlight’. ‘It is usual to compare attention with an illuminating HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE 133 light,’ wrote Husserl.10 He also used the expressions ‘mental glance’ and ‘glancing ray’, referring to the directing of the ego towards something and the way it is deflected back. Our attention can shift, for instance, from something in the external world to something in our own consciousness, and can also move in the opposite direction. Attention, then, is also a movement. The shifts it involves occur within the field of consciousness: ‘Ultimately it extends [the range of the unitary notion of attention] as far as the concept: consciousness of something.’11 This is not, however, simply a matter of two things coinciding in the same field. Paying attention and being conscious are closely linked. A minimum level of attention is needed to be conscious of something, and attention depends on consciousness: they are two sides of the same coin. Merleau-Ponty describes this relationship as follows: ‘For it to gain possession of the knowledge brought by attention, it is enough for it to come to itself again, in the sense in which a man is said to come to himself again after fainting. On the other hand, inattentive or delirious perception is a semi-torpor, describable only in terms of negations, its object has no consistency, the only objects about which one can speak being those of waking consciousness.’12 Attention is a kind of activity (‘paying attention’ implies focusing and selecting), but also a state (‘being focused, alert, etc.’). As a state it contrasts with being unfocused, sleepy or distracted. William James, one of the founders of modern psychology, gave a clear description of what attention involves: ‘Focalisation, concentration, of consciousness are of its essence. It HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 13 JAMES, William: The Principles of Psychology, The University of Chicago/Encyclopaedia Britannica, Chicago/London, 1952. Pag. 261. Paying attention and the spiral of attention 125-144 134 JOSEP MARÍA ESQUIROL JOSEP MARÍA ESQUIROL JOSEP MARÍA ESQUIROL implies withdrawal from some things in order to deal effectively with others, and is a condition which has a real opposite in the confused, dazed, scatter-brained state which in French is called distraction, and Zerstreutheit in German.’13 Attention means leaving behind a state characterised by a certain lack of focus, sleepiness or immersion in the flow of things. In relation to the idea of sleepiness, attention is an awakening; seen as a movement away from lack of focus, distraction and flow, attention is an activity that arises from the self, an effort that consists in taking oneself out of the stream of the impersonal in order to come to a stop. Extracting ourselves from this flow to come to a stop is harder than we might think, though. We are in the habit of letting ourselves drift, and grow accustomed to not making an effort and to the ease this brings. Of course, to come to a stop does not mean to hesitate. It is the condition that makes attention possible. We can perceive things, or at least perceive them precisely, only when we come to a stop. To become aware of the progress of the sun, or the rhythm of our own breathing, we must come to a halt. Oddly, this stillness makes us visionaries. It is not those who are in the greatest hurry or who are most mobile that see most, it is those who are capable of coming to a stop. In our increasingly fast-paced society this is something we can experience very intensely on the rare occasions when we manage to make ourselves pause. Given that we spend most of our time immersed in the impersonal in a thousand different ways, when we extract ourselves from this flow we are at first disconcerted (indeed, we often find it hard to tolerate solitude and silence), but then we begin to see. Needless to say, we need not take pause in any special place. For this purpose, all places are privileged: the profound is to be HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE 135 found in the everyday. Neither do we need to travel to remote and exotic locations. 14 HEIDEGGER, Martin: Being and Time, MACQUARRIE, John & ROBINSON, Edward (trans.), Harper & Row, New York, 1962. Pags. 137-138. Paying attention and the spiral of attention Anyone unable to see what is surprising and beautiful in their immediate surroundings will not find these qualities in far-off lands either. Familiarity all too often immerses us in routine, and to a large degree the everyday is the river of inattention that sweeps us along. In his analysis of the mode of being of what is ‘ready-to-hand’ in Being and Time, Heidegger writes: ‘The readiness-to-hand which belongs to any such region beforehand has the character of inconspicuous familiarity, and it has it in an even more primordial sense than does the Being of the ready-to-hand.’14 What I am most interested in underlining in this text is the fact that these ‘regions’ that surround and are familiar to us do not attract our attention. The same applies to ‘things’, which means that familiarity (habit, routine, etc.) results in a certain degree of inattention and leads us to become dispersed among things. This probably happens because this approach is not too tiring and we need not expend much energy. We like to let ourselves go with the flow. All of this highlights the dangers inherent in the everyday. The most serious of these concern what we no longer perceive due to the state of inattention we are drawn into. Even when we are immersed in the everyday, though, this situation can be turned around. We can stop, and when we do the simple and commonplace character of the familiar can be revealed to us in all its strangeness and profundity. In addition to the possibilities of awakening and taking pause, we also need to examine the idea of receiving, or, more accurately, of emptying ourselves in order to receive. Although this idea contrasts with the image of attention as a spotlight, there is no contradiction in saying that attention involves a kind of heightening and broadening of our capacity to perceive. In order to receive one HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 136 JOSEP MARÍA ESQUIROL JOSEP MARÍA ESQUIROL must have free space available and a very flexible ‘opening’. Otherwise, how is the other to enter? Contrary to what is often thought attention does not imply rigidity. Rigidity of any kind hinders perception. This misunderstanding is nevertheless widespread, particularly in the field of education. According to Simone Weil: ‘To pay attention, it is necessary to know how to set about it. 15 WEIL, Simone: Waiting on God, CRAUFURD, Emma (trans.), Routledge and Keagan, London, 1951. Pag. 54. 16 LEWIS, Clive: An Experiment in Criticism, Cambridge University Press, Cambridge, 1961. Pag. 16 . Paying attention and the spiral of attention Most often attention is confused with a kind of muscular effort. If one says to one's pupils: "Now you must pay attention," one sees them contracting their brows, holding their breath, stiffening their muscles. If after two minutes they are asked what they have been paying attention to, they cannot reply. They have been concentrating on nothing. They have not been paying attention. They have been contracting their muscles.’15 The effort of attention does not require that any muscle be contracted. Attention is a tension (paying attention is like drawing a bow), but this tension does not involve muscular rigidity (Rodin’s famous sculpture, The Thinker, is therefore not very lifelike: all of the effort made by the figure is being applied to tense his muscles). It is a different kind of tension that must come into play when we pay attention, and it must be a flexible tension like that of the archer’s bow. Together with flexibility and tension, an emptying is also necessary. An emptying and a detachment from the self must be achieved. Our thoughts must be suspended so that our minds are more available and penetrable. We must put aside our own ‘baggage’ (at least momentarily) so that we can move away from our own centre. Attention requires that we do not allow ourselves to be diluted in the impersonal; that we do not cling to what is ours; and that we do not fill ourselves with easy certainties. We must avoid becoming dispersed in everything, but let go of ourselves a HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE PHILOSOPHY AS ATTENTIVE GAZE 137 bit so that we can be more authentically ourselves. The act of paying attention is somewhat paradoxical: the effort the subject must make does not involve being more present, but rather a reduction or emptying, and an opening towards the other. The subjective intensity of attention involves making a space available to receive something or allow entry to the object that is the focus of attention. Not paying attention means remaining closed or too impenetrable to the influence of the other. Particularly helpful in illustrating this point are C. S. Lewis’ reflections on how to look at paintings (that is, how to direct our attention at them), which are found in an essay on the experience of reading. Lewis says that when we look at a painting most of us tend to make ‘use’ of it in some way. In other words, we do something with the painting instead of opening ourselves to it so that it can do something in us. In particular, we make use of paintings when we do not give them the attention they deserve: ‘We must not let loose our own subjectivity upon the pictures and make them its vehicles. We must begin by laying aside as completely as we can all our own preconceptions, interests, and associations. We must make room for Botticelli's Mars and Venus, or Cimabue's Crucifixion, by emptying out our own. After the negative effort, the positive. We must use our eyes. We must look, and go on looking till we have certainly seen exactly what is there. We sit down before the picture in order to have something done to us, not that we may do things with it. The first demand any work of any art makes upon us is surrender. Look. Listen. Receive. Get yourself out of the way.’16 The passage describes very well what it means to pay attention: ‘look, and go on looking till we have certainly seen exactly what is there’ and at the same time receive. Lewis says the same about both painting and literature. All the examples he gives underscore the HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 138 JOSEP MARÍA ESQUIROL JOSEP MARÍA ESQUIROL fact that only true attention can take us outside ourselves, and in so doing allow us to learn. In this light, attention is seen as a struggle against self-absorption. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE There is no other struggle as worthwhile as this one can end up being. This perspective also allows us to understand that insufficient attention implies insufficient respect. When we focus on ourselves – on our own ideas, interests, opinions and desires – we become incapable of receiving the other, and therefore incapable of respecting it. p p g The need for flexibility must be stressed. There is a kind of attention that in the end does not deserve to be described as such, and, in fact, hinders perception. It is important to realise that there are no models or standards for defining attention. The image of the spotlight is probably a good one, but so are the ideas of flexibility and receiving. The fact that a spotlight is directed at something specific suggests concentration. In contrast, the notion of receiving involves opening up to the other. Could it be that a certain kind of concentration is inflexible and allows only limited opening to the other? In my view, we should not give up either image: together they show us how rich an activity paying attention is. Sometimes we need to focus in order to pay attention; on other occasions we need to open ourselves up. Sometimes I experience a form of attention with virtually no object. Nevertheless, this state keeps me open and alert to what is around me and to its demands. Other times, attention is more focused, and involves looking at and examining something specific. At times, despite a great deal of opening, we perceive nothing; on other occasions, too much focus yields the same result. It may even be the case that optimal attention involves striking a balance between these two approaches, and that too great an emphasis on either diminishes our capacity to see. In any case, flexibility is always essential. Even when focusing our attention, we must take an unhurried approach, freeing ourselves of any pressures and especially of any preconceived HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE 139 notions. Throwing ourselves into the task of paying attention will be utterly counterproductive. The best form of attention arises from a state of relative calm. Attention is not a kind of spiritual determination with which we nail objects down: its application should be a mild and serene process. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 17 WEIL, Simone: op. cit., pag. 52. 18 ROUSSEAU, Jean-Jacques: ‘The Discourses’ and Other Early Political Writings, GOUVERICH, V. (ed.), Cambridge University Press, Cambridge, 1997. PHILOSOPHY AS ATTENTIVE GAZE I do not see attention as a form of strict and continuous abstraction. Indeed, I would count those who are self-absorbed among the distracted as surely as those who are scatterbrained. Neither has achieved the right kind of attention. Attention should be firm but mild. We should work to acquire the flexibility needed to shift our attention from one object to another as required by the course of events. The secret is to achieve a form of attention that is steady without being unyielding, and flexible without being slack. Given this understanding of attention, my meaning will now be clear when I speak of how it can benefit us in terms of our growth as individuals. True knowledge comes from looking attentively and systematically in the right direction. Patience in paying attention leads to significant insights and discoveries. As I have already mentioned, attention is learned through exercise: paying attention is habit-forming. It is a skill at which we become proficient by virtue of constant exercise. The effort we make today may bear fruit tomorrow in a way that is completely unforeseeable. Weil also has something to say on this point: ‘If we concentrate our attention on trying to solve a problem of geometry, and if at the end of an hour we are no nearer to doing so than at the beginning, we have nevertheless been making progress each minute of that hour in another more mysterious dimension. Without our knowing or feeling it, this apparently barren effort has brought more light into the soul. The result will one day be discovered in prayer. Moreover it may very likely be felt besides in some department of the intelligence in no way connected with mathematics. Perhaps he who made the unsuccessful effort will one day be able to grasp the beauty of a line of Racine more vividly on 140 JOSEP MARÍA ESQUIROL account of it.’17 Not surprisingly, Weil believes that the fundamental goal of education should be precisely to ‘exercise attention’. The acuity attention can be applied in countless different ways. It allows us to perceive specific aspects of physical reality, and can also be applied in the moral sphere. It can detect what relates to beauty, or to technical efficiency, and also what is concerned with goodness or justice. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE The value of things and actions is revealed to the attentive gaze, and what is just or good takes on a natural primacy from such a perspective. Painting aspires to be recognised as beautiful; just action aspires to be preferred over a lie… Attention is fundamental to our sense of morality, but this does mean that we should not also recognise the value of moral reasoning. I do believe, however, that there is something crucial to our sense of morality that does not emerge from the lengthy and arduous application of reason. This view is one I think many share, among them Rousseau. Leaving aside the question of whether or not Rousseau contradicted himself on this point, I will refer only briefly to what he says in his Discourse on the Origin and Foundations of Inequality Among Men,18 where he speaks of compassion, which he understands as repugnance at seeing a fellow-creature suffer. Rousseau argues that compassion is a natural feeling that even beasts show signs of. He stresses that this feeling precedes the exercise of reason, and therefore is not a product of it. For Rousseau, the conclusion that I should feel repugnance in the face of the suffering of other creatures is not one arrived at after lengthy digressions: it is a feeling I experience naturally and spontaneously. Nevertheless, might this feeling not depend on a minimum level of attention? Rousseau gets to the heart HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE 141 of the matter when he criticises the philosopher, shut away in his room, who ‘only has to put his hands over his ears and to argue with himself a little’ to ignore the fact that a murder is being committed with impunity beneath his window. This so-called philosopher pays no attention to what is going on around him. Instead, he uses the exercise of reason to isolate and distract himself. Rousseau continues in an ironic tone: ‘Savage man has not this admirable talent’.19 Clearly he is referring not to the capacity to pay attention, but to the ability to turn off one’s attention so as to better evade what it might reveal. Uncivilised man is doubtless unacquainted with any ethical theory, but he does have the capacity to pay attention, to look with care, and this is precisely what is most important. 19 ROUSSEAU: op. cit., pag. 153. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE Civilised man, on the other hand, can study and learn about wonderful philosophical systems and sophisticated moral theories, but his ability to pay attention may be atrophied or intentionally put aside, leaving him incapable of contact with the world around him. The pseudo-philosopher covers his ears, avoids looking out the window and goes off on tangents, and in so doing ceases to pay attention to his situation. I am particularly interested in Rousseau's observation that philosophy (false philosophy) isolates us from things and from others. This kind of philosophy turns off our natural compassion, cutting us off from the world. Attention, on the other hand, connects us to the world, to its density and novelty (as Arendt would say). The atrophy of our sense of morality follows naturally from the atrophy of our capacity to be in the world and recognise its continuous novelty. In my view, Arendt is absolutely right when she says that one of the most important characteristics of totalitarianism is its practical identification of control, security, absence of novelty and rejection of the world. There are certain parallels between the distractions of the 142 JOSEP MARÍA ESQUIROL JOSEP MARÍA ESQUIROL pseudo-philosopher and the experience of mass man in totalitarian regimes. In such regimes, the individual's knowledge and vision of the world tend to be governed exclusively by the coherence of an ‘ideology’, rather than by experience or contact with the reality of the world. The masses leave reality and the world behind because novelty is a natural part of that world; the coherence of the prevailing theory is more comforting and safer than continually dealing with the novelty of the world and the demands it makes. The resulting isolation means life without the world and without identity: ‘Self and world, capacity for thought and experience are lost at the same time.’20 There are many ways to isolate oneself, some more sophisticated than others. There are theories that insulate us from the world and its demands under the pretext of sheltering and protecting us (though these may more accurately be described as ‘ideologies’ in the worst sense of the word).21 In Husserlian terms, ideology evacuates the meaning inherent in the world of life and absolutises logical relation. The latter, cut adrift from reality, can be adopted by individuals without their capacity for reflection or experience of the world ever coming into play. 21 Ideology, understood literally as the logic of an idea, is, according to Arendt, the backdrop to totalitarianism. And what Arendt says about political totalitarianism parallels an observation made by Levinas about the philosophical tradition of the West: “Philosophy itself is identified with the substitution of ideas for persons, the theme for the interlocutor, the interiority of the logical relation for the exteriority of interpellation.” LEVINAS, Emmanuel: Totality and Infinity. An Essay on Exteriority, LINGIS, Alphonso (trans.), Duquesne University Press & Martinus Nijhoff, Pittsburgh & The Hague, 1969. Pag. 88. 20 ARENDT, Hannah: The Origins of Totalitarianism, Harcourt Brace, New York, 1973. Pag. 477. 21 HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE Individuals can apply the logical system without thought and without reference to the world. Attention connects us to the world, and this link is our best vaccine against dogmatism, slogans, ideologies and languages that HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE 143 seem technical but are in fact empty. This connection also safeguards us against the atrophy of our moral feeling. Without it and without our experience of things and others, everything tends to become contrived. Words lack meaningful content; what we say does not respond to anything or anyone (for we are no longer capable of listening or of feeling that we are engaged in a dialogue, and no longer grasp the requirements of each situation). In the absence of this connection, theories focus on evasion, mere play or security, rather than pursuing a sense of direction and meaning. In making these points I have two aims in mind: first, to indicate the parallel between the role I am assigning to attention and that which other authors such as Rousseau and Scheler have attributed to moral feeling; and second, to highlight the close relationship between attention, contact with the world, and reflection, which is also highly significant in the moral sphere. References ARENDT, Hannah: The Origins of Totalitarianism, Harcourt Brace, New York, 1973. BACHELARD, Gaston: The Poetics of Reverie. Childhood, Language, and the Cosmos, RUSSELL, Daniel (trans.), Bacon Press, Boston, 1969. ELLUL, Jacques: The Humiliation of the Word, Eerdmans, Michigan, 1985. HEIDEGGER, Martin: Being and Time, MACQUARRIE, John & ROBINSON, Edward (trans.), Harper & Row, New York, 1962. HUSSERL, Edmund: Ideas: General Introduction to Pure Phenomenology,W. R. BOYCE GIBSON, W.R., (trans.), George Allen/Humanities Press, London/New York. 1969. JAMES, Willim: The Principles of Psychology, The University of Chicago/Encyclopaedia Britannica, Chicago/London, 1952. References ARENDT, Hannah: The Origins of Totalitarianism, Harcourt Brace, New York, 1973. BACHELARD, Gaston: The Poetics of Reverie. Childhood, Language, and the Cosmos, RUSSELL, Daniel (trans.), Bacon Press, Boston, 1969. ELLUL, Jacques: The Humiliation of the Word, Eerdmans, Michigan, 1985. HEIDEGGER, Martin: Being and Time, MACQUARRIE, John & ROBINSON, Edward (trans.), Harper & Row, New York, 1962. HUSSERL, Edmund: Ideas: General Introduction to Pure Phenomenology,W. R. BOYCE GIBSON, W.R., (trans.), George Allen/Humanities Press, London/New York. 1969. JAMES, Willim: The Principles of Psychology, The University of Chicago/Encyclopaedia Britannica, Chicago/London, 1952. References ARENDT, Hannah: The Origins of Totalitarianism, Harcourt Brace, New York, 1973. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144 PHILOSOPHY AS ATTENTIVE GAZE BACHELARD, Gaston: The Poetics of Reverie. Childhood, Language, and the Cosmos, RUSSELL, Daniel (trans.), Bacon Press, Boston, 1969. ELLUL, Jacques: The Humiliation of the Word, Eerdmans, Michigan, 1985. HEIDEGGER, Martin: Being and Time, MACQUARRIE, John & ELLUL, Jacques: The Humiliation of the Word, Eerdmans, Michigan, 1985. HEIDEGGER, Martin: Being and Time, MACQUARRIE, John & ROBINSON, Edward (trans.), Harper & Row, New York, 1962. HUSSERL, Edmund: Ideas: General Introduction to Pure Phenomenology,W. R. BOYCE GIBSON, W.R., (trans.), George Allen/Humanities Press, London/New York. 1969. JAMES, Willim: The Principles of Psychology, The University of Chicago/Encyclopaedia Britannica, Chicago/London, 1952. JOSEP MARÍA ESQUIROL 144 LACAN, Jacques: The Four Fundamental Concepts of Psychoanalysis, SHERIDAN, Alan (trans.), Penguin Books, London, 1994. LEVINAS, Emmanuel: Totality and Infinity. An Essay on Exteriority, LINGIS, Alphonso (trans.), Duquesne University Press & Martinus Nijhoff, Pittsburgh & The Hague, 1969. LEWIS, Clive: An Experiment in Criticism, Cambridge University Press, Cambridge, 1961. MERLEAU-PONTY, Maurice: “Eye and Mind”, in The Primacy of Perception: And Other Essays on Phenomenological Psychology, the Philosophy of Art, History and Politics, EDIE, James (trans.), the Philosophy of Art, History and Politics, EDIE, James (trans.), Northwestern University Press, Evanston, 1964. p y f y ( ) Northwestern University Press, Evanston, 1964. Phenomenology of Perception, SMITH, Colin (trans.), Northwestern University Press, Evanston, 1964. Phenomenology of Perception, SMITH, Colin (trans.), R l d L d /N Y k 1962 y Phenomenology of Perception, SMITH, Colin (trans.), Routledge, London/New York, 1962. The Visible and the Invisible, LINGIS, Alphonso (trans.), Northwestern University Press, Evanston, 1968. ROUSSEAU, Jean-Jacques: ‘The Discourses’ and Other Early Political Writings, GOUVERICH, V. (ed.), Cambridge University Press, Cambridge, 1997. Political Writings, GOUVERICH, V. (ed.), Cambridge University Press, Cambridge, 1997. SCOTTI ERIUGENAE, Iohannis: Periphyseon (De divisione naturae), Liber Primus, SHELDON-WILLIAMS (ed.), The Dublin Institute for Advanced Studies, Dublin, 1978. WEIL, Simone: Waiting on God, CRAUFURD, Emma (trans.), Routledge and Keagan, London, 1951. WITTGENSTEIN, Ludwig: Culture and Value, WINCH, Peter (trans.), Basil Blackwell, Oxford, 1980. Logical Investigations, vol. I, FINDLAY, J. N. (trans), Humanity Books, New York, 2000. Philosophical Investigations, ANSCOMBE, Elizabeth (trans.), Basil Blackwell, Oxford, 1978. SCOTTI ERIUGENAE, Iohannis: Periphyseon (De divisione naturae), Liber Primus, SHELDON-WILLIAMS (ed.), The Dublin Institute for Advanced Studies, Dublin, 1978. WEIL, Simone: Waiting on God, CRAUFURD, Emma (trans.), Routledge and Keagan, London, 1951. WITTGENSTEIN, Ludwig: Culture and Value, WINCH, Peter (trans.), Basil Blackwell, Oxford, 1980. Logical Investigations, vol. I, FINDLAY, J. N. (trans), Humanity Books, New York, 2000. PHILOSOPHY AS ATTENTIVE GAZE Humanity Books, New York, 2000. y Philosophical Investigations, ANSCOMBE, Elizabeth (trans.), Basil Blackwell, Oxford, 1978. HASER. Revista Internacional de Filosofía Aplicada, nº 8, 2017, pp. 125-144
https://openalex.org/W4307254879	https://research-repository.st-andrews.ac.uk/bitstream/10023/27777/1/Sowinski_2023_CC_Conformational_tuning_CC.pdf	English	null	Conformational tuning improves the stability of spirocyclic nitroxides with long paramagnetic relaxation times	null	2,022	cc-by	8,208	1 Department of Chemistry, UiT The Arctic University of Norway, 9037 Tromsø, Norway. 2 Hylleraas Center for Quantum Molecular Sciences, UiT The Arctic University of Norway, 9037 Tromsø, Norway. 3 SUPA, School of Physics and Astronomy and BSRC, University of St Andrews, North Haugh, St Andrews KY16 9SS, UK. 4These authors contributed equally: Bjarte A. Lund, Anna-Luisa Warnke. ✉email: marius.m.haugland@uit.no COMMUNICATIONS CHEMISTRY \| (2023) 6:111 \| https://doi.org/10.1038/s42004-023-00912-7 \| www.nature.com/commschem Results and discussion S h i X ll Synthesis, X-ray crystallography, and conformational analysis. Our initial strategy for the synthesis of substituted spir- ocyclohexyl piperidine nitroxides relied on classical enolate chemistry (Fig. 2a). Spirocyclohexyl piperidinone 3 was prepared from the commercially available 2,2,6,6-tetramethylpiperidinone according to a literature procedure35. Deprotonation of 3 with lithium diisopropylamide (LDA), followed by alkylation with methyl iodide, resulted in the mono-methylated product 4 in moderate yield. Subsequent oxidation with m-chloroperbenzoic acid (m-CPBA) provided nitroxide radical 5 in 84% yield. As radical 5 is crystalline, we investigated its three-dimensional structure by X-ray crystallography to reveal the conformational effect of a single substituent on the nitroxide ring. The solid-state structure (Fig. 2b) indicates that the methyl group adopts an axial orientation, with the nitroxide ring preferring a twist-boat con- formation. Indeed, twist-boat conformations have also previously been observed in crystal structures of spirocyclic piperidinyl nitroxides29,36. The twist-boat conformation permits the spir- ocyclic scaffold to maintain an overall open arrangement, despite the presence of the additional methyl substituent. N FG R R O 1 R R Functional groups Ring size/ saturation -Substituents N N Open conformation Closed conformation O O N OH O O N OH O O H2N O Ref. 31 Ref. 32 N O R Scaffold tuning 1 N O R 2 This work: - Higher preference for closed conformations - Improved reduction stability - Long Tm preserved O a c b d Fig. 1 Context and motivation for this work. a Structural factors affecting nitroxide stability. b Proposed equilibrium between open and closed conformations of spirocyclohexyl nitroxides. c Examples of conformational shifts in spirocyclic nitroxides31,32. d This work. N FG R R O 1 R R Functional groups Ring size/ saturation -Substituents a N N Open conformation Closed conformation O O O N O R Scaffold tuning N O R This work: - Higher preference for closed conformations - Improved reduction stability - Long Tm preserved b d N N Open conformation Closed conformation O O b a b Open conformation N O R Scaffold tuning N O R This work: - Higher preference for closed conformations - Improved reduction stability - Long Tm preserved d d N OH O O N OH O O H2N O Ref. 31 Ref. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 mobile ethyl substituents, which effectively occupy a larger spatial volume surrounding the spin center25,26. N N itroxides (also known as aminoxyl or nitroxyl radicals) are suited for innumerable applications across the chemical sciences1,2. These stable organic radicals are, for example, used as oxidants in synthetic organic chemistry3, mediators for radical polymerization4–7, redox-active components for materials science8,9, and potential therapeutic agents10. Nitroxides have also found a unique place of signiﬁcance in spectroscopy. Due to the localized radical distribution, small molecular size, and readily tunable properties through structural modiﬁcation, nitroxides are highly suitable for use as observable probes in electron para- magnetic resonance (EPR) spectroscopy11–13 and EPR imaging14. Moreover, nitroxides are used in nuclear magnetic resonance (NMR) spectroscopy as dynamic nuclear polarization (DNP) transfer agents15,16, paramagnetic relaxation enhancement (PRE) probes17, and as contrast agents in magnetic resonance imaging (MRI)18,19. Beyond their impact on reduction stability, α-substituents also have a critical inﬂuence on Tm. The rotation of methyl groups is a signiﬁcant contributor to the relaxation of nitroxides, especially at temperatures above 70 K27. Thus, the commonly used tetramethyl- substituted nitroxides require the use of cryogenic temperatures for DEER. This effect is also present in tetraethyl-substituted nitr- oxides, where a lower energy barrier for methyl rotation leads to increased relaxation rates at even lower temperatures28. In spir- ocyclic systems, however, the lack of methyl groups signiﬁcantly enhances Tm, even at higher temperatures29,30. Thus, the design of nitroxides for in-cell applications is often a compromise between high reduction stability and long Tm. The conformation of spirocyclohexyl nitroxides is an impor- tant consideration: it has been proposed that such scaffolds exist as an equilibrium between open and closed conformations, where the former is preferred in solution (Fig. 1b)30,31. Closed con- formations are, however, expected to have higher stability in reducing environments due to more effective steric shielding of the nitroxide. Previous investigations have demonstrated that disubstituted ﬁve-membered pyrrolidinyl nitroxides (e.g., 1, Fig. 1c) can adopt semi-open conformations with improved reduction stability32. Moreover, six-membered spirocyclohexyl nitroxides can be forced to adopt more stable semi-open or even closed conformations by the introduction of substituents on the cyclohexane rings (e.g., 2)31,33,34. Within the ﬁeld of EPR spectroscopy, double electron-electron resonance spectroscopy (DEER), also known as pulsed electron double resonance (PELDOR), is a powerful tool for the investi- gation of biomolecular structure20. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 DEER experiments enable the measurement of nanometer-scale distances and angles between two spin centers (referred to as spin labels), which have been incorporated into biomolecular systems via site-directed spin labeling (SDSL)13,21,22. The resolution of DEER measurements is, among other things, inﬂuenced by the temperature-dependent relaxation parameter Tm, the phase memory time, which should be as high as possible22. For imaging and for modern spectroscopy applications, moreover, nitroxides should possess a sufﬁciently high aqueous solubility and stability towards the reducing environments typi- cally found within cells22. In the presence of reducing agents such as ascorbate or glutathione, nitroxides are known to undergo reduction to diamagnetic hydroxylamines, which are not detect- able by EPR23. Several structural factors inﬂuence nitroxide sta- bility towards reduction (Fig. 1a). The size and saturation of the nitroxide ring has a signiﬁcant impact: saturated ﬁve-membered pyrrolidinyl nitroxides are more stable than the unsaturated ﬁve- membered pyrrolinyl nitroxides, while six-membered piperidinyl nitroxides are more prone to reduction22. Electronic factors also inﬂuence the reduction resistance of nitroxides: electron-donating substituents enhance stability, while electron-withdrawing moi- eties decrease it24. Finally, the α-substituents shielding the para- magnetic center are a crucial factor. Replacing the most common methyl substituents with spirocyclohexyl groups results in a slight increase in the stability in reducing environments24. However, stability is signiﬁcantly enhanced by more conformationally Here, we report that structural modiﬁcation of the nitroxide rings itself can cause a conformational shift to provide nitroxides in fully closed conformations (Fig. 1d). The additional steric shielding obtained through this strategy results in a dramatic increase in the stability of these nitroxides towards reduction by ascorbate. Moreover, the beneﬁcial Tm of spirocyclic systems is largely preserved, resulting in a scaffold that ﬁnds a balance between improved stability and beneﬁcial relaxation properties. These ﬁndings are of great signiﬁcance for the future design of new nitroxide-based spin labels and imaging agents. Conformational tuning improves the stability of spirocyclic nitroxides with long paramagnetic relaxation times Mateusz P. Sowiński 1, Sahil Gahlawat 1,2, Bjarte A. Lund 1,4, Anna-Luisa Warnke 1,4, Kathrin H. Hopmann 1, Janet E. Lovett 3 & Marius M. Haugland 1✉ Nitroxides are widely used as probes and polarization transfer agents in spectroscopy and imaging. These applications require high stability towards reducing biological environments, as well as beneﬁcial relaxation properties. While the latter is provided by spirocyclic groups on the nitroxide scaffold, such systems are not in themselves robust under reducing condi- tions. In this work, we introduce a strategy for stability enhancement through conformational tuning, where incorporating additional substituents on the nitroxide ring effects a shift towards highly stable closed spirocyclic conformations, as indicated by X-ray crystallography and density functional theory (DFT) calculations. Closed spirocyclohexyl nitroxides exhibit dramatically improved stability towards reduction by ascorbate, while maintaining long relaxation times in electron paramagnetic resonance (EPR) spectroscopy. These ﬁndings have important implications for the future design of new nitroxide-based spin labels and imaging agents. 1 ARTICLE Results and discussion S h i X ll 32 1 2 O c Scaffold tuning R This work: This work: - Higher preference for closed conformations - Improved reduction stability - Long Tm preserved We next aimed to force the spirocyclohexyl moieties into closed conformations by further increasing the steric pressure through additional α-functionalization of ketoamine 4. However, this highly sterically hindered substrate proved unreactive towards further alkylation, either upon deprotonation with LDA and treatment with various alkylating agents (e.g., MeI, Me2SO4), or Fig. 1 Context and motivation for this work. a Structural factors affecting nitroxide stability. b Proposed equilibrium between open and closed conformations of spirocyclohexyl nitroxides. c Examples of conformational shifts in spirocyclic nitroxides31,32. d This work. Fig. 1 Context and motivation for this work. a Structural factors affecting nitroxide stability. b Proposed equilibrium between open and closed conformations of spirocyclohexyl nitroxides. c Examples of conformational shifts in spirocyclic nitroxides31,32. d This work. COMMUNICATIONS CHEMISTRY \| (2023) 6:111 \| https://doi.org/10.1038/s42004-023-00912-7 \| www.nature.com/commschem 2 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 under Stork enamine alkylation conditions. Conversely, starting from ketoamine 3, two methylene substituents were directly incorporated through condensation with formaldehyde (Fig. 2a)37. Although dienone 6 could be isolated, it polymerized in a matter of hours. Thus, it was immediately subjected to hydrogenation to afford dimethyl spirocyclohexyl ketoamine 7 in excellent yield as a mixture of diastereomers (dr 1:1). Subsequent oxidation with m-CPBA resulted in two diastereomers of dimethylated nitroxide 8, which was recrystallized to enable structural investigation by X-ray crystallography. We obtained a solid-state structure for the trans-diastereomer of 8, where the nitroxide ring adopts a chair conformation with one methyl substituent in an axial and the other in an equatorial orientation. The equatorial methyl group induces a conformational change of the proximal spirocyclohexyl moiety, resulting in a semi-open arrangement. Thus, we proceeded to reduce ketone 8 to alcohol 9, which was obtained in excellent yield as a mixture of two diastereomers (dr 1:1), which were separable by column chromatography. The cis,cis- diastereomer of 9 was a crystalline solid, with the relative stereochemistry conﬁrmed by X-ray crystallography (Fig. 2b). To our delight, cis,cis-9 exhibits a closed conformation, presumably due to the combined steric pressure exerted by the two equatorial methyl substituents. The nitroxide ring adopted a chair conforma- tion, placing the alcohol functionality in an axial position. Kinetic studies—stability toward reduction. Results and discussion S h i X ll The stability of nitroxides towards reduction was measured by continuous-wave (CW) X-band EPR spectroscopy as a function of decay of the characteristic triplet signal in the presence of sodium ascorbate (1.4 equivalents). These conditions permitted the observation of meaningful decay rates at nitroxide concentrations high enough to be reliably measured on a benchtop EPR spectrometer. The time-dependent decay curves of the novel nitroxides 5, 8, and cis,cis-9 are shown in Fig. 3a. To conﬁrm our hypothesis, these substituted nitroxides were compared with the non-methylated spirocyclohexyl nitroxides 10 and 11. The reduction kinetics of the well-established tetramethyl-substituted TEMPOL (12) and tetraethyl-substituted TEEPOL (13), were included as bench- marks for the piperidinyl nitroxides that are least and most resistant towards reduction, respectively25. Second-order rate constants k were derived by ﬁtting decay data to a second-order kinetic rate law (see the SI for more details), and are included in Fig. 3c. Deviations from ideal second-order behavior were apparent, especially for rapidly reducing nitroxides, for which reliable rate constants could not be extracted. The non-ideal reduction kinetics are consistent with previous ﬁndings that the mechanism of nitroxide reduction by ascorbate is reversible and proceeds through a complex mechanism23. The two diastereomers of 8 were not readily separable by chromatography; moreover, we were concerned about possible diastereomeric interconversion via keto-enol tautomeric equilibria. N H O 3 LDA; CH3I N H O 6 NaOH, HCHO, THF/H2O (1:1) H2 (5 atm.), Pd/C THF, - 44% N H O 4 N O O 5 m-CPBA CH2Cl2, 84% EtOAc 70% (2 steps) N H O 7 N O O 8 m-CPBA, K2CO3 CH2Cl2, 92% N O 9 OH a NaBH4, EtOH, 99% b 5 trans-8 cis,cis-9 Fig. 2 Synthetic route and solid-state structures of nitroxides. a Synthesis strategies for obtaining substituted spirocyclohexyl nitroxides 5, 8, and 9. b Crystal structures of solid-state nitroxides 5, trans-8, and cis,cis-9 obtained by X-ray crystallography. N H O 3 LDA; CH3I N H O 6 NaOH, HCHO, THF/H2O (1:1) H2 (5 atm.), Pd/C THF, - 44% N H O 4 N O O 5 m-CPBA CH2Cl2, 84% EtOAc 70% (2 steps) N H O 7 N O O 8 m-CPBA, K2CO3 CH2Cl2, 92% a b 5 trans-8 cis,cis-9 b The presence of functional groups is known to inﬂuence the reduction rate of nitroxides25. Results and discussion S h i X ll In line with previous studies, the presence of an alcohol vs a ketone functionality has a negligible impact on the rate of reduction for the conditions and nitroxide scaffolds examined in this work. It is known that spirocyclohexyl- substituted piperidinyl nitroxides show slightly higher stability towards reduction than tetramethyl-substituted nitroxides, but not higher than tetraethyl-substituted radicals26,38,39, a trend that is also clear from the decay curves in Fig. 3a. However, we were trans-8 cis,cis-9 5 Fig. 2 Synthetic route and solid-state structures of nitroxides. a Synthesis strategies for obtaining substituted spirocyclohexyl nitroxides 5, 8, and 9. b Crystal structures of solid-state nitroxides 5, trans-8, and cis,cis-9 obtained by X-ray crystallography. Fig. 3 Kinetic decay studies of nitroxides. a Second-order kinetic reduction curves of nitroxides (2 mM) with sodium ascorbate (1.4 eq.) in PBS buffer/ DMSO (1:1) at 291 K. Data points are the average of three runs, with error bars representing 2 × standard deviation. b Structures of nitroxides for kinetic studies. c Second-order rate constants k [M−1 s−1] for 2 mM piperidinyl nitroxides, derived from decay curves by ﬁtting to a second-order kinetic rate law. See the SI, Figs. S11, S12, and Table S1 for further details. Fig. 3 Kinetic decay studies of nitroxides. a Second-order kinetic reduction curves of nitroxides (2 mM) with sodium ascorbate (1.4 eq.) in PBS buffer/ DMSO (1:1) at 291 K. Data points are the average of three runs, with error bars representing 2 × standard deviation. b Structures of nitroxides for kinetic studies. c Second-order rate constants k [M−1 s−1] for 2 mM piperidinyl nitroxides, derived from decay curves by ﬁtting to a second-order kinetic rate law. See the SI, Figs. S11, S12, and Table S1 for further details. Fig. 3 Kinetic decay studies of nitroxides. a Second-order kinetic reduction curves of nitroxides (2 mM) with sodium ascorbate (1.4 eq.) in PBS buffer/ DMSO (1:1) at 291 K. Data points are the average of three runs, with error bars representing 2 × standard deviation. b Structures of nitroxides for kinetic studies. c Second-order rate constants k [M−1 s−1] for 2 mM piperidinyl nitroxides, derived from decay curves by ﬁtting to a second-order kinetic rate law. See the SI, Figs. S11, S12, and Table S1 for further details. Fig. 3 Kinetic decay studies of nitroxides. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 ARTICLE Fig. 4 Relaxation time measurements. a Spin echo decay curves for nitroxides cis,cis-9, 11, 12, and 13 at 50 and 100 K. b Temperature-dependent Tm values for nitroxides cis,cis-9, 11, 12, and 13 measured by spin echo decay. The method for the extraction of Tm values is described in the SI, Fig. S13. Fig. 4 Relaxation time measurements. a Spin echo decay curves for nitroxides cis,cis-9, 11, 12, and 13 at 50 and 100 K. b Temperature-dependent Tm values for nitroxides cis,cis-9, 11, 12, and 13 measured by spin echo decay. The method for the extraction of Tm values is described in the SI, Fig. S13. similar echo decay to the non-methylated spirocyclohexyl nitroxide 11, which exhibits the longest Tm. Tetramethyl nitroxide 12 has a slightly shorter Tm even at 50 K, but tetraethyl nitroxide 13 shows signiﬁcantly more rapid relaxation. This has previously been ascribed to an increased local proton concentration in the tetraethyl substituents43. However, we note that spirocyclohexyl nitroxides also have an increased local proton concentration, and that the line shape of the echo decay of 13 suggests there are multiple mechanisms of spin echo dephasing in operation for the tetraethyl system at 50 K. pleased to observe a signiﬁcant decrease of almost one order of magnitude in the rate of reduction of dimethylated spirocyclohexyl nitroxide cis,cis-9 compared to non-methylated nitroxide 11. This effect can be ascribed to the conformational effect on the spirocyclohexyl rings caused by the additional steric pressure from the two methyl substituents in 9, causing the compound to prefer a closed conformation, similar to that observed in the solid-state X-ray crystal structure (Fig. 2b), that more effectively shields the radical from reducing agents. The reduction rate of mono-methylated ketone nitroxide 5, however, does not differ signiﬁcantly from that of the non-methylated version 10. This is also in agreement with the open conformation adopted in the crystal structure of this compound (Fig. 2b). Surprisingly, the dimethylated ketone 8, which was used as a mixture of diastereomers, is reduced more rapidly than all other spirocyclohexyl nitroxides. While the trans-diastereomer of 8 adopts a semi-open conformation in the solid state (Fig. 2b), the preferred conformation of the other diastereomer is unknown. Thus, at this point, the conformation of 8 cannot be related to its reactivity. We next investigated Tm across a range of temperatures. A plot of Tm as a function of temperature (Fig. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 4b) clearly shows how methyl rotation signiﬁcantly increases relaxation in tetramethyl nitroxide 12 (from 80 K) and tetraethyl nitroxide 13 (from 60 K). The earlier onset of methyl rotation in tetraethyl nitroxides is explained by a lower energy barrier of methyl rotation in these systems44. It is not clear, however, why the Tm apparently increases again, and becomes longer than the tetramethyl nitroxide 12 as the temperature rises above ca. 120 K. This unexpected behavior is in line with observations in our previous work, and suggests further complications connected with the use of tetraethyl nitroxides45. The origins of this effect will be further investigated in the future. Conversely, spirocyclohexyl nitroxides cis,cis-9 and 11 exhibit low Tm dephasing rates even at higher temperatures, with dimethylated spirocyclohexyl nitroxide cis,cis- 9 having relaxation rates similar to the non-methylated analog 11 up to ca. 100 K. Additional dephasing beyond this temperature is presumably driven by rotation of the methyl substituents on the nitroxide ring. The spin echo decay curves at 100 K (Fig. 4a) illustrate how distinct behavior of different methyl environments have a critical impact on Tm at this temperature, which can be easily achieved using liquid nitrogen cooling. The diversity of the impact of methyl substituents on Tm between cis,cis-9 and 12 may be a function of the distance between the paramagnetic center and the methyl groups, or different rates of methyl rotation44. The alignment of the group with the anisotropic spin tensor of the electron may be an additional contributing factor. Relaxation time measurements. For potential applications as spin labels for DEER or polarization transfer agents in DNP NMR, nitroxides should also ideally exhibit sufﬁciently long relaxation times (Tm) to enable precise measurement of long distances, and efﬁcient polarization transfer for signal enhance- ment, respectively20,40. It is well-known that the presence of methyl groups shortens Tm due to methyl rotation, especially at temperatures above 70 K27. Although the requirement for using cryogenic temperatures that rely on liquid helium increases the operating costs of EPR spectrometers, contemporary instruments now minimize liquid helium consumption by the use of circu- lating liquid helium pumps41. Nevertheless, for reasons of cost and sustainability, it is desirable to preserve extended Tm into temperatures that can be acquired using liquid nitrogen, espe- cially for DNP NMR applications42. For Tm measurements, nitroxides were dissolved in 1:1 PBS buffer/glycerol, in which concentrations of 50 µM were easily achieved. Results and discussion S h i X ll a Second-order kinetic reduction curves of nitroxides (2 mM) with sodium ascorbate (1.4 eq.) in PBS buffer/ DMSO (1:1) at 291 K. Data points are the average of three runs, with error bars representing 2 × standard deviation. b Structures of nitroxides for kinetic studies. c Second-order rate constants k [M−1 s−1] for 2 mM piperidinyl nitroxides, derived from decay curves by ﬁtting to a second-order kinetic rate law. See the SI, Figs. S11, S12, and Table S1 for further details. COMMUNICATIONS CHEMISTRY \| (2023) 6:111 \| https://doi.org/10.1038/s42004-023-00912-7 \| www.nature.com/commschem 3 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 For cis,cis-9 there are two low- energy conformations: a closed structure with equatorial methyl groups (as also observed in the solid-state crystal structure, Fig. 2b), and an open conformation with both methyl groups in axial positions (Fig. 5b). These structures are the predominantly popu- lated conformations in solution, in an approximate ratio of 1:2. Notably, the two most stable conformers have very similar energies, resulting in a change in their order of relative stability when using B3LYP-D3 instead of PBE0-D3BJ (see SI, Fig. S14). Thus, the calculations support our hypothesis that methyl substituents at the 3- and 5-positions of the nitroxide ring exert a steric pressure that can increase the preference for the closed conformation. they may be even more stable towards reduction than cis,cis-9. The calculated conformational energies and Boltzmann distributions of ketone nitroxides 10, 5, and 8 present a more complicated picture (Fig. 6 and SI, Fig. S16). Due to reduced torsional strain, six-membered rings with two sp2-hybridized centers in a 1,4-relationship can readily access low-energy twist- boat conformations58. Reduced strain in the transition states reported for conformational interconversion further indicates that such systems will rapidly interconvert at room temperature59. Indeed, the non-methylated ketone nitroxide 10 preferentially adopts an open twist-boat conformation, which is favored over a chair conformation by 1.7 kcal/mol (Fig. 6a and SI, Fig. S16). In mono-methylated nitroxide 5 (Fig. 6b), a small energetic preference is found for an open twist-boat conformation that places the methyl substituent in an axial orientation, as observed in the solid-state crystal structure of this compound (Fig. 2b). Nitroxide 5, however, also populates several other low- energy open and semi-open structures, but the steric pressure exerted by a single methyl substituent is apparently insufﬁcient to induce any signiﬁcant population of closed conformations. The trans-diastereomer of dimethylated nitroxide 8 prefers a semi-open chair conformation, as observed in the X-ray structure (Fig. 2b), but can also access two closed structures only 0.5 kcal/mol higher in energy (Fig. 6c). For the cis-8 diastereomer, a preference is found for a semi-open twist-boat conformation, but this compound also signiﬁcantly populates a closed chair conformation that is only 0.2 kcal/mol higher in energy (Fig. 6d). COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 Fig. 5 Computational investigation of the conformational distribution of alcohol nitroxides. Distribution of calculated conformers in open, semi-open, and closed conformations for nitroxides 11 (a), cis,cis-9 (b), cis,trans-9 (c), and trans,trans-9 (d). The most highly populated conformers are shown for each nitroxide, along with the calculated Gibbs free energies (in kcal/mol, relative to the most stable conformation) and Boltzmann distribution (parentheses, in percentage) (298 K, PBE0-D3BJ/def2-TZVPP[IEFPCM, water])46–56. See the SI, Fig. S15, for all calculated conformations and energies. Fig. 5 Computational investigation of the conformational distribution of alcohol nitroxides. Distribution of calculated conformers in open, semi-open, and closed conformations for nitroxides 11 (a), cis,cis-9 (b), cis,trans-9 (c), and trans,trans-9 (d). The most highly populated conformers are shown for each nitroxide, along with the calculated Gibbs free energies (in kcal/mol, relative to the most stable conformation) and Boltzmann distribution (parentheses, in percentage) (298 K, PBE0-D3BJ/def2-TZVPP[IEFPCM, water])46–56. See the SI, Fig. S15, for all calculated conformations and energies. open conformation, by 5.0 kcal/mol, if all nitroxide ring substituents are equatorial and 2.7 kcal/mol if substituents are axial (Fig. 5d and SI, Fig. S15). The increased preference for closed conformations of these other diastereomers of 9 suggests that they may be even more stable towards reduction than cis,cis-9. comparison (SI, Fig. S14). First, Gibbs free energies and Boltzmann distributions were computed for open, semi-open, and closed con- formations of alcohol nitroxides 11 and 9 (Fig. 5 and SI, Fig. S15). Conformers were identiﬁed where both the central nitroxide- containing ring and spirocyclohexyl substituents adopt chair conformations. Interconversion between these conformers corre- sponds to a ring-ﬂip of either the nitroxide-containing heterocycle or the spirocyclohexyl rings, via intermediate twist-boat and chair structures. We expect the identiﬁed conformations to be freely interconverting in solution at room temperature; as the nitroxide functionality is approximately planar, nitroxides 11 and 9 are structurally comparable to highly sterically crowded cyclohex- anones, which have an estimated energy barrier for conformational interconversion of 16.7 kcal/mol57. For the non-methylated nitr- oxide 11 (Fig. 5a), the open conformation with an equatorial hydroxyl group is favored over the corresponding closed con- formation by 2.2 kcal/mol, in line with the expectation that this compound favors an open conformation. This contrasts with the dimethylated analog 9, which can exist as the diastereomers cis,cis- 9, trans,trans-9, and cis,trans-9. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 After ﬂash freezing in liquid nitrogen, samples were cooled to 50 K and spin echo decay curves were recorded using pulsed Q-band EPR spectroscopy. The spin echo decays of nitroxide cis,cis-9, 11, 12 and 13 at this temperature are shown in Fig. 4a. Dimethylated spirocyclohexyl nitroxide cis,cis-9 has a DFT calculations. To assess the impact of the additional nitroxide ring substitution across the conformational equilibrium, a com- putational analysis using density functional theory (DFT, PBE0- D3BJ/def2-TZVPP[IEFPCM])46–56 was performed. The robustness of computed results was checked by applying B3LYP-D3 for COMMUNICATIONS CHEMISTRY \| (2023) 6:111 \| https://doi.org/10.1038/s42004-023-00912-7 \| www.nature.com/commschem 4 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 ARTICLE COMMUNICATIONS CHEMISTRY \| (2023) 6:111 \| https://doi.org/10.1038/s42004-023-00912-7 \| www.nature.com/commschem COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 Overall, the calculated conformational energies for nitroxides 5, 8, and 10 indicate that the introduction of methyl substituents does increase the occupancy of semi-open and closed conformations also for ketone spirocyclohexyl nitroxides, although this does not translate into increased stability towards reduction (cf. Fig. 3a). A possible explanation may be found in a higher thermodynamic electron afﬁnity of 5 and 8 (vide infra). The calculations also show that the relative stereochemical conﬁguration of the ring substituents is important. The diastereomer cis,trans-9 (Fig. 5c) mostly populates semi-open and closed conformations, with the former, lowest-energy structure demonstrating that an equatorial methyl group is more effective at forcing the spirocyclohexyl ring to adopt a closed position than an axial group, for nitroxide rings in a chair conformation. In line with this, the last possible diastereomer, trans,trans-9, displays a strong preference for the closed over the To assess any thermodynamic contribution to reduction stability, we next calculated nitroxide electron afﬁnities (EAs). 5 MMUNICATIONS CHEMISTRY \| (2023) 6:111 \| https://doi.org/10.1038/s42004-023-00912-7 \| www.nature.com/commschem 5 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 ARTICLE Fig. 6 Computational investigation of the conformational distribution of ketone nitroxides. Distribution of calculated conformers in open, semi-open, and closed conformations for nitroxides 10 (a), 5 (b), trans-8 (c), and cis-8 (d). The most highly populated conformers are shown for each nitroxide, along with the calculated Gibbs free energies (in kcal/mol, relative to the most stable conformation) and Boltzmann distribution (parentheses, in percentage) (298 K, PBE0-D3BJ/def2-TZVPP[IEFPCM, water])46–56. See the SI, Fig. S16, for all calculated conformations and energies. Fig. 6 Computational investigation of the conformational distribution of ketone nitroxides. Distribution of calculated conformers in open, semi-open, and closed conformations for nitroxides 10 (a), 5 (b), trans-8 (c), and cis-8 (d). The most highly populated conformers are shown for each nitroxide, along with the calculated Gibbs free energies (in kcal/mol, relative to the most stable conformation) and Boltzmann distribution (parentheses, in percentage) (298 K, PBE0-D3BJ/def2-TZVPP[IEFPCM, water])46–56. See the SI, Fig. S16, for all calculated conformations and energies. Energies were calculated for optimized geometries of all possible conformations of the hydroxylamine anions formed by single- electron reduction of nitroxide radicals (SI, Fig. S17), and the adiabatic EA was taken as the energy difference between the most stable conformation of the nitroxide (Figs. 5, 6) and the most stable conformation of the hydroxylamine anion (SI, Fig. S17) for each compound. The EAs (Fig. Methods G l et ods General methods and materials are described in the Supplementary Information (Page 2, SI). General methods and materials are described in the Supplementary Information (Page 2, SI). Chemical synthesis. Detailed synthetic procedures and characterization data for all nitroxides and intermediates are described in the Supplementary Information. X-ray crystallography. Crystals of compounds 5, 8, and cis,cis-9 were grown from diethyl ether by the slow evaporation method. Crystals were glued to glass ﬁbers and mounted on the goniometer. Data were collected using a D8 Venture system with a Cu-anode d (λ = 1.54178 Å). See the SI, Figs. S1–S3 for the ellipsoids. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 7) are generally higher for ketone nitroxides compared to the alcohols. This may be due to an electronic contribution from the electron-withdrawing carbonyl groups. For the alcohol nitroxides, dimethylated spirocyclohexyl nitroxide cis,cis-9, and the non-methylated analog 11 have identical EAs, which supports our assumption that the stabiliza- tion achieved by the introduction of methyl groups is a kinetic effect. By comparison, tetramethyl-substituted nitroxide 12 has a slightly higher apparent EA, while the EA of tetraethyl analog 13 is considerably lower. We ascribe this trend to the electronic repulsion between the hydroxylamine anion and the α-substi- tuents, which is reduced in 12 and increased in 13, and constitutes a thermodynamic factor that disfavors the reduction of nitroxides. The ketone nitroxide cis-8, however, has a signiﬁcantly higher EA than all other nitroxides, which counter- acts the presumed stability in more highly populated semi-open and closed conformations. This can explain the experimental ﬁnding (Fig. 3a) that nitroxide 8 (as a mixture of both diastereomers) is reduced more rapidly than the other spirocy- clohexyl nitroxides. substituents on the nitroxide ring can induce spirocyclohexyl nitr- oxides to favor closed conformations. Our results indicate that only methyl substituents in an equatorial position on chair conformations of the central nitroxide ring can maximize the steric pressure to fully enforce a closed conformation of the nearby spirocyclohexyl groups. As indicated by kinetic decay studies with CW-EPR, this con- formational biasing signiﬁcantly increases the stability of spir- ocyclohexyl nitroxides towards reduction by ascorbate. Moreover, the beneﬁcial extended phase memory times (Tm) exhibited by spir- ocyclohexyl nitroxides in pulsed EPR are generally preserved despite the introduction of methyl substituents on the nitroxide ring, thereby striking an excellent compromise between reduction stability and extended Tm in these scaffolds. These balanced properties are of great potential utility in nitroxides intended for applications such as bio- compatible spin labels, imaging probes, or DNP NMR polarization transfer agents. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 11 cis,cis-9 N O HO N O HO N O O 5 N O O cis-8 trans-8 N O O N O O 10 N O HO N O HO N O O N O O N O O N O HO N O HO N O HO N O HO 12 13 + e G = 54.6 kcal/mol + e G = 54.6 kcal/mol + e G = 55.5 kcal/mol + e G = 52.4 kcal/mol + e G = 57.6 kcal/mol + e G = 60.3 kcal/mol + e G = 57.7 kcal/mol + e G = 56.9 kcal/mol N O O Fig. 7 Computational investigation of nitroxide electron afﬁnities. Calculated adiabatic electron afﬁnities (EAs) for nitroxides 5, 8, cis,cis-9, and 10–13 (298 K, PBE0-D3BJ/def2-TZVPP[IEFPCM, water])46–56. EAs were taken as the energy difference between the most stable nitroxide conformer and the most stable hydroxylamine anion conformer for each compound. 11 cis,cis-9 N O HO N O HO N O HO N O HO N O HO N O HO N O HO N O HO 12 13 + e G = 54.6 kcal/mol + e G = 54.6 kcal/mol + e G = 55.5 kcal/mol + e G = 52.4 kcal/mol N O O 5 N O O cis-8 trans-8 N O O N O O 10 N O O N O O N O O + e G = 57.6 kcal/mol + e G = 60.3 kcal/mol + e G = 57.7 kcal/mol + e G = 56.9 kcal/mol N O O s-9 N O HO N O O cis,cis-9 5 Fig. 7 Computational investigation of nitroxide electron afﬁnities. Calculated adiabatic electron afﬁnities (EAs) for nitroxides 5, 8, cis,cis-9, and 10–13 (298 K, PBE0-D3BJ/def2-TZVPP[IEFPCM, water])46–56. EAs were taken as the energy difference between the most stable nitroxide conformer and the most stable hydroxylamine anion conformer for each compound. 5. Lamontagne, H. R. & Lessard, B. H. Nitroxide-mediated polymerization: a versatile tool for the engineering of next generation materials. ACS Appl. Polym. Mater. 2, 5327–5344 (2020). Kinetic studies. Sodium ascorbate solution in PBS buffer/DMSO (1:1 v/v) was used as a reduction agent. Ascorbate solution was added to the nitroxide solution and the peak height of the low-ﬁeld line of the nitroxide triplet CW-EPR spectrum was measured as a function of time. Each kinetic run was repeated three times on the same day using the same ascorbate solution. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 Detailed procedures and data ﬁtting are described in the Supplementary Information. y ( ) 6. Tebben, L. & Studer, A. Nitroxides: applications in synthesis and in polymer chemistry. Angew. Chem. Int. Ed. 50, 5034–5068 (2011). y 6. Tebben, L. & Studer, A. Nitroxides: applications in synthesis and in polymer chemistry. Angew. Chem. Int. Ed. 50, 5034–5068 (2011). 7. Hawker, C. J., Bosman, A. W. & Harth, E. New polymer synthesis by nitroxide mediated living radical polymerizations. Chem. Rev. 101, 3661–3688 (2001). 8. Hansen, K.-A. & Blinco, J. P. Nitroxide radical polymers – a versatile material class for high-tech applications. Polym. Chem. 9, 1479–1516 (2018). DFT calculations. All calculations were performed on complete molecular systems without any truncations using Gaussian16 (Revision B.01)56. The systems were fully relaxed, and no symmetry constraints were imposed. For the geometry optimizations, the DFT hybrid functional PBE049,50, which contains 25 percent Hartree–Fock exchange, was used along with the D3BJ55 dispersion correction (additional calcu- lations with B3LYP47-D3 provide similar trends, see Figure S14). The basis set used for geometry optimizations and frequency calculations was def2-TZVPP46,48,52. Solvation effects were included using the polarizable continuum model (IEFPCM)54 with the parameters of water (ε = 80). Reported Gibbs free energies include thermal corrections computed at 298 K. See the SI, Fig. S18, for the optimized geometries. Hartree energies of all calculated conformers are shown in Table S2. 9. Ratera, I. & Veciana, J. Playing with organic radicals as building blocks for functional molecular materials. Chem. Soc. Rev. 41, 303–349 (2012). 10. Lewandowski, M. & Gwozdzinski, K. Nitroxides as antioxidants and anticancer drugs. Int. J. Mol. Sci. 18, 2490 (2017). 11. Keana, J. F. W. Newer aspects of the synthesis and chemistry of nitroxide spin labels. Chem. Rev. 78, 37–64 (1978). 12. Haugland, M. M., Anderson, E. A. & Lovett, J. E. Elect 12. Haugland, M. M., Anderson, E. A. & Lovett, J. E. Electr Resonance Vol. 25 (Royal Society of Chemistry, 2016). Resonance Vol. 25 (Royal Society of Chemistry, 2016). 13. Hubbell, W. L., Caﬁso, D. S. & Altenbach, C. Identifying conformational changes with site-directed spin labeling. Nat. Struct. Biol. 7, 735–739 (2000). 14. Hyodo, F. et al. Pulsed EPR imaging of nitroxides in mice. J. Magn. Reson. 197, 181–185 (2009). Conclusion h k Electron paramagnetic resonance (EPR) spectroscopy. Room temperature CW- EPR measurements were acquired using an X-band Afﬁrmo microESR Benchtop EPR Spectrometer (now marketed as the Bruker microESR). See the SI, Figures S4- S10 for X-band CW-EPR spectra of the nitroxides. Bruker Elexsys E580 with high powered (150 W) Q-band (34 GHz) and an ER 5106QT-2w cylindrical resonator was used for relaxation parameter investigations. Detailed parameters and proce- dures are described in the Supplementary Information. In this work, we have successfully developed a new and reliable synthetic route to obtain a series of novel 3,5-substituted spir- ocyclohexyl piperidinyl nitroxides. Investigation of the conformations of these compounds by X-ray crystallography and DFT calculations conﬁrmed our hypothesis that the introduction of additional COMMUNICATIONS CHEMISTRY \| (2023) 6:111 \| https://doi.org/10.1038/s42004-023-00912-7 \| www.nature.com/commschem 6 ARTICLE Data availability 15. Kubicki, D. J. et al. Rational design of dinitroxide biradicals for efﬁcient cross- effect dynamic nuclear polarization. Chem. Sci. 7, 550–558 (2016). Copies of the NMR spectra are included in Supplementary Data 1 (Figs. S19–S46). The X-ray crystallographic coordinates for structures reported in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition numbers 2214625, 2214626, and 2214627. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Moreover, CIF ﬁles and SCXRD reports for nitroxides 5, trans-8, and cis,cis-9 are included in Supplementary Data 2–7. Coordinates of all optimized geometries and their energies are given in Supplementary Data 8 (XYZ ﬁle). Additional raw data is available through the DataverseNO repository at https://doi.org/10.18710/UQMMZE60. 16. Levien, M., Hiller, M., Tkach, I., Bennati, M. & Orlando, T. Nitroxide derivatives for dynamic nuclear polarization in liquids: the role of rotationa diffusion. J. Phys. Chem. Lett. 11, 1629–1635 (2020). 17. Clore, G. M. & Iwahara, J. Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem. Rev. 109, 4108–4139 (2009). 18. Soule, B. et al. The chemistry and biology of nitroxide compounds. Free Radic. Biol. Med. 42, 1632–1650 (2007). Received: 3 March 2023; Accepted: 24 May 2023; Received: 3 March 2023; Accepted: 24 May 2023; 19. Shah, S. A. et al. Nitroxide‐enhanced MRI of cardiovascular oxidative stress. 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Spirocyclic nitroxides as versatile tools in modern natural sciences: from synthesis to applications. Part I. Acknowledgements 31. Stevanato, G. et al. Open and closed radicals: local geometry around unpaired electrons governs magic-angle spinning dynamic nuclear polarization performance. J. Am. Chem. Soc. 142, 16587–16599 (2020). The authors thank Dr. D. G. Mazhukin for helpful suggestions for the preprint version of this work. M.M.H. and A.-L.W. thank the Tromsø Research Foundation and UiT Centre for New Antibacterial Strategies (CANS) for a start-up grant (TFS project ID: 18_CANS). K.H.H. and S.G. thank the Research Council of Norway (No. 300769) and Sigma2 (No. nn9330k and nn4654k), and the European Union’s Horizon 2020 research and inno- vation program under the Marie Skłodowska-Curie grant agreement No 859910. J.E.L. thanks Drs Hassane El Mkami and Robert I. Hunter for technical assistance, the BBSRC (BB/T017740/1) and the Wellcome Trust (099149/Z/12/Z) for the Q-band EPR spec- trometer, the Royal Society for a University Research Fellowship and Research Grant RG120645 for the benchtop spectrometer. B.A.L. thanks The Research Council of Norway for the Centre of Excellence and project grants (Grant Nos. 262695 and 274858). performance. J. Am. Chem. Soc. 142, 16587–16599 (2020). 32. Kirilyuk, I. A. et al. Synthesis of 2,5-Bis(spirocyclohexane)-substituted nitroxides of pyrroline and pyrrolidine series, including thiol-speciﬁc spin label: an analogue of MTSSL with long relaxation time. J. Org. Chem. 77, 8016–8027 (2012). 33. Jing, Y., Mardyukov, A., Bergander, K., Daniliuc, C. G. & Studer, A. Synthesis of bulky nitroxides, characterization, and their application in controlled radical polymerization. Macromolecules 47, 3595–3602 (2014). p y 34. Jing, Y., Tesch, M., Wang, L., Daniliuc, C. G. & Studer, A. 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Conceptualization: M.M.H.; funding acquisition: M.M.H., J.E.L., and K.H.H.; methodology: M.M.H., J.E.L., K.H.H., S.G., and M.P.S.; investigation: M.P.S., S.G., A.-L.W., B.A.L., and J.E.L.; formal analysis: M.P.S., S.G., B.A.L., A.-L.W., M.M.H., J.E.L., K.H.H.; software: S.G., K.H.H., and J.E.L.; valida- tion: M.P.S., S.G., B.A.L., M.M.H., K.H.H., and J.E.L.; data curation: M.P.S., S.G., and B.A.L.; visualization: M.P.S., M.M.H., and S.G.; writing—original draft: M.P.S., M.M.H., and S.G.; writing—review & editing: K.H.H., A.-L.W., J.E.L., and B.A.L.; project administration: M.M.H.; resources: M.M.H., J.E.L., and K.H.H.; supervision: M.M.H., K.H.H., and J.E.L. 36. Kiesewetter, M. K., Corzilius, B., Smith, A. A., Grifﬁn, R. G. & Swager, T. M. Dynamic nuclear polarization with a water-soluble rigid biradical. J. Am. Chem. Soc. 134, 4537–4540 (2012). 37. Malievskii, A. D., Shapiro, A. B., Yakovleva, I. V. & Koroteev, S. V. 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Peptide and protein dynamics and low-temperature/DNP magic angle spinning NMR. J. Phys. Chem. B 121, 4997–5006 (2017). 43. Huber, M. et al. Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location. Biophys. Chem. 94, 245–256 (2001). COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-00912-7 Old and new synthetic approaches to spirocyclic nitroxyl radicals. Molecules 26, 677 (2021). Funding g Open access funding provided by UiT The Arctic University of Norway (incl University Hospital of North Norway). 40. Zagdoun, A. et al. Large molecular weight nitroxide biradicals providing efﬁcient dynamic nuclear polarization at temperatures up to 200 K. J. Am. Chem. Soc. 135, 12790–12797 (2013). 41. Zhang, Y., Zhou, F., Li, J., Kang, J. & Zhang, Q. Application and research of new energy-efﬁciency technology for liquid ring vacuum pump based on turbulent drag reduction theory. Vacuum 172, 109076 (2020). References 22. 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If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 49. Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation made simple. Phys. Rev. Lett. 77, 3865–3868 (1996). 50. Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation made simple [Phys. Rev. Lett. 77, 3865 (1996)]. Phys. Rev. Lett. 78, 1396–1396 (1997). 51. Adamo, C. & Barone, V. Toward reliable density functional methods without adjustable parameters: the PBE0 model. J. Chem. Phys. 110, 6158–6170 (1999). 52. Weigend, F. & Ahlrichs, R. Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: design and assessment of accuracy. Phys. Chem. Chem. Phys. 7, 3297 (2005). y y y 53. 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https://openalex.org/W2947338569	https://www.scielo.br/j/reeusp/a/Gz9P835ZfNnxwWdndy9gPvm/?lang=en&format=pdf	English	null	Social determinants of a Quilombola Community and its interface with Health Promotion	Revista da Escola de Enfermagem da USP	2,019	cc-by	6,418	Social determinants of a Quilombola Community and its interface with Health Promotion Determinantes Sociais de uma comunidade quilombola e a interface com a Promoção da Saúde Determinantes sociales de una comunidad quilombola y la interfaz con la promoción de la salud How to cite this article: d d How to cite this article: Durand MK, Heidemann ITSB. Social determinants of a Quilombola Community and its interface with Health Promotion. Rev Esc Enferm USP. 2019;53:e03451. DOI: http://dx.doi.org/10.1590/S1980-220X2018007703451 ORIGINAL ARTICLE ORIGINAL ARTICLE DOI: http://dx.doi.org/10.1590/S1980-220X2018007703451 Michelle Kuntz Durand1,2 Objective: Understanding the relationship of social determinants in the Health Promotion of quilombola women. Method: This was a participatory study developed through the Research Itinerary of Paulo Freire, which comprises three stages: thematic investigation, codification and decoding, and critical unveiling. These stages were developed in Culture Circles from April to June 2016. Twenty themes were investigated, coded and decoded into eight themes, revealing the theme of women and their relationship with the Social Determinants. Results: Ten women from a quilombola community in Santa Catarina participated. The Health Promotion of this population and its interface with Social Determinants were understood. Health, religiosity, racial issues, social and community networks emerged as potentializing community empowerment and solidification. Conclusion: An accentuated correlation of racial issues and fragility combined with health care, education and information, and above all the distancing of vulnerable communities from comprehensive and equitable health was observed, highlighting the imminent need to strengthen Health Promotion strategies. www.ee.usp.br/reeusp Study type This is a qualitative, action-participant research study. The Research Itinerary of Paulo Freire was used as a meth- odological reference, which consists of three intertwined dialectical moments: thematic investigation, codification and decoding, and critical unveiling(11-13), developed in Culture Circles.h The Culture Circle was named by Freire and represents a dynamic moment of dialogue and knowledge; it is an open space which enables different exchanges, mutual respect and the construction of distinct knowledge. Everyone advances through the process of action-reflection-action and “exits” different from how they enter, as limiting situations of reality are revealed and they reflect on it, and then they decode it and visualize it(13,14). Integrating the two approaches of Health Promotion and Social Determinants of Health can contribute to under- standing and challenging health disparities with a view to social well-being, since this integration focuses on the role of inequality reduction policy and distances itself from tra- ditional health discourse based on the individual(5). In addition, Paulo Freire’s Research Itinerary scheme is demonstrated by colors on a moving wheel, as shown in Figure 1, below. The symbolism of a rose is represented in the Thematic Research refers to the ingenuity during the course of the study in generating themes. The codification lilacs, still in a moment of magic, transitioning to gray to represent the exercise of decoding, where the exercise of a critical and reflexive look unfolds. The green in the Critical Unveiling symbolizes the freedom of thought provided by the dialogical encounters culminating in awareness, as represented by the yellow in remitting to light and knowl- edge. It is also perceived that red transits as a symbol of energy, passion and provocation within the circle, represent- ing the constant dialogical problematizations provided by the encounters and linked with the continuous process of action-reflection-action.h It is recognized that risk factors(6) are not equally dis- tributed among populations. Certain subgroups, particularly those with low socioeconomic status, have been found to have higher proportions of risk factors and consequently suffer from precarious health. f Communities with social vulnerability are inserted in this context; a concept that refers to the capacity to struggle and to the recovery of individuals and social groups to confront the health issue(7). Social vulnerability implies in characteristics, resources and abilities inherent in the sub- jects or groups, which may be scarce or inadequate to take advantage of the opportunities available in society. 2 Rev Esc Enferm USP · 2019;53:e03451 METHOD In Brazil, the Health Promotion Policy had a long and conflicting process until its promulgation in 2006, and its redefinition in 2014: it reinforced the fundamental val- ues of solidarity, happiness, ethics, respect for diversity, humanization, responsibility, justice and social inclusion, and adopted the principles of equity, social participation, autonomy, empowerment, intersectoriality, intrasectori- ality, sustainability, comprehensiveness and territoriality. This Policy highlights the Social Determinants of Health (SDH) as a possibility to reduce inequities and promote equity in health(4). INTRODUCTION brought forth a relevant interpretation for the liberated in their journey, struggle and independence, conquering other contexts and perspectives(9). Health Promotion aims to recognize the inspirations and needs of individuals so that it is possible to change the environment in which they transit and consequently reach a full state of individual and collective physical, mental, and social well-being, going beyond a healthy lifestyle, as it encompasses an overall well-being(1-2). h Quilombola communities are marked by historical pro- cesses of discrimination and exclusion, and experience a mar- ginalized socioeconomic reality in relation to the Brazilian population in general(10). Thus, it is fundamental to combine studies of Social Determinants to Health Promotion, espe- cially of socially vulnerable populations. The conception of Health Promotion, although con- temporary, still prevails as a term with ambiguities and contradictions. This dichotomy is linked to the presence of two different discourses: one with an emphasis on modifying individual behaviors, and another that seeks to advance towards a liberating perspective by strength- ening the population’s participation in confronting social determinants(3). In this sense, we seek to perform a work with a group of quilombola women due to their organization and lead- ership within a scenario of struggling to overcome social inequities, in order to understand the relationship of Social Determinants in Health Promotion of these women. www.ee.usp.br/reeusp Social determinants of a Quilombola Community and its interface with Health Promotion Social determinants of a Quilombola Community and its interface with Health Promotion Social determinants of a Quilombola Community and its interface with Health Promotion INTRODUCTION Ivonete Teresinha Schülter Buss Heideman1,3 1 Universidade Federal de Santa Catarina, Programa de Pós-Graduação em Enfermagem, Florianópolis SC, Brazil. 2 Universidade Federal do Paraná, Departamento de Enfermagem, Curitiba, PR, Brazil. 3 Universidade Federal de Santa Catarina, Departamento de Enfermagem, Florianópolis, SC, Brazil. 3 Universidade Federal de Santa Catarina, Departamento de Enfermagem, Florianópolis, SC, Brazil. DESCRIPTORS DESCRIPTORS Social Determinants of Health; Health Promotion; Women; African Continental Ancestry Group; Personal Autonomy; Public Health Nursing. Received: 03/12/2018 Approved: 08/23/2018 Corresponding author: Michelle Kuntz Durand Rua Nereu Ramos, 1441D/ apto. 601 – Centro CEP 89801-021 – Chapecó, SC, Brazil michakd@hotmail.com 1 Rev Esc Enferm USP · 2019;53:e03451 www.ee.usp.br/reeusp Study type hi Critical Unveiling took place in the sixth meeting, in which two significant themes related to the Social Determinants of quilombola women and access to health were unveiled. h This study was conducted in a linear and participative way; therefore, it was possible to explore the different par- ticipations of each member and to value the richness in loco, mutual care and the various ways of learning and apprehen- sion. Thus, the importance of being open to the new and diverse ways of thinking and building health was ratified. hi The participants were introduced in the first meeting, as well as the proposal and the study objectives along with an explanation of the study methodology to be developed in the Culture Circles. i i The themes were recorded in a field notebook, where the observations and information learned during the experiences with the participants were inserted. Audio recorders were also used after consent was given by the participants, and the recorded themes were reproduced by the researcher through directed transcription. In the second meeting we needed to reconfirm and plan the dates and times of the meetings, for which we agreed would be 2 hours before the manual work of the participants. The group reception and their interest in the study were already observed on this day. We also com- pleted the individual socioeconomic questionnaire in this initial phase with the objective of getting to know them and understand their way of thinking about quilombola health and living.h www.ee.usp.br/reeusp Study type Thus, this relationship will determine the degree of impact on individuals’ quality of life(8). In this way, the “quilombo rem- nant” population stands out as a vulnerable ethnic group, especially females, who face an environment of discrimina- tion, prejudice and disrespect, constantly having to revin- dicate their rights and citizenship. l The Culture Circles took place in the Quilombola Community called Morro do Fortunato, municipality of Garopaba, Santa Catarina state. The Community is located in a rural area, with difficult access both to health services and basic human needs. This community was elected by approaching the state’s quilombola communities and con- versing with their leaders, who indicated it as a positive and representative population, with strong receptivity and acceptance of ethnic and racial studies and research. In order to conceptualize the theme in question, the word “quilombo” stands out because it has greater recogni- tion and notoriety, since the term is based on Bantu ety- mology, which means “warrior camp in the forest”. It was popularized in Brazil to support organizations instituted by insurgents against the slavery regime and its resistances, vindications and battles against slavery; this expression also 2 www.ee.usp.br/reeusp Durand MK, Heidemann ITSB Figure 1 – Research Itinerary scheme represented in Colors. Figure 1 – Research Itinerary scheme represented in Colors. The Culture Circles had the participation of 10 quilom- bola women aged between 24 and 54 years old, living in the Morro do Fortunato Quilombola Community. Women who resided in other adjacent quilombola communities were not included in this study.h It is noteworthy that in the decoding phase it was nec- essary to deepen two of the generated themes, which were decoded as transversal to the others: health themes and quilombola women. Topics that lacked further reflection and discussion were decoded in the fifth meeting, which led to the unveiling. The thematic investigation occurred over the course of six Culture Circle meetings from April to June 2016. The Research Itinerary stages were carried out during these three months. The meetings took place with the “women’s group”, held weekly at the Association’s Headquarters in which the women of the Community have been meeting for 8 years. It was agreed that the meetings related to the research would be on the days of the women’s group. Ethical aspects This study was evaluated and approved by the Quilombola community and by the Human Research Ethics Committee of the Federal University of Santa Catarina under Opinion No. 1.466.641 in 2016, pursuant to Resolution 466/2012 of the National Health Council, respecting the ethical prin- ciples of autonomy, beneficence, non-maleficence and justice. The anonymity of the participants (named by codenames chosen by themselves) was respected, and their participa- tion in the study was authorized by signing the Free and Informed Consent Form. The third meeting was the moment in which we began a survey for generating themes. An activity of clipping and collage of journals was proposed, based on the Social Determinants of Health and related to quilombola living. In the fourth encounter we experienced the second phase of the Culture Circle (codification and decoding), in which the 20 themes generated by the women in the Thematic Investigation were selected, which were then inserted onto cards and discussed with the group, requesting that each one choose the one which interested them the most. Rev Esc Enferm USP · 2019;53:e03451 Social Determinants and race issues of Quilombola women In decoding this theme, the group was asked about the concept of health, and the reflections began to become productive: Although often masked in everyday life, racist and seg- mented discourse is still present in the women’s speeches when they tell about certain experiences. It is often camou- flaged and perhaps linked to cultural practices, yet prejudice is perceived by subtle but recurrent euphemisms: Health for me is living well. Health is everything! It’s healthy eating. It is to live in peace, with much love for one another. He- re in this smiling person! This is happiness and which I consider health. And family. Mother, father (...) (Cheirosa). It comes from ancestors (...). Now we are not so reproached. But there are places we go and they look at us a little strange. Atleast employment is good (Amorosa). This was a crucial point revealed in the speeches and unveiled in the group, representing an expanded form of thinking and conceptualizing health, and relating it to Social Determinants. The relationship that the participants have with healthy lifestyles, happiness and well-being is perceived, having the family in harmony as a foundation. The participants report that employment opportunity as “good”, but in the course of the dialogues they verbalize, although still in a naive and unnoticed way, that color and race are often linked to underemployment, which for the most part do not require professional qualification and/or financial resources to enter the labor market. This is reported in the following speech: Another health photo here ... a doctor ... (Querida). Health centered on the figure of the health professional refers to a still naive, biomedical view, and a reduced view of its expanded concept. Some provocations were produced, generating reflections and questions about health and the factors that promote it. RESULTS After selecting the women’s cards of interest, the gen- erating themes were reduced to eight, which were then worked on in two different groups. The proposal was that each group of participants mediated by the researcher and/or the research assistant would discuss the selected themes so that these were later decoded in a large group. Ten women aged between 24 and 54 years belonging to the Morro do Fortunato community participated in this study. In relation to schooling, only one did not study, five finished elementary school and four had completed high school (one is ending this year). Eight of the women have children: two have one son, two have two children, three 3 Rev Esc Enferm USP · 2019;53:e03451 Social determinants of a Quilombola Community and its interface with Health Promotion Although the option for spiritualism was not revealed in the course of the discussions, closeness/similarities with this religious current emerged in the dialogue Circles. have three children and one of the women has four chil- dren. Regarding their professions, one is a clerk, three of the women are vendors, one is a general service assistant, one is an artisan and four are stay-at-home mothers. Regarding their residence time in the Morro do Fortunato, five have always lived there. The shortest residence time was six months, and the longest time was 26 years. g g g A closeness between faith and being healthy was observed, describing the thinking as a strength for healing: One day I also had high blood pressure, but I thought, ‘I don’t accept that’ (...). I took care with salt and food and put it on my head that I would not take medicine at all until I was well. I spent the whole week with only a little salt in my food, lots of salad, green leaves and I was good ... Take care of your food to see if you will need to take medicine ... And another thing, you have to believe that you will be well. After all, do you have faith or not? (Cheirosa). Twenty raised themes were generated over the course of the Culture Circles, subsequently codified and decoded into eight of interest for the group, and further reduced to four which are discussed below. 4 Rev Esc Enferm USP · 2019;53:e03451 Social determinants and the health of Quilombola women The relationship of spirituality as a strategy for healing becomes even more evident in statements of blessings and other religious beliefs. Alternatives of complementary treat- ments were discussed in the Culture Circles, which are based on faith and belief as a treatment and cure: The concept of Health Promotion linked to Social Determinants allows us to have an expanded view of living well, which brings us to the extension of what is thought and proposed in health. hl Thus, new reflections were proposed in the course of the Culture Circle meetings regarding the concept of health connected only to the absence of disease and/or health sys- tems, as in Morgana’s speech which refers to care only being centered on the professional figure: There are some people here who still believe and you have to respect them. In the past there were those who participated in consulta- tions with the “benzedeira”. Since we did not have a doctor, the consultation was always with her. I don’t know if they still have it, but there used to be more. She was wearing an outfit, she blessed me, she gave tea and cured a lot of people (Batalhadora). i The Community Health Agent came here only once during my entire pregnancy. It was a risky pregnancy. I lost my baby, and the Agent only came here once, and made a file. This is something that I don’t see here (Morgana). When thinking about religion, one perceives a strong relation with care practices and ways of thinking about health, and reaches an instigating contemplation in facing the reality of the participants, in other words an understand- ing of being black and how much the religious differences influence their health issues, struggles and organization, moral issues/preconceptions, their living in community and the strengthening of quilombola communities, meaning that they experience a valuation of cultural issues and customs such as health and healing. Or still, when viewing a photo of a man in a white apron, they quickly report: Another health photo here ... a doctor ... (Querida). DISCUSSION It is understood that as the dialogical process advances, new concerns and conceptions emerge, and so the issues are reflected and broadened by the participants. Therefore, the themes of health, religiousness, race and community rela- tions discussed in this study need to be debated and defined with the perspective of being used with more coherence and understanding, contributing to improving the quality of life of the women studied in the community.h More so in the past. I think the elders were really united (Batalhadora). It’s not so much anymore today. (Apaixonada). And Violeta added: It is everyone for themself (Violeta). The participants highlighted the difficulty in commu- nicating and complained they could feel more present and support themselves more because they were geographically close and all of them familiar with each other. They men- tioned that daily activities are increasingly individual and become a reason for separation between them: The concept of health transcendent to the absence of disease presents an expanded view and is linked to healthy habits, leisure and living in harmony, to religiosity, to hap- piness and to the family, perceives the individual in their singularity and in the real context in which they live. This concept is linked to health promotion(15) and advances when influenced by movements to reduce social inequalities and inequities, contemplating prerequisites in its concept to actu- ally achieve it. In the past everything was done together, from washing clothes in the fountain, doing everything together and communicating (Apaixonada). A new reflection begins to emerge in the course of the debate, and with this they perceive a different way of char- acterizing themselves. They come to understand that rela- tionships accompany temporal changes, and as a result they undergo changes that can be seen from a new perspective: Women today are overwhelmed. In the old days she did not have so much to do. Today she works at home and out. At that time she A new reflection begins to emerge in the course of the debate, and with this they perceive a different way of char- acterizing themselves. Social Determinants and the religiosity of Quilombola women Among the determinants that stood out strongly in the meetings was religiosity. On the sociodemographic question- naire, 70% of the participants put the Catholic religion as their religious option, one (10%) is Evangelican, one (10%) is Wicca and Celtic, and one refers to following all religions. For us this goes into the university. Even because I don’t unders- tand much why my profession is to be a cleaning lady and I can’t leave it, but as it goes inside the university, for us it is pride, it is gratifying (Flor). www.ee.usp.br/reeusp Durand MK, Heidemann ITSB The dialogue in the Culture Circles made it possible to reflect on the theme of racism. This theme begins to be unveiled and perceived in facing the barriers that they find in their routines, thus enabling a new way to perceive, and with that to be strengthened: was washing clothes by day at the fountain and today she also spends the day working. How are you going to wash clothes at the fountain at night? (Apaixonada). Although they perceive the increasing role of women in everyday spaces, there is still a gender inequality in the com- munity. The transition from empowerment to an overload in the women’s role is identified by these women as a still ambiguous achievement, which is revealed in the follow- ing lines: Racism also generates difficulty to access ... Even to get a better job, to leave illegal work. That’s why they end up having jobs as a clea- ning lady, or bricklayer. Husbands working without a formal labor contract. Her daughter, for example, started going to college, but it was in Tubarão (city) and it was very difficult, so she gave up. So all this is difficult access ... Even to have a better future (Flor). We respect men (Cheirosa). And Violeta added: But who battles the most, in the fight here (...) are the women (Violeta). fi The participants begin to perceive themselves as women strengthened by the adversities that the issue of race makes them experience. The discussions in the circles culminate with the unveiling of a very present issue in vulnerable popu- lations, namely prejudice: The woman has to run back, sweating (...). There are somethings I do here which are for men and women. You do not have to wait for men (Apaixonada). Social Determinants and the religiosity of Quilombola women The reflections worked out in the meetings incite provo- cations to the reciprocity of the group and mutual affection between them. A new perception then emerges, allowing to overcome the alienation linked to comparing past experi- ences and enabling an analysis of the current reality and the astuteness in facing the dynamicity of life and its real strengths, as expressed in speech: The difficulty of access and racism show how much we are war- riors. Let’s sidestep the difficulties and touch/experience life in a good mood (Flor). The dialogue in the Circles stimulated reflection and articulation of the SDH on racial issues. Racism appears beyond discrimination, but it has a split between the issue of job opportunities, as well as access to health services. This theme comes to be seen as a form of strengthening based on resistance and social struggle in the history of the Morro do Fortunato quilombola community’s constitution. Really, girls. We have our faults, but we also have many qua- lities. I think we even have more quality than defects. And we are united (...) (Batalhadora). The position of the studied women in the study on Social and Community Networks are directed to the past as a com- munication difficulty in the present time, as they find the temporal changes in the Culture Circles and their influence on the communication form, perceiving the union, differ- ences and the inclusion that transits the group. www.ee.usp.br/reeusp Social Determinants of Health and Social and Community Networks A potentiality unveiled in the course of this study refers to the structuring of social networks developed by the group. Although not yet recognized as an important characteristic linked to living in a community, the participants initially demonstrate that they do not consider themselves united and manifest this adjective as something inherent in the past, which is discussed in the Culture Circle meetings: More so in the past. I think the elders were really united (Batalhadora). It’s not so much anymore today. (Apaixonada). And Violeta added: It is everyone for themself (Violeta). A potentiality unveiled in the course of this study refers to the structuring of social networks developed by the group. Although not yet recognized as an important characteristic linked to living in a community, the participants initially demonstrate that they do not consider themselves united and manifest this adjective as something inherent in the past, which is discussed in the Culture Circle meetings: CONCLUSIONh The development of this study sought to understand the relationship of Social Determinants and Health Promotion of this socially vulnerable community, having the experience in the meetings with the quilombola women’s group as a guiding factor. We highlight the Social Determinants of Health related to employment, living and working conditions, religiosity, family, social networks, customs, female empowerment, rac- ism and access to health services. The concept of health was comprehensively and holistically debated, making it possible to articulate it with social, psychological, political, racial, cul- tural and historical issues, reinforcing its expanded concept, and not merely the absence of diseases.h Therefore, it reinforces that religiosity and belief in alternative therapies have a relevant perspective in health care. By means of popular knowledge, the traditional health system is no longer seen as a segmented care prac- tice, evolving towards an expanded understanding of the health and disease process, and acquiring an important meaning within the health-disease process in order to pro- vide answers to what is inexplicable within the biomedical model of healthcare(18). h There is a strong perceived correlation between race relations and fragility combined with care, education, health information, and above all distancing of vulner- able communities from reaching comprehensiveness and equity, highlighting the imminent need to promote practi- cal strategies of health promotion programs linked to these populations. h The participants highlighted the relationship of color and race racism/prejudice with underemployment and dif- ficulties of social insertion into society. Clarity is ratified along with research on social and racial-ethnic inequalities that permeate Brazilian society, since there is usually a great chasm that separates well-to-do groups from subalternized groups, especially historically stigmatized, oppressed and marginalized groups such as blacks and indigenous people, among others(19). The Research Itinerary of Paulo Freire enables sparking topics of interest to the participants, which were concomi- tantly revealed to the dialogical process. The methodology entails to encompass the preconceptions established by the group as it progresses, and with this to promote new and productive reflections. This process, characterized in the scope of participatory research, as well as providing opportunities for knowledge exchange, enables participants to become actors of their realities and access new oppor- tunities for action. h Black Brazilians internalize their belonging to a social minority from an early age. 6 Rev Esc Enferm USP · 2019;53:e03451 DISCUSSION They come to understand that rela- tionships accompany temporal changes, and as a result they undergo changes that can be seen from a new perspective: Living with or without health is not limited to organic, natural and objective evidence, nor to a state of equilib- rium, but it is strongly linked to the sociocultural charac- teristics and meanings that each subject attributes to their living process(15). Health is not only seen as absence of disease, it departs from the reductionist concept and focuses on the professional Women today are overwhelmed. In the old days she did not have so much to do. Today she works at home and out. At that time she 5 Rev Esc Enferm USP · 2019;53:e03451 Social determinants of a Quilombola Community and its interface with Health Promotion figure, enables discussions focused on the conception of expanded health-disease and its articulations with the liv- ing process(1). emphasizing community living as a propulsion strategy and visibility to the feminine role(22). Empowerment emerges as a powerful conception and evaluation of practices which promote female autonomy to overcome the inequality of power in which women, especially black women, find themselves. h It has been reported that it is difficult for health profes- sionals(16), including nurses, to address all issues related to the health/disease process, and their interest in including other social, cultural and spiritual influences in care practices is necessary. Thus, it is necessary to highlight the need to foster strengthening of individual and collective capacities through the multiple Social Determinants, which condition and pro- mote health and well-being(23). Investigating care directs the individual or the family to seek various possibilities related to their beliefs and/or spirituality in a real religious synergy. This spirituality is often found with a healer, “benzedeira” and/or other popular thera- pies, being the opposite of what is found in the traditional health system: a person with similar experience, who under- stands the needs of the patient with empathy(17). Therefore, health must be seen beyond the biological perspective, also encompassing the conception of SDH as a foundation for well-being. h www.ee.usp.br/reeusp DESCRIPTORES Determinantes Sociales de la Salud; Promoción de la Salud; Mujeres: Grupo de Acendencia Continental Africana; Autonomía Personal; Enfermería en Salud Pública. RESUMEN Ob C Objetivo: Comprender la relación de los determinantes sociales en la Promoción de la Salud de mujeres quilombolas. Método: Investigación participativa, desarrollada por medio del Itinerario de Investigación de Paulo Freire, que comprende tres etapas: investigación temática, codificación y desvelamiento crítico. Dichas etapas fueron desarrolladas en los Círculos de Cultura, de abril a junio de 2016. Fueron investigados 20 temas generadores, codificados y descodificados en ocho, develando la temática mujer y la relación con los Determinantes Sociales. Resultados: Participaron 10 mujeres de un comunidad quilombola del Estado de Santa Catarina. Se comprendieron la Promoción de la Salud de esa población y su interfaz con los Determinantes Sociales. La salud, religiosidad, temas raciales, redes sociales y comunitarias surgieron como potencializadores del empoderamiento y fortalecimeinto comunitario. Conclusión: Se nota la acentuada correlación de los temas raciales con la fragilidad en la asistencia, la educación y la información y, especialmente, el alejamiento de comunidades vulnerables de una salud íntegra y ecuánime, destacándose la necesidad eminente de fortalecer estrategas de Promoción de la Salud. DESCRITORES Determinantes Sociais da Saúde; Promoção da Saúde; Mulheres; Grupo com Ancestrais do Continente Africano; Autonomia Pessoal; Enfermagem em Saúde Pública. REFERENCES 1. Malta DC, Moraes Neto OL, Silva MMA, Rocha D, Castro AM, Reis AAC, et al. National Health Promotion Policy (PNPS): chapters of a journey still under construction. Ciênc Saúde Coletiva [Internet]. 2016 [cited 2018 May 31];21(6):1683-94. Available from: http://www. scielo.br/pdf/csc/v21n6/en_1413-8123-csc-21-06-1683.pdf 1. Malta DC, Moraes Neto OL, Silva MMA, Rocha D, Castro AM, Reis AAC, et al. National Health Promotion Policy (PNPS): chapters of a journey still under construction. Ciênc Saúde Coletiva [Internet]. 2016 [cited 2018 May 31];21(6):1683-94. Available from: http://www. scielo.br/pdf/csc/v21n6/en_1413-8123-csc-21-06-1683.pdf 2. Teixeira MB, Casanova A, Oliveira CCM, Ensgtrom EM, Bodstein RCA. Avaliação das práticas de promoção da saúde: um olhar das equipes participantes do Programa Nacional de Melhoria do Acesso e da Qualidade da Atenção Básica. Saúde Debate [Internet]. 2014 [citado 2017 nov. 28];38(n.esp.):52-68. Disponível em: http://www.scielo.br/pdf/sdeb/v38nspe/0103-1104-sdeb-38-spe-0052.pdf 2. Teixeira MB, Casanova A, Oliveira CCM, Ensgtrom EM, Bodstein RCA. Avaliação das práticas de promoção da saúde: um olhar das equipes participantes do Programa Nacional de Melhoria do Acesso e da Qualidade da Atenção Básica. Saúde Debate [Internet]. 2014 [citado 2017 nov. 28];38(n.esp.):52-68. Disponível em: http://www.scielo.br/pdf/sdeb/v38nspe/0103-1104-sdeb-38-spe-0052.pdf 3. Heidemann ITSB, Cypriano CC, Gastaldo D, Jackson S, Rocha CG, Fagundes E. Estudo comparativo de práticas de promoção da saúde na atenção primária em Florianópolis, Santa Catarina, Brasil e Toronto, Ontário, Canadá. Cad Saúde Pública [Internet]. 2018 [citado 2018 maio 30];34(4):e00214516. Disponível em: http://www.scielo.br/pdf/csp/v34n4/1678-4464-csp-34-04-e00214516.pdf 4. Albuquerque TIP, Sá RMPF, Araújo Junior JLAC. Perspectivas e desafios da “nova” Política Nacional de Promoção da Saúde: para qual arena política aponta a gestão? Ciênc Saúde Coletiva [Internet]. 2016 [citado 2018 maio 30];21(6):1695-706. Disponível em: http://www. scielo.br/pdf/csc/v21n6/1413-8123-csc-21-06-1695.pdf 5. Jackson SF, Birn AE, Fawcett SB, Poland B, Schultz JA. Synergy for health equity: integrating health promotion and social determinants of health approaches in and beyond the Americas. Rev Panam Salud Pública [Internet]. 2013 [cited 2018 Mar 05];34(6):473-80. Available from: https://www.scielosp.org/scielo.php?script=sci_arttext&pid=S1020-49892013001200015&lng=en&nrm=iso&tlng=en 6. Gaspar T, Balancho L. Fatores pessoais e sociais que influenciam o bem-estar subjetivo: diferenças ligadas estatuto socioeconômico. Ciênc Saúde Coletiva [Internet]. 2017 [citado 2018 maio 29];22(4):1373-80. Disponível em: http://www.scielo.br/pdf/csc/v22n4/1413-8123- csc-22-04-1373.pdf 7. Prestes CRS, Paiva VSF. Psychosocial approach and health of black women: vulnerabilities, rights and resilience. Saude Soc [Internet]. 2016 [cited 2018 May 29];25(3):673-88. Available from: http://www.scielo.br/pdf/sausoc/v25n3/en_1984-0470-sausoc-25-03-00673.pdf 8. Paula CEA, Silva AP, Bittar Cléria ML. Legislative vulnerability of minority groups. Ciênc Saúde Coletiva [Internet]. 2017 [cited 2018 May 31];22(12):3841-8. Available from: http://www.scielo.br/scielo.php?pid=S1413-81232017021203841&script=sci_arttext&tlng=en 9. Leite IB. The Brazilian quilombo: ‘race’, community and land in space and time. RESUMO RESUMO Objetivo: Compreender a relação dos determinantes sociais na Promoção da Saúde de mulheres quilombolas. Método: Pesquisa participativa, desenvolvida por meio do Itinerário de Pesquisa de Paulo Freire, que compreende três etapas: investigação temática, codificação e descodificação e desvelamento crítico. Essas etapas foram desenvolvidas nos Círculos de Cultura, de abril a junho de 2016. Foram investigados 20 temas geradores, codificados e descodificados em oito, desvelando a temática mulher e a relação com os Determinantes Sociais. Resultados: Participaram 10 mulheres de uma comunidade quilombola catarinense. Compreenderam-se a Promoção da Saúde desta população e sua interface com os Determinantes Sociais. A saúde, religiosidade, questões raciais, redes sociais e comunitárias emergiram como potencializadoras do empoderamento e fortalecimento comunitário. Conclusão: Percebe-se a acentuada correlação das questões raciais e a fragilidade junto à assistência, à educação e à informação em saúde e, principalmente, o distanciamento de comunidades vulneráveis de uma saúde íntegra e equânime, destacando-se a necessidade eminente de fortalecer estratégias de Promoção da Saúde. CONCLUSIONh This classification is exalted, while the opportunities are inserted as a minimum condi- tion of survival linked to low levels of education as a factor of social and political exclusion, still being preponderant for conserving the status quo of Brazilian society, as pointed out by deep inequalities(20).fi This study’s contributions are directed to health pro- fessionals, especially to nursing, who have strong commu- nication with vulnerable populations, extending to other areas and public policies to reach joint action strategies. Regarding the relevance of Paulo Freire’s methodological proposal, it is understood that it was entirely pertinent and timely, since it has made contributions that were still empirical in the course of the meetings, but which certainly materialized as a result of this dialogical and participatory proposal. Official history omits the protagonism and the insub- mission of black women who, in facing the subordination, segregation and vulnerability, maintained resilient move- ments for maintaining and strengthening their culture(21). An empowered person or group is one who performs, by themself, changes and actions which lead them to evolve and grow stronger(22). Emancipatory movements are articulated to the notion of self-esteem of social groups, mainly refer- ring to their individual and psychological dimension(23). It reinforces the imminent need for dialogical exchanges and an expansion of Health Promotion as a strategy for com- munity empowerment.h It is recommended that other communities in socially vulnerable situations be accompanied and “heard”, and that new participatory research be developed in order to approach and promote results which are concomitant to the studied groups, thereby enabling closer approximation of academia with the realities highlighted. The feminist empowerment practices studied in a litera- ture review pondered the contexts in which women live and reflected on the reproductive and domestic role they occupy, 6 Rev Esc Enferm USP · 2019;53:e03451 www.ee.usp.br/reeusp Durand MK, Heidemann ITSB REFERENCES J Peasant Studies. 2015;42(6):1225-40. 10. Cardoso CS, Melo LO, Freitas DA. Condições de saúde nas Comunidades Quilombolas. Rev Enferm 2018 [citado 2018 mar. 05];12(4):1037-45. Disponível em: https://periodicos.ufpe.br/revistas/revistaenfermagem/artic 11. 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Educação em saúde e promoção da saúde: impacto na qualidade de vida do idoso. Saúde Debate [Internet]. 2015 [citado 2018 maio 29];39(105):480-90. Disponível em: http://www.scielo.br/pdf/sdeb/v39n105/0103-1104-sdeb-39-105-00480.pdf 16. Tavares F. Rediscutindo conceitos na antropologia da saúde: notas sobre os agenciamentos terapêuticos. Mana [Internet]. 2017 [citado 2018 maio 30];23(1):201-28. Disponível em: http://www.scielo.br/pdf/mana/v23n1/1678-4944-mana-23-01-00201.pdf 17. Araújo JA. Racismo, Violência e Direitos Humanos: pontos para o debate. Rev Interdiscip Direitos Humanos [Internet]. 2014 [citado 26 out. 2016];2(2):75-96. Disponível em: http://www2.faac.unesp.br/ridh/index.php/ridh/article/view/177/93 18. Santos RC, Silva MS. Condições de vida e itinerários terapêuticos de quilombolas de Goiás. Saúde Soc [Internet]. 2014 [citado 2019 jan. 11];23(3):1049-63. Disponível em: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0104-12902014000301049&lng=en&nrm= iso&tlng=pt 19. Montoro T, Ferreira C. Mulheres negras, religiosidades e protagonismos no cinema brasileiro. Galáxia (São Paulo) [Internet]. 2014 [citado 02 fev. 2017];27(1):145-59. Disponível em: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1982-25532014000100012 20. Portella DDA, Araújo EM, Rocha WJSAF, Chaves JM, Oliveira DD. Homicídios dolosos, tráfico de drogas e indicadores sociais em Salvador, Bahia, Brasil. Ciênc Saúde Coletiva [Internet]. 2017 [citado 2018 maio 30]. Disponível em: http://www.cienciaesaudecoletiva.com.br/ artigos/homicidios-dolosos-trafico-de-drogas-e-indicadores-sociais-em-salvador-bahia-brasil/16153 21. Roso A, Romanini M. Empoderamento individual, empoderamento comunitário e conscientização: um ensaio teórico. Psicol Saber Soc. 2014;3(1):83-95. 22. Saldanha MPA, Gonçalves HS. Práticas de empoderamento feminino na América Latina. Rev Estud Soc [Internet]. 2016 [citado 2018 mar. 05];56(1):80-90. Disponível em: http://www.scielo.org.co/pdf/res/n56/n56a07.pdf 23. Pinheiro DGM, Scabar TG, Maeda ST, Fracolli LA, Pelicioni MCF, Chiesa AM. Health promotion competencies: challenges of formation. Saúde Soc [Internet]. 2015 [cited 2018 May 29];24(1):180-8. Available from: http://www.scielo.br/scielo.php?pid=S0104- 12902015000100180&script=sci_arttext&tlng=en This is an open-access article distributed under the terms of the Creative Commons Attribution Licen This is an open-access article distributed under the terms of the Creative Commons Attribution License. 8 Rev Esc Enferm USP · 2019;53:e03451 Rev Esc Enferm USP · 2019;53:e03451 www.ee.usp.br/reeusp
https://openalex.org/W3124723660	https://figshare.com/articles/journal_contribution/Differential_i_in_vitro_i_interactions_of_the_Janus_kinase_inhibitor_ruxolitinib_with_human_SLC_drug_transporters/13699236/1/files/26312781.pdf	English	null	Differential <i>in vitro</i> interactions of the Janus kinase inhibitor ruxolitinib with human SLC drug transporters	Xenobiotica	2,021	cc-by	368	Differential in vitro interactions of the Janus kinase inhibitor ruxolitinib with human SLC drug transporters HEK-MOCK cells 4-Di-ASP 4-Di-ASP + Ruxo DBF DBF + Ruxo 6-CF 6-CF + Ruxo DCF DCF + Ruxo 8-FcA 8-FcA + Ruxo 0 50 100 150 200 NS NS NS NS NS fluorescence (AU) Figure S1: Lack of major analytical interaction of ruxolitinib (Ruxo) with fluorescent probes used as substrates for transporters. HEK-MOCK cells plated in 96 wells were either pre- treated or not with 100 µM ruxolitinib for 3 h; they were next incubated with the reference fluorescent probes 4 Di-ASP, DCF, DBF, 6-CF or 8-FcA (each at 10 µM) for 5 min. Probe- related fluorescence was then determined by spectrofluorimetry; data are expressed as arbitrary unit (AU)/wells and are the mean ± SEM of at least three assays. NS, not statistically significant. Differential in vitro interactions of the Janus kinase inhibitor ruxolitinib with human SLC drug transporters HEK-MOCK cells 4-Di-ASP 4-Di-ASP + Ruxo DBF DBF + Ruxo 6-CF 6-CF + Ruxo DCF DCF + Ruxo 8-FcA 8-FcA + Ruxo 0 50 100 150 200 NS NS NS NS NS fluorescence (AU) Figure S1: Lack of major analytical interaction of ruxolitinib (Ruxo) with fluorescent probes used as substrates for transporters. HEK-MOCK cells plated in 96 wells were either pre- treated or not with 100 µM ruxolitinib for 3 h; they were next incubated with the reference fluorescent probes 4 Di-ASP, DCF, DBF, 6-CF or 8-FcA (each at 10 µM) for 5 min. Probe- related fluorescence was then determined by spectrofluorimetry; data are expressed as arbitrary unit (AU)/wells and are the mean ± SEM of at least three assays. NS, not statistically significant. Figure S1: Lack of major analytical interaction of ruxolitinib (Ruxo) with fluorescent probes used as substrates for transporters. HEK-MOCK cells plated in 96 wells were either pre- treated or not with 100 µM ruxolitinib for 3 h; they were next incubated with the reference fluorescent probes 4 Di-ASP, DCF, DBF, 6-CF or 8-FcA (each at 10 µM) for 5 min. Probe- related fluorescence was then determined by spectrofluorimetry; data are expressed as arbitrary unit (AU)/wells and are the mean ± SEM of at least three assays. NS, not statistically significant.
https://openalex.org/W2280048501	http://ejournal.ikado.ac.id/index.php/teknika/article/download/22/22	Indonesian	null	Studi Pembentukan Huruf Font Dengan Kurva Bezier	Teknika	2,014	cc-by-sa	2,892	Studi Pembentukan Huruf Font Dengan Kurva Bezier Agung Haryono, S.Kom., M.Kom. Jurusan Teknologi Informasi Institut Sains Terapan dan Teknologi Surabaya Jl. Ngagel Jaya Tengah 73-77, Surabaya andreasagung88@gmail.com ABSTRAK Kurva bezier (baca : bes-ee-ay) merupakan sebuah kurfa parametrik yang sering diterapkan pada komputer grafis dan dalam bidang-bidang berhubungan lainnya. Kurva bezier sangat dikenal karena kemampuannya untuk menghasilkan kurva yang halus. Penelitian ini bertujuan untuk mengetahui peran kurva bezier dalam pembentukan huruf-huruf Font dan mencari perbedaan peran kurva bezier dalam pembentukan Font TrueType dan Font PostScript. Sedangkan manfaat dari penelitian ini adalah mengenalkan pada masyarakat cara kerja dan peran kurva bezier dalam pembentukan huruf-huruf Font. Font TrueType dan font PostScript merupakan font vektor yang sama-sama menggunakan kurva bezier untuk menggambarkan kontur-kontur glyph. Font TrueType menggunakan kurva bezier kuadratik yaitu kurva bezier berordo 2, Font PostScript menggunakan kurva bezier kubik yaitu kurva bezier berordo 3. Kata kunci : Kurva bezier, font TrueType, font PostScript. 1. PENDAHULUAN Dalam dunia tipografi, Font adalah sebuah kumpulan karakter lengkap yang mempunyai satu ukuran dan gaya. Sedangkan dalam dunia komputer, Font merupakan file data elektronik yang menganduk sebuah kumpulan dari elemen penulisan (glyph), karakter-karakter atau simbol-simbol. Saat ini terdapat lebih dari ratusan ribuan jenis Font di dunia. Sistem operasi ternama di dunia, Microsoft Windows, yang mampu menampung hingga 4000 Font dalam sistemnya, menggunakan TrueType sebagai format Font digital. 2. METODOLOGI PENELITIAN Telah diketahui pada latar belakang bahwa kurva bezier sangat berperan penting dalam pembentukan Font outline pada TrueType dan PostScript. Untuk mengenal lebih lanjut apa itu kurva bezier, Font TrueType dan PostScript, makan akan dijabarkan pada sub bab -sub bab dibawah ini. Jurnal TEKNIKA 69 Jurnal TEKNIKA A. Kurva Bezier Pada akhir tahun 1950, komputer sudah mampu digunakan untuk membuat kalkulasi dan desain automobil. Sayangnya waktu itu tidak ada program yang mampu untuk membuat dan mewakili penggambaran kurva pada sebuah automobil secara efektif. Seorang insinyur asa Perancis yang bekerja pada Renault, Pierre Bezier, menerapkan sebuah kurva khusus untuk menyelesaikan masalah ini. Meskipun kurva ini dinamakan seperti itu, bukan berarti Pierre Bezier adalah orang yang menemukannya karena pada tahun yang sama P.De Castelju juga menerapkan kurva yang sama dalam perancangan tubuh automobil. Kurva parametrik ini telah diketahui sejak lama, namun tidak banyak orang yang mengenalnya. Baru pada tahun 1962, Pierre Bezier mempublikasikan kurva ini. Kurva bezier terdiri dari beberapa titik pembentuknya yang berjumlah n+1 sehingga terdapat titik-titik P0 sampai dengan Pn. Titik P0 merupakan titik awal kurva dimana terdapat kurva akan berangkat daru titik tersebut dan melengkung ke arah Pn dan berhenti di titik tersebut.Semua ini dilakukan dengan t sebagai presentase waktu tempuh kurva dari titikP0 dan Pn yaitu dari t=0 hingga t=1. Jadi ketika t=0 garis kurva berada tepat pada titik P0 dan ketika t=1 garis kurva berada tepat pada titik Pn. Perlu diketahui bahawa t bukanlah lamanya waktu yang ditempuh untuk membuat sebuah kurva. Variabel t menunjukkna seberapa jauh kurva bezier yang diwakili oleh fungsi B(t) bergerak dari P0 hingga P1. Sebagai contoh, ketika t=0,25 fungsi B(t) berada pada seperempat jalan dari titik P0 ke titik P1. Adapun persamaan untuk kurva bezier ordo satu (bezier linier) adalah : B(t) = Pn + t(P1-Pn) = (1-t) Pn + tP1 . t € [0.1] Jika diperhatikan rumus-rumus kurva bezier yang berurutan dari ordo satu sampai tiga menganut kaidah segitiga pascal. Setiap tingkat ordo satu pada kurva bezier berbanding lurus dengan baris-baris segitiga Pascal yaitu setiap ordo n akan memiliki koefisien- koefisien pada paris n+1 pada segitiga Pascal. Jurnal TEKNIKA 70 B. Font Font merupakan salah satu bentuk seni yang dibuat hak ciptanya untuk dibeli atau dijual. Saat ini terdapat lebih dari ratusan ribuan jenis Font didunia. Seringkali dalam komputerisasi orang-orang mengatakan Font ketika istilah typeface lebih dikatakan. Contoh dari typeface antara lain Times, Courier dan Arial karena memasukkan banyak ukuran dan banyak gaya. Sedangkan contoh Font antara lain bold (cetak tebal), italic (cetak miring), bold italic (cetak miring dan tebal), underline (bergaris bawah), outline (hanya mempunyai garis luar), dan shadow (berbayangan). Dalam definisi, Font merupakan keluarga typeface, sebagai contoh Arial 12-point italic, maka itu adalah dengan ukuran 12-point bercetak miring dari typeface Arial. Typeface telah diketahui sendiri merupakan karakter-karakter khusus dan unik yang mempunyai banyak ukuran dan banyak gaya. Dengan demikian typeface memiliki banyak Font dengan berbagai ukuran dan didsain dengan kesatuan gaya. Terdapat tiga macam format Font pada komputer antara lain Font bitmap, outline dan stroke. Perbedaan format tersebut bergantung pada komponen-komponen dalam pembentukan Font. • Font TrueType TruetType merupakan Font vektor atau Font berformat outline yang digunakan pada sistem operasi Microsoft Windows dan Apple Computer. Pada Font vektor, sebuah seri dari koordinasi menggambarkan sebuah kontur karakter, jadi perubahan pengskalaan sederhana secara efektif mengecilkan atau memperbesar karakter. Sejak dikenalkan pada publik pada tahun 1991, Font TrueType telah menjadi teknologi Font yang dominan untuk digunakan sehari-hari. Karena Font TrueType tidak membutukan bahasa pencetakan khusus, Font TrueType menggantikan peran Font PostScript pada dunia percetakan. Terdapat ribuan Font TrueType yang tersedia di dunia dan cukup banyak diantaranya yang disediakan cuma-cuma pada web. Banyak dari Font ini telah ditinjau dan diubah ke dalam sumber-sumber lain. Sementara sebagian Font dapat diproses dengan baik, pembuatan Font TrueType yang kurang baik dapat mengandung error yang dapat mengakibatkan kerusakan pada komputer. Font yang didesain secara profesional dapat menghabiskan banyak biaya namun biasanya membutuhkan hint yang cukup berat dan telah diuji pada berbagai ukuran dan sudut pada kualitas optimal. 71 Jurnal TEKNIKA 71 Jurnal TEKNIKA D. Prosedur Pengumpulan Data Penelitian ini menggunakan prosedur tertentu dalam proses pengumpulan data. Prosedur pengumpulan data pada penelitian ini dijabarkan sebagai berikut : a. Mencari file-file yang diperlukan pada penelitian ini yaitu file ttf dan file pfb. Hal ini dilakukan dengan mencari file-file kemudian mengunduh tersebut pada web-web internet. Namun pencarian dengan internet tidak perlu dilakukan jika sistem operasi yang digunakan telah memiliki cukup banyak Font untuk dijadikan data. Sebagai keterangan, khusus untuk file berekstensi pfb pencairan dilakukan dengan internet karena Windows tidak menyediakan file-file tersebut. b. Membuka file –file berekstensi ttf dan pfb dengan program file Font editor yaitu Font Creator Professional Editor 5.6 dan FontLab Studio 5. Kedua program tersebut memiliki perbedaan dalam kapasitas file yang dapat dibuka. Font Creator hanya mampu membuka file-file berekstensi ttf sedangkan FontLab Studio mampu membuka file-file baik berekstensi ttf maupun file berekstensi pfb. c. Mendapatkan titik-titik koordinat pada glyph karakter untuk setiap contoh file Font baik yang berekstensi ttf dan pfb. Setelah mengetahui titik-titik koordinat yang menunjukkan titik-titik penting pada kurva, langkah selanjutnya adalah mencatat titik-titik tersebut pada sebuah tabel. Setiap tabel hanya mewakili satu glyph karakter pada Font tertentu. c. Mendapatkan titik-titik koordinat pada glyph karakter untuk setiap contoh file Font baik yang berekstensi ttf dan pfb. Setelah mengetahui titik-titik koordinat yang menunjukkan titik-titik penting pada kurva, langkah selanjutnya adalah mencatat titik-titik tersebut pada sebuah tabel. Setiap tabel hanya mewakili satu glyph karakter pada Font tertentu. • Font PostScript PostScript merupakan Font outline atau Font berbasis vektor yang mendeskripsikan karakter dengan konstruksi matematis yaitu kurva bezier. Karena dikonstruksi oleh rumus matematika, seperti Font TrueType, Font PostScript juga memungkinkan dilakukan perubahan ukuran, penggambaran karakter dalam berbagai ukuran dan dalam resolusi tinggi. Karakter-karakter disimpan pada tabel bitmap yang merepresentasikan setiap karakter untuk setiap ukuran. PostScript sendiri adalah bahasa pemrograman yang digunakan untuk mengubah instruksi pencetakan grafis yang rumit ke printer digital. PostScript dikenalkan pada tahun 1984 oleh Adobe. Pada waktu itu terdapat dua tipe Font yang berbeda yaitu tipe 1 dan tipe 3. Dari kedua tipe tersebut, Tipe 1 merupakan tipe Font yang memiliki format yang lengkap. Penelitian tentang fungsi kurva bezier dalam pembuatan Font TrueType dan PostScript ini dilakukan dengan cara mengumpulkan beberapa paper dari internet. Selain itu, pengambilan data juga dilakukan dengan mengumpulkan contoh-contoh glyph pada suatu Font beserta titik-titik koordinatnya. Hal ini dilakukan dengan cara membuka file Font TrueType yang berekstensi ttf dan file Font PostScript yang berkekstensi pfb dengan dua program file Font editor berbeda. Penggunaa dua program ini hanyalah ditujukan untuk mempermudah memperoleh data untuk analisis. Batas- batas dalam penelitian ini adalah sebagai berikut : a. Peran kurva bezier ordo tertentu dalam pembentukan Font TrueType dan PostScript. b. Analisa titik-titik kurva bezier pada rancangan glyph karakter pada Font TrueType dan PostScript. c. File-file Font TrueType dan file-file Font PostScript yang hanya berlaku pada sistem operasi Microsoft Windows yaitu file ttf untuk Font TrueType dan file pfb untuk Font PostScript. d. Font PostScript yang dijabarkan dan dianalisis pada penelitian in hanyalah Font PostScript Tipe 1. 72 Jurnal TEKNIKA A. Algoritma De Casetljau Tujuan dari algoritma ini adalah menentukan titik tengah dari dua titik terdekat kemudian menyatukan beberapa titik tengah satu sama lain. Berikut ini adalah algoritma singkatnya : a. Tentukan nilai t untuk setiap bilangan real dari 0 sampai 1. t adalah bilangan konstan. b. Tentukan Pi untuk setiap nilai t dengan i=0,...,n c. Untuk setiap j=0 lakukan perhitungan rumus kurva bezier. d. Hubungkan menjadi kurva g(t)=P[n](t). Berikut adalah gambar dari analisa algoritma de castlejau pada kurva bezier : Berikut adalah gambar dari analisa algoritma de castlejau pada kurva bezier : Gambar Analisa algoritma de Casteljau pada kurva bezier Gambar Analisa algoritma de Casteljau pada kurva bezier Pada gambar, nilai t adalah 0,4. 10 merupakan segmen garis dari 00 dan 01, 11 merupakan segmen garis dari 01 dan 02, dan seterusnya hingga 14 adalah segmen garis dari 04 dan 05. Titik-titik baru tersebut diberi nomor dengan ketentuan 1i. E. Prosedur Penganalisaan Data Setelah melakukan pengumpulan data, langakh selanjutnya adalah penganalisaan data. Penelitian ini menggunakan prosedur tertentu dalam proses penganalisaan data. Prosedur penganalisaan data pada penelitian ini dijabarkan sebagai berikut : Setelah melakukan pengumpulan data, langakh selanjutnya adalah penganalisaan data. Penelitian ini menggunakan prosedur tertentu dalam proses penganalisaan data. Prosedur penganalisaan data pada penelitian ini dijabarkan sebagai berikut : a. Menginputkan pada program sederhana beberapa contoh titik yang merupakan bagian kurva pada glyph. Program sederhana merupakan program open source yang didapatkan dari internet yang kemudian dimodifikasi untuk keperluan penelitian ini. b. Menganalisis gambar kurva yang terbentuk pada program. c. Mendapatkan gambar kurva pada glyph yang sebenarnya. Hal ini dilakukan dengan cara memperbesar gambar glyph yang merupakan output dari program Jurnal TEKNIKA 73 Font editor. Pembesaran gambar difokuskan hanya pada beberapa bagian kurva dari contoh glyph yang sesuai dengan bagian kurva yang diujikan pada program. Font editor. Pembesaran gambar difokuskan hanya pada beberapa bagian kurva dari contoh glyph yang sesuai dengan bagian kurva yang diujikan pada program. Font editor. Pembesaran gambar difokuskan hanya pada beberapa bagian kurva dari contoh glyph yang sesuai dengan bagian kurva yang diujikan pada program. d. Membandingkan gambar kurva yang didapatkan dari program dan gambar kurva yang didapatkan dari hasil pembesaran gambar glyph yang sebenarnya. e. Menyimpulkan perbandingan untuk Font TrueFont dan Font PostScript secara keseluruhan berdasarkan hasil penelitian yang dilakukan. e. Menyimpulkan perbandingan untuk Font TrueFont dan Font PostScript secara keseluruhan berdasarkan hasil penelitian yang dilakukan. B. Font TrueType Garis tepi dari sebuah karakter atau biasa disebut glyph dalam Font TrueType dibuat dari bagian-bagian garis lurus dan kurva-kurva kuadratik Bezier. Kurva-kurva matematik yang lebih simpel ini sedikit efisien dalam prosesnya daripada kurva kubik bezier yang lazimnya terdapat pada PostScript dan digunakan pada Font Type 1. Jurnal TEKNIKA Jurnal TEKNIKA 74 Meskipun demikian, sebagian besar bentuk membutuhkan lebih banyak titik untuk menggambarkan dengan kurva kuadratik daripada kurva kubik. TrueType Collection (TTC) adalah ekstensi dari format TrueType yang memungkinkan sejumlah Font dikombinasikan menjadi satu file, menciptakan ruang penyimpanan yang kokoh untuk koleksi-koleksi Font yang hanya menggunakan glyph- glyph yang berbeda pada beberapak karakter. Format Font TrueType dasar tersusun dari beberapa tabel yang ditentukan pada header. Sebuah nama tabel dapat tersusun atas 4 kata. Dalam file TTC, mengandung sebuah ttcf tabel yang menyatakan berapa jumlah Font di dalam koleksi. Sejumlah Font di dalam koleksi menggunakan tabel glyph yang sama. Font TrueType regular berekstensi ttf, sementara TrueType Collection berekstensi ttc. Font TrueType disusun dengan kurva bezier kuadratik. Kurva bezier kuadratik merupakan kurva bezier berordo 2 yang memiliki satu titik off-curve sebagai titik kontrol. Namun ada beberapa area kurva yang memiliki 2 titik off-curve. Hal ini disebabkan oleh dua kurva kuadratik yang saling berurutan sehingga terlihat seperti kurva bezier kubik atau kurva bezier ordo 3. Meskipun dipenuhi oleh titik-titik yang berbeda, setiap kontur pada glyph dapat dibagi menjadi beberapa segmen kurva. Setiap segmen kurva diawali dengan titik on- curve dan diakhiri juga dengan titik on-curve. Setiap segmen bisa memiliki 1 titik off- curve maupun 2 titik off-curve. Jika terdapat 2 titik on-curve yang berurutan maka segmen tersebut membentuk kurva bezier linier yang berupa garis lurus. Perbedaan tempat dari titik-titik kontrol sangat berpengaruh pada tingkat kelengkungan kurva. Jika titik kontrol tidak jauh dari kurva maka kurva terbentuk tidak terlalu melengkung, jika titik kontrolnya terletak lebih jauh dari kurva sehingga kurva yang terbentuk terlihat lebih melengkung. C. Font PostScript Jika Font TrueType menggunakan kurva bezier kuadratik, glyph-glyph pada Font PostScript digambarkan dengan kurva bezier kubik. Satu set glyph pada Font PostScript dapat diubah-ubah ukurannya melalui transformasi matematika sederhana yang kemudian dapat dikirim ke printer PostScript. Karena data dari Font PostScript khususnya pada Tipe 1 merupakan deskripsi dari garis-garis tepi (outline) dari sebuah glyph maka Font ini juga disebut Font outline. Jurnal TEKNIKA 75 Jurnal TEKNIKA Pada sistem operasi Macintosh, Font PostScript terdiri dari file Suitcase dan file Printer. Hanya ada boleh satu file suitcase untuk seluruh keluarga Font yang menyediakan hingga 4 file printer yaitu reguler, italic, bold dan bold italic. Pada sistem operasi Windows, Font PostScript dapat terdiri dari 2 atau 3 file. Satu set 3 file terdiri dari file PFB yang mengandung dara garis tepi, AFM yang mengandung informasi tentang spacing dan kerning, INF yang mengandung informasi tambahan yang berguna untuk instalasi. Selama instalasi berlangsung, dengan program Adobe Type Manager (ATM), Windows menghasilan file PFM yang berbasis pada file AFM dan file INF. Adobe Type Manager merupakan program yang harus dipasang pada Windows terlebih dahulu sebelum melakukan instalasi Font PostScript. Namun tidak pada semua tipe Windows harus melakukan instalasi ini. ATM dibutuhkan pada sistem operasi Windows 95, 98 NT dan Windows ME. Namun pada sistem operasi Windows 2000, XP dan Vista tidak perlu melakukan instalasi ATM. File PFB tidak jauh beda denga file TTF. File PFB juga terdiri dari beberapa tabel yang mengandung informasi tersimpan pada header. Glyph-glyphI karakter tersusun pada tabel memiliki informasi sendiri-sendiri. Jumlah glyph juga sangta banyak, bermacam-macam dan berlainan bentuk, sama halnya seperti file TTF pada Font TrueType. Kurva-kurva bezier pada Font PostScript tidaklah sama dengan kurva-kurva bezier pada Font TrueType karena Font PostScript menggunakan kurva bezier kubik sedangkan Font TrueType menggunakan kurva bezier kuadratik. Selain itu kurva-kurva pada glyph Font PostScript cukup konsisten. Pada glyph Font PostScript, kurva hanya terdiri dari kurva bezier linier yang berupa garis lurus dan kurva bezier kubik yang memiliki 2 titik kontrol. D. Perbandingan Font TrueType dengan Font PostScript Banyak hal yang telah dijabarkan dan dianalisis menganai kedua Font tersebut sehingga dapat disimpulkan adanya beberapa perbedaan dan persamaan di antara Font TrueType dan Font PostScript. Persamaan dari kedua Font adalah keduanya sama-sama merupakan Font outline. Glyph-glyph yang dibentuk pada kedua Font tersebut disusun berdasatrkan garis-garis tepi atau kontur yang dibuat berdasarkan kurva matematika. Kurva matematika yang dipakai adalah kurva bezier. Hal ini berlaku untuk keduanya. Jadi A. Kesimpulan Hasil dari penelitian ini dapat disimpulkan sebagai berikut : 1. Font TrueType dan font PostScript merupakan font vektor yang sama-sama menggunakan kurva bezier untuk menggambarkan kontur-kontur glyph. 2. Font TrueType menggunakan kurva bezier kuadratik yaitu kurva bezier berordo 2 yang membutuhkan dua titik yang merupakan titik ujung kurba dan satu titik kontrol. 3. Font PostScript menggunakan kurva bezier kubik yaitu kurva bezier berordo 3 yang membutuhkan dua titik yang merupakan titik ujung kurva dan dua titik kontrol kurva. Jurnal TEKNIKA Jurnal TEKNIKA 76 tidak hanya Font TrueType saja yang menggunakan kurva bezier tetapi juga Font PostScript. Meskipun keduanya sama-sama menggunakan kurva bezier, jenis kurva bezier yang digunakan pada kedua Font tersebut berbeda. Font TrueType menggunakan kurva bezier kuadratik yang menggunakan 3 titik untuk membentuk kurva, sedangkan PostScript menggunakan kurva bezier kubik yang menggunakan 4 titik untuk membentuk kurva. Perbandingan Font TrueType dan Font PostScript dapat dilihat pada tabel dibawah. Font TrueType Font PostScript Format Font Outline / Vektor Outline / Vektor Pengembang Microsoft dan Apple Adobe System Ekstensi file pada Windows .TTF .PFB dan .PFM Jenis Kurva Bezier Kaudratik (ordo 2), membutuhkan 3 titik yaitu 2 titik on-curve dan 1 titik off-curve Kubik (ordo 3), membutuhkan 4 titik yaitu 2 titik on-curve dan 2 titik off-curve Font TrueType Font PostScript Format Font Outline / Vektor Outline / Vektor Pengembang Microsoft dan Apple Adobe System Ekstensi file pada Windows .TTF .PFB dan .PFM Jenis Kurva Bezier Kaudratik (ordo 2), membutuhkan 3 titik yaitu 2 titik on-curve dan 1 titik off-curve Kubik (ordo 3), membutuhkan 4 titik yaitu 2 titik on-curve dan 2 titik off-curve B. Saran Saran untuk penelitian lanjutan di masa mendatang adalah perlu adanya program yang lebih kompleks dalam penggambaran kurva-kurva bezier sehingga dapat membentuk kontur-kontur glyph secara keseluruhan. Jurnal TEKNIKA 77 77 REFERENSI [1] Computer, Apple, Inc. 1993. Inside Macintosh : Text, Addison Weasly. [2] Prautzsch, Harmut, Wolfgang Boehm, Marco Paluszny, Bezier and B-Spline Techniques, Springer-Verlag Berlin Heidelberg New York, 2002 [3] Solomon, David, Curves and Surfaces for Computer Graphics, Springer Verlag, August 2006 [3] Solomon, David, Curves and Surfaces for Computer Graphics, Springer Verlag, August 2006 [4] Kennedy, Jhon. A Brief Introduction To Bezier Curves. Mathematics Department. Santa Monica College. [4] Kennedy, Jhon. A Brief Introduction To Bezier Curves. Mathematics Department. Santa Monica College. [5] Garfield, Simon. Just My Type : A Book about Fonts, Profile Books LTD ,Great Britain, 2010 78 Jurnal TEKNIKA
https://openalex.org/W2745163626	https://philpapers.org/archive/RIGLFE-2.pdf	English	null	Looking for Emergence in Physics	DOAJ (DOAJ: Directory of Open Access Journals)	2,017	cc-by	5,854	JOANA RIGATO JOANA RIGATO JOANA RIGATO Champalimaud Center for the Unknown - Lisbon joana.rigato@neuro.fchampalimaud.org 1 Acknowledgments: I thank Margarida Telo da Gama, Robert Bishop and Pranab Das for helpful discussions an comments on earlier drafts of this paper. LOOKING FOR EMERGENCE IN PHYSICS1 Despite its recent popularity, Emergence is still a field where philosophers and physicists often talk past each other. In fact, while philosophical discussions focus mostly on ontological emergence, physical theory is inherently limited to the epistemological level and the impossibility of its conclusions to provide direct evidence for ontological claims is often underestimated. Nevertheless, the emergentist philosopher’s case against reductionist theories of how the different levels of reality are related to each other can still gain from the assessment of paradigmatic examples of discontinuity between models in physics, even though their implications must be handled with care. Phenomenology and Mind, n. 12 - 2017, pp. 174-183 DOI: 10.13128/Phe_Mi-21116 Web: www.fupress.net/index.php/pam © The Author(s) 2017 CC BY 4.0 Firenze University Press ISSN 2280-7853 (print) - ISSN 2239-4028 (on line) © The Author(s) 2017 CC BY 4.0 Firenze University Press ISSN 2280-7853 (print) - ISSN 2239-4028 (on line) Looking for Emergence in Physics Emergentism, in its various forms, is the view according to which there are features of1 reality (properties, objects or laws) that are irreducible to the lower-level basis from which they emerge, in the sense that they are more than just the result of the combination of the system’s parts and their interactions. These features are paradoxically (or so it seems) both dependent on and autonomous from their emergence base, i.e. from the lower-level that brings them about. They are dependent in the sense that they cannot exist unless the lower-level structure is in place, but they are autonomous insofar as that structure does not suffice for the novel features that arise to be fully explained and predicted. This inexplicability and unpredictability is, of course, in the eyes of the beholder and amounts to what is commonly known as epistemological emergence. Still, metaphysical or ontological emergence can be assumed to underlie this apparent underivability, in which case there is a radical discontinuity in the hierarchical organization of reality, whereby the causal effects of emergent phenomena in the world cannot even in principle be accounted for by the causal powers of the lower-level structure on which they depend (Kim, 1999; O’Connor & Wong, 2005).i 1. Introduction y g This type of theory has been developed in very many areas, from different scientific branches to philosophy. In the latter field, its study ranges from metaphysics and the philosophy of mind (in which the focus has been mainly on the relationship between mental/conscious entities and their physiological substrate – Kim, 1993; Searle, 1992), to philosophy of science in general (where the focus is on whether certain theories are reducible to others or not – Bedau, 1997), to philosophy of physics (where emergence seems to be a good conceptual tool for explaining nonlinear phenomena – Andersen, 1972) and philosophy of biology (where the main interest is top-down causation in self-organizing systems – Arp, 2008). i What is common in all these fields is the opposition to the reductionist ideal of a Lego world where the elements of the bottom-most domain provide necessary and sufficient conditions for the phenomena taking place at higher levels of organization. According to reductionism, the lower-level properties and laws of a system determine its upper-levels properties and laws. The instantiation of the former necessitates the instantiation of the latter. This implies, as a consequence, that the scientific domain that explains the lower-level occurrences is in principle sufficient to explain the upper-level ones (Sober, 1999). Emergentist views all counter this 175 Joana Rigato Joana Rigato perspective with various examples that are used as evidence for the existence of upper-level phenomena that challenge microphysical explanation, not only in practice, but allegedly in principle. p p However, needless to say, there is no uniformity in the way the concept of emergence is used and in the candidates that are accepted as good examples of emergent phenomena (Bedau & Humphreys, 2008). p y In this paper, I chose to focus on the debate about emergence going on in philosophy of physics, hoping to show its relevance to the general discussion. A commendable tendency in the past years in philosophy of science has been to develop accounts of emergence that move away from armchair metaphysics and anchor philosophical analyses in scientific theory and practice. yi y p Philosopher and physicist Robert Bishop, for example, has been working in questions of Emergence and Complex Systems for many years. 1. Introduction Alone (2005, 2009) or together with theoretical physicist Harald Atmanspacher (2006), Bishop developed an account of what he calls “contextual emergence”, which is a relation between different levels of description (in its epistemological form) or between domains of reality (in its ontological form), whereby the description, properties or behaviors of the lower domain provide some necessary but no sufficient conditions for the novelty existing at the upper-level. The remaining conditions must be provided by the context, which includes the stability conditions of the emergent states and observables (i.e. the conditions that guarantee their existence and persistence), which are not given by lower-level descriptions. Bishop uses several examples as evidence for the ubiquity of contextual emergence, from the domain of quantum chemistry to that of human society. All of them have to do with scale transformations: how the laws of microphysics give rise to the laws and properties of the macro world. Bishop is in good company, as voices have been rising in the attempt to tell philosophers and unexamined reductionists that real-world science does not actually have any models that drill down from many-body physics to some mythical microphysical state and that, in fact, productive scientific models largely ignore such thinking altogether. According to nobel laureate Robert Laughlin, for example, the idea that one might in principle deduce the goings- on in the domains of chemistry, biology and other special sciences from a complete knowledge of particle physics is totally unfounded and, even though reductionism is a belief that is central to much of physical research, “the safety that comes from acknowledging only the facts one likes is fundamentally incompatible with science. Sooner or later it must be swept away by the forces of history” (Laughlin & Pines, 2000, p. 264). Let us now look at some examples of emergence in physics that have been put forward in the literature. The examples challenge the reductionist ideal from different fronts: first, the need for singular limits, for example in the transition from quantum to classical mechanics (section 2.1), is used to call into question the idea that a macro-physical state can be derived from a micro-physical equation. Second, the case of criticality, which is an example of universality (section 2.2), questions the assumption that a macro-physical state has a unique microphysical basis, which is commonly taken as a consequence of a reductionistic world. 2. Emergence in Physics 2.2. When a Macro State Is Compatible With Multiple Micro States Looking for Emergence in Physics Looking for Emergence in Physics The transition from quantum to classical mechanics is a mysterious one. Mathematically, these two realms are separated by singular limits (mathematical expansions in which some quantities are assumed to tend either to zero or to infinity), which means that the transition between the formalism that describes the behavior of particles at the quantum level (the Hamiltonian dynamics) and the equations used in the field of many-body physics is discontinuous. The behavior before and after that transition is qualitatively different and has to be described by a totally distinct equation. And in order to move from one equation to the other, Planck’s constant is assumed to tend to zero, which is a mathematical trick that departs from reality (where it is actually non-zero). Hence, between the classical and the quantum domains there is a radical epistemological gap which can be bridged only with the help of a formal artifact. This can be illustrated with the example of molecular shape. Isomers are molecules that share identical chemical formulas but have different spatial arrangements which give them very different properties. According to Bishop, these are good candidates as examples of contextually emergent phenomena since the specific structure into which a certain quantum description (the so-called Hamiltonian) will evolve at the chemical level cannot be deduced from quantum mechanical data alone. Even though QM [i.e. quantum mechanics] contains necessary conditions in terms of nucleons, electrons and their properties, fundamental force laws and so forth, observables relevant for molecular structure do not exist in the domain of QM. For such observables to obtain, an additional context not given by QM must be specified (Bishop, 2009, p. 177). Only with the help of heuristic formal procedures, like assuming the nucleus of the atom to be stationary and infinitely larger than the electron mass, can one derive the equation encoding molecular shape.2 This means that the chemical context (the stability conditions of a “clamped nucleus” together with the ratio of the electron mass over the nucleus mass tending to zero) must be fed into the mathematical treatment of the quantum mechanical information. It is thanks to these constraints that come from “outside” the quantum realm that the quantum correlations between nuclei and electrons are broken and classical position and momentum observables, as well as molecular shape, can arise. 2.1. When a Macro State Cannot Be Derived From a Micro Equation 2 This “clamped-nucleus” assumption is part of the so called Born-Oppenheimer “approximation”. Mathematically, it corresponds to an asymptotic series expansion in which the parameter ε (= electron mass/ nuclear mass) diverges to zero, that is, the nuclear mass is assumed to be infinitely large with respect to the electron mass. 1. Introduction Finally, the phenomenon of liquidity (section 2.3) highlights the role of stability conditions which are often not provided by the underlying emergence base. 176 3 As with the previous cases, this phenomenon too is described as the result of assuming a variable to be infinite: viz. the number of particles or the correlation lengths between them (the distance over which one particle can influence another). 4 The process, developed by Kadanoff, Fisher, and Wilson (cf. Batterman, 2002), is based on an iterated transformation of the Hamiltonian of each system, by which as one gradually changes scale, more and more fine- grained information is lost and the resulting function ends up being the same for all the elements of the universality class in question (a value that is called a “fixed point”). 5 Laughlin and Pines (2000) provide various examples. Here are two more: “The Josephson quantum is exact because of the principle of continuous symmetry breaking. The quantum Hall effect is exact because of localization. Neither of these things can be deduced from microscopics and both are transcendent, in that they would continue to be true and to lead to exact results even if the Theory of Everything were changed” (p. 261). Looking for Emergence in Physics Several natural phenomena share with the previous case this feature of being theoretically dependent on mathematical tricks, such as the postulation of the infinite or null value of certain observables that we know to be finite. They are cases in which the appearance of the macro property is not a mere quantitative derivation from a smaller scale to a larger scale, but rather a qualitative transformation which can be explained and predicted (at least on the basis of the models and theories presently available to us) only through the artificial normalization of singular limits. i g According to Robert Batterman, a leading figure in Philosophy of Physics, emergence happens precisely there where singular limits cause our theories to break down. And as a matter of fact, our most important physical theories are asymptotically related in pairs: 177 Joana Rigato Joana Rigato Lim1/c → 0 (special relativity) → Newtonian mechanics Limλ →0(wave optics) → ray optics λ → 0 p y p Limh → 0 (quantum mechanics) → classical mechanics. This can be interpreted as an indicator of the inadequacy of our theories and models, or instead as a “source of information”. Batterman has been arguing for the latter attitude for several years now: This can be interpreted as an indicator of the inadequacy of our theories and models, or instead as a “source of information”. Batterman has been arguing for the latter attitude for several years now: If it were not for the singularities that appear in our theories and models we would have no understanding of the emergence at different scales of distinct and apparently “protected” states of matter (2011, p. 1040). The “protected” states of matter that Batterman is referring to are what Laughlin and Pines (2000) call “protectorates”, which are stable states of matter which are not only mathematically underivable from more fundamental equations without the help of singular limits, but are also insensitive to changes at the micro-level. These protectorates are the units of the phenomenon physicists call “universality”, which is what philosophers dub “multiple realizability”. One example of such a phenomenon is thermodynamic criticality. 3 As with the previous cases, this phenomenon too is described as the result of assuming a variable to be infinite the number of particles or the correlation lengths between them (the distance over which one particle can influe another). h d l d b d ff h d l ( f ) b d d 3. From Epistemology to Ontology in Physics Looking for Emergence in Physics Looking for Emergence in Physics But are emergent phenomena exhausted by cases such as these, where we find radical discontinuities between theories? What can physics tell us regarding properties that do not look so mysterious to us? Liquidity, for example, is a very familiar property (or cluster of properties) which we may resist considering emergent. In spite of its in practice unpredictability and novelty with respect to the properties of the components of the liquid taken in isolation (Weisskopf, 1977), the macroscopic properties of liquids (like viscosity or surface tension) and their causal powers seem straightforwardly derivable from the laws governing chemical bonds and other microscopic states and events. What we see at the level of the liquid is not something over and above the goings-on at the level of the molecules and their interactions. So reducibility seems possible, almost unavoidable. However, condensed-matter physicists reply, it is not that simple. p The stability of liquids depends on temperature, which cannot be derived from the particle’s interactions. Even though in the philosophical literature temperature is still cited as a good example of reduction (it is taken to be nothing but the mean translational kinetic energy of molecules in a system), it is actually considered to be a case of emergence by most condensed- matter physicists. Temperature is a property that arises out of two mathematical transitions (from particle mechanics to statistical mechanics, and from there to thermodynamics), the calculation of which depends upon mathematical limits (e.g. the thermodynamic limit, which assumes the container of a gas to be infinitely large) as well as on stability conditions which are not available in the underlying domain, such as thermodynamic equilibrium.i p p qi establishing the stability conditions upon which the liquid depends, that is, without taking into account the macro conditions that make it so that some laws of interaction rather than others apply. Molecular shape and criticality are considered to be good candidates for emergence because of the irreducibility of their macro description to the underlying quantum properties. This happens also in the case of liquids, since they cannot exist unless there is a certain sort of symmetry breaking induced by temperature, which in turn depends on conditions that can be provided only at the macro level. Therefore, if molecular shape and criticality are emergent, liquids should be considered to be so as well. 2.3. When Macro Context is as Crucial as Micro Structure Looking for Emergence in Physics l The critical point of a fluid is a state in which liquid and vapor can coexist, and it is determined by a specific temperature and pressure (which is different from fluid to fluid).3 Surprisingly, once they reach their specific critical point, all fluids (as well as magnets) behave in an identical manner, even if their properties are radically different in other phases and even if the values of their critical points are as diverse as 1,040.85oC/270 atm for sulfur and -239.95oC/12.8 atm for hydrogen. This macroscopic similarity beyond microscopic differences has been mathematically accounted for by the renormalization group theory (Batterman, 2002, 2011, 2014). This mathematical technique (for which Kenneth Wilson won the Nobel Prize) shows how the molecular details that are specific to each fluid are irrelevant for the macroscopic behavior that it shares with all other fluids.4 Batterman’s conclusion (2014, p. 15) is that this concrete method for explicating a process whereby higher order patterns arise that are not derivable from micro-structure can be considered as evidence against reductionism. In the words of Laughlin, who uses examples such as these in his battle against the reductionistic framework often used in physics, it is obvious at the eyes of solid-state physicists, chemists and biologists that nature is filled with phenomena that are insensitive to microphysical variability, the behavior of which is determined by higher organizing principles, and that we may confidently call emergent.5 This is one of the reasons why “predicting protein functionality or the behavior of the human brain from [quantum mechanical] equations is patently absurd” (Laughlin & Pines, 2000, p. 260). 178 Looking for Emergence in Physics All physics does is designing models that are quantitative, predictive and falsifiable. Whether those models correspond to the actual objective truth is something physics cannot tell us. Such an instrumentalist approach, which is the physicist’s default standpoint, can make it hard on the philosopher to extract useful information from physical theory and practice for her metaphysical speculations. Of course, philosophers of a realist inclination would consider it legitimate to move from epistemological facts about physical theorizing to facts about the world. However, they would be moving alone. Physicists would hardly approve of such an extrapolation, which would hence be missing the safety net provided by the scientific method and the credibility that comes with intersubjective agreement. of knowledge of the details and intricacies of that lower level, its parts and the relations obtaining between them. Our theories may be incomplete. obtaining between them. Our theories may be incomplete. If the epistemic irreducibility we found in the cases described in the previous section were to express a deeper ontological irreducibility, that would mean that there is a spontaneous and unexplained symmetry breaking at a certain point in the evolution of the system, whereby new properties with new causal powers come about. Isomers with different boiling points and densities, critical points in which new visible phenomena such as opalescence take place (the fluid becomes opaque and colored), temperature with different effects on macroscopic bodies (such as melting). If our epistemic limits express true ontological irreducibility, these examples, as well as many others, which might be more or less familiar and more or less complex, all seem to be cases of causally new and irreducible, hence emergent, macro features. However, it is very hard to apply the epistemological/ontological distinction to physics. Physics does not have the pretense of knowing reality. All physics does is designing models that are quantitative, predictive and falsifiable. Whether those models correspond to the actual objective truth is something physics cannot tell us. Such an instrumentalist approach, which is the physicist’s default standpoint, can make it hard on the philosopher to extract useful information from physical theory and practice for her metaphysical speculations. Of course, philosophers of a realist inclination would consider it legitimate to move from epistemological facts about physical theorizing to facts about the world. However, they would be moving alone. Looking for Emergence in Physics What the aforementioned examples show is that the reductionist ideal of macro properties being derivable from microscopic features and laws is not grounded in scientific practice. Scale transformations are highly problematic and many aspects of reality seem to simply pop up when a certain threshold of complexity is crossed, which mathematically corresponds to unphysical singular limits. But is this not a merely epistemic matter? Even if we cannot predict upper-level phenomena on the basis of our lower-level knowledge, this does not imply that we are dealing with ontologically irreducible features. As a matter of fact, besides the misunderstandings caused by the lack of agreement regarding the definition of emergence to which I alluded briefly in the introduction, another major source of confusion, especially in the scientific community, is the lack of clarity concerning the distinction between epistemological and ontological forms of emergence. Epistemological emergence is a relation existing between theories or models of the world. Ontological emergence is a relation existing between objects or properties in the world. Even though the latter implies the former, the inverse is not true. Epistemological emergence is no guarantee for ontological emergence. The impossibility of reducing a certain theory, with which we explain a certain upper-level domain, to a lower-level theory may be due only to our lack 179 Joana Rigato Joana Rigato of knowledge of the details and intricacies of that lower level, its parts and the relations obtaining between them. Our theories may be incomplete. If the epistemic irreducibility we found in the cases described in the previous section were to express a deeper ontological irreducibility, that would mean that there is a spontaneous and unexplained symmetry breaking at a certain point in the evolution of the system, whereby new properties with new causal powers come about. Isomers with different boiling points and densities, critical points in which new visible phenomena such as opalescence take place (the fluid becomes opaque and colored), temperature with different effects on macroscopic bodies (such as melting). If our epistemic limits express true ontological irreducibility, these examples, as well as many others, which might be more or less familiar and more or less complex, all seem to be cases of causally new and irreducible, hence emergent, macro features. However, it is very hard to apply the epistemological/ontological distinction to physics. Physics does not have the pretense of knowing reality. 6 This is, of course, an extreme example of the renowned Heisenberg uncertainty relations. Looking for Emergence in Physics So to imagine a universal calculation of the evolution of an ideal “system of the world” just sounds plainly absurd. Third, because the theories we have that describe and explain macro properties on the basis of molecular properties are statistical in nature. They do not express the sort of one-to-one causality relations we would like a laplacean demon to have access to. This means that a really carefully imagined omniscient being would have to have a theory set that is fully coherent across scales, which is something we are nowhere near to achieving and cannot even know is possible. A philosopher may tend to react to such arguments with dismay and call attention again to the hypothetical nature of the laplacean demon that need not suffer from the physical limitations of our brains, theories and actual computers, but the dialogue with the physicist will likely have come to a halt. It seems like the philosopher and the physicist are talking past each other. The former aims at inferences about the ontology underlying our theories, which the latter will never be able to provide. In principle reductionism is impossible to prove and so is non-derivability. Even if we ignored the distance between the epistemological and the ontological levels, we still could not use as arguments what science might be able to reveal in the future, but only what it is able to verify right now. And even that is extremely difficult to generalize. Every microphysical law, which is an abstract construct formulated by theoreticians in as simple and context-free a way as possible, is tacitly implying that its application depends on the absence of outside influences (influences from upper levels of organization). What happens in a laboratory, then, is the testing of such abstract physical laws in equally aseptic environments, carefully designed to exclude any disturbing factor. However, outside these controlled setups, things get very messy. Even though the results of the experiments often corroborate the laws we wish to test, they cannot confirm their applicability to real-case scenarios where the boundaries between organization levels are loose and causal interactions between them much more likely (Dupré, 2001).i y p Hence, it would be fallacious to infer from the results of experimental scientific research such a strong metaphysical assumption as reductionism, which would require us to be able to ascertain with profound detail what happens in increasingly complex and ever changing contexts. 4. Weakening the Reductionist’s Case Looking for Emergence in Physics Physicists would hardly approve of such an extrapolation, which would hence be missing the safety net provided by the scientific method and the credibility that comes with intersubjective agreement. But philosophers do not give up easily on what might be a fruitful dialogue. Even if physics only provides us with models of the world, one can still hypothesize a laplacean demon, a universal and omniscient calculator, whose complete and truthful knowledge of a certain system might be sufficient to explain all its macro properties. Could such a calculator predict the formation of a certain molecule in a fluid with such and such initial and boundary conditions? Granted, the conceivability of this omniscient being is traditionally used as an argument for universal determinism, not ontological reductionism, the truth of which it assumes from the start. Still, this theoretical exercise serves as a way to flesh out what can otherwise seem too abstract a hypothesis and allow physicists to more easily explicate their case against it.i g Unfortunately, they will likely find the idea of a laplacean demon to be inapplicable to current physical science for several reasons. First, because in the world of particles at the subatomic scale, classical physics does not apply. Only once a certain measurement is made, is the system in a well determinate state; before, in general, it is considered to be in what is called a superposition of states. There is no way of knowing with absolute certainty all the information that fully characterizes a physical system: if we know precisely where a particle is located – its position in a certain spatial coordinate –, we will miss all knowledge about its momentum (in the same coordinate), and vice-versa.6 The laplacean demon, therefore, cannot do his job in the quantum realm. q Second, because the in practice impossibility of calculating all the information contained in any macro system, not to mention the whole universe, is considered by physicists to be an in principle impossibility. It is presently established that no computer can ever accurately solve the equations describing the total energy of a system with more than ten particles at the quantum level (Laughlin & Pines, 2000, p. 160), because the interactions, which grow with the 6 This is, of course, an extreme example of the renowned Heisenberg uncertainty relations. 180 Looking for Emergence in Physics factorial of N (number of particles), are intractable. Looking for Emergence in Physics Evidence for reductionism should consist in the verification that the behavior of all complex systems (from chemical, to biological, to neurological, to psychological, to social), in real-case situations, can be fully explained by microphysical laws, which is something that cannot even be done at a molecular level. While one single case attesting to the failure of the universal claim of reductionism would suffice to falsify it, the same argument cannot be applied to emergentism, which is committed only to the existence of some irreducible phenomena. We would not need to survey the whole natural realm in order to prove it true; one relevant case of irreducibility would be enough. In this sense, even if the epistemological emergence of many-body properties and the radical mathematical discontinuity between theories at different levels cannot prove the truth of ontological emergentism, they do come in handy as circumstantial evidence against the ontological reducibility of the macro to the micro. g y In short, given the impossibility of using physics to prove their ontological claims, what both reductionists and emergentists must do is try to make the case for the higher implausibility of their rival position. That is much easier for a weaker, existential claim (emergentism) than for a strong, universal one (reductionism). And while the empirical evidence we have surveyed in section 2 cannot prove the truth of ontological emergence, it can shed strong doubts on its alternative and thus make the case for emergentism stronger. 181 Joana Rigato Joana Rigato 5. Conclusion Let us sum up. All the examples used in this paper consist in systemic properties that are qualitatively different from the properties of the parts. They can be calculated once we know the stability conditions that allow them to persist, but the singular limits that separate the theories that describe them render it impossible to explain the whole only on the basis of the parts. Nevertheless, the fact that we cannot know whether this epistemic irreducibility corresponds to an ontological gap rather than to mere limitations of our models prevents us from being able to assert conclusively whether these are cases of ontological emergence or not. In the end, the move from the epistemic to the ontological level of analysis is a matter of personal preference and intuition. Looking for Emergence in Physics Physics is silent about what is really there and so all it can do to help the emergentist’s case is tell her that ontological emergence is not an absurd anti-scientific hypothesis. It is actually plausible, if our theories are true, since the way our models relate to each other is exactly what one should expect if ontological emergence were the case. Brian MacLauglin has famously said: Given the advent of quantum mechanics and these other scientific theories, there seems not a scintilla of evidence that there are emergent causal powers or laws (1992, p. 23). Given the advent of quantum mechanics and these other scientific theories, there seems not a scintilla of evidence that there are emergent causal powers or laws (1992, p. 23). As we have seen, this statement is highly questionable. Despite the advent of quantum mechanics and other scientific theories that allow us to explain the behavior of the smallest portions of matter we know, there is much more than a few “scintillas of evidence” that there are emergent causal powers and laws in the world. And they might be much more common than usually supposed, even though reductionism cannot be disproven. 5. Conclusion REFERENCES Anderson, P.W. (1972). More is Different: Broken Symmetry and the Nature of the Hierarchical Structure of Science. In M.A. Bedau & P. Humphreys (Eds.) (2008). Emergence. Contemporary Readings in Philosophy and Science. Cambridge, MA: MIT Press, 221-229. Arp, R. (2008). Emergence in Biology. 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Reduction and multiple realizability. www.robertbatterman.org. Bedau, M. (1997). Weak Emergence. Noûs, 3, 375-399. Bedau, M.A. & Humphreys, P. (2008). Emergence. Contemporary Readings in Philosophy and Science. Cambridge, MA: MIT Press. Bedau, M.A. & Humphreys, P. (2008). Emergence. Contemporary Readings in Philosophy and Science. Cambridge, MA: MIT Press. hop, R.C. (2005). Patching physics and chemistry together. Philosophy of Science, 72, 710-722. Bishop, R.C. (2005). Patching physics and chemistry together. Philosophy of Science, 72, 710-722. Bishop, R.C. (2009). Whence chemistry? Reductionism and neoreductionism. Studies in History and Philosophy of Modern Physics, 41, 171-177. Bishop, R.C. & Atmanspacher, H. (2006). Contextual emergence in the description of properties. Foundations of Physics, 36, 1753-1777. f y Dupré, H. (2001). Human nature and the limits of science. Oxford: Clarendon Press. Dupré, H. (2001). Human nature and the limits of science. Oxford: Clarendon Press. Ki J (1993) S i d Mi d S l t d E C b id C b id U i it P Dupré, H. (2001). Human nature and the limits of science. Oxford: Clarendon Press. Kim, J. (1993). Supervenience and Mind: Selected Essays. Cambridge: Cambridge University Press. ( ) k f h l h l d Kim, J. (1993). Supervenience and Mind: Selected Essays. Cambridge: Cambridge University Pre Kim, J. (1999). Making Sense of Emergence. Philosophical Studies, 95, 3-36. (Eds.) (2008). Emergence. Contemporary Readings in Philosophy and Science. Cambridge, MA: MIT Press, 19-59. REFERENCES g g p Laughlin, R.B. & Pines, D. (2000). The Theory of Everything. In M.A. Bedau & P. Humphreys (Eds.) (2008). Emergence. Contemporary Readings in Philosophy and Science. Cambridge, MA: MIT Press, 259-268. g g p Laughlin, R.B. & Pines, D. (2000). The Theory of Everything. In M.A. Bedau & P. Humphreys (Eds.) (2008). Emergence. Contemporary Readings in Philosophy and Science. Cambridge, MA: MIT Press, 259-268. McLaughlin, B. (1992). 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https://openalex.org/W1578129620	https://www.intechopen.com/citation-pdf-url/46387	English	null	Stem Cells in Pancreatic Cancer	InTech eBooks	2,014	cc-by	13,075	Chapter 2 Chapter 2 Stem Cells in Pancreatic Cancer Cristiana Pistol Tanase, Ana-Maria Enciu, Maria Linda Cruceru, Laura Georgiana Necula, Ana Iulia Neagu, Bogdan Calenic and Radu Albulescu Cristiana Pistol Tanase, Ana-Maria Enciu, Maria Linda Cruceru, Laura Georgiana Necula, Ana Iulia Neagu, Bogdan Calenic and Radu Albulescu Additional information is available at the end of the chapter http://dx.doi.org/10.5772/57530 Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/57530 http://dx.doi.org/10.5772/57530 1. Introduction Pancreatic cancer is the fourth most frequent cause of cancer-related deaths; it also represents one of the most aggressive cancer types, with a high incidence of distant metastasis and mortality [1]. The detection of pancreatic cancer at early stages, the prediction of the potential resectability, or the response to therapy are the current major challenges in improving the clinical outcome of pancreatic ductal adenocarcinoma (PDAC) [2]. The main issue against successful therapy is represented by the absence of early diagnostic and prognostic markers, as well as the unresponsiveness to radiation and chemotherapies [3]. Among other factors that contribute to the lack of success in the therapy of pancreatic malignancies, cancer stem cells (CSCs) appear to have a major role. Cancer is characterized by cellular heterogeneity; CSCs, which represent a distinct subpopulation of cells, seem to be responsible for tumor initiation and persistency, due to their properties of self-renewal and multilineage differentiation. CSCs are considered as best candidates responsible for tumorigenesis, metastasis, and chemo-and radio-resistance [4]. Understanding and properly addressing the challenge represented by CSCs appears as a logical, yet difficult task in anti-cancer strategies. © 2014 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. © 2014 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. 2.1. Pancreatic cancer stem stells (CSCs) phenotyping and isolation Cancer stem cells from epithelial tissues were identified for the first time in breast cancer in 2003, when Al-Hajj et al. reported that a distinct population of cells, CD44+CD24−/low 30 ncreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment epithelial-specific antigen (ESA+), develops tumors in immunodeficient mice [5]. In pancreatic cancer, the presence of CSCs was reported in 2007 by Li C et al, who showed that CD44+CD24+ESA+cells possess highly tumorigenic potential [6]. Similar to other types of cancer, pancreatic tumor cells apparently grow around a population of CSCs which are capable of promoting tumor growth and progression through many mechanisms, including alteration of adjacent stromal cells and evasion of conventional therapies [7]. Therefore, their identification, isolation and further in vitro studies represent the field that provided the most important breakthroughs in pancreatic cancer. The phenotypic characterization of CSCs is an ongoing process, however, there are some biomarkers that are recognized as significant for the stemness phenotype: CD133, Nestin, Notch1-4, Jagged 1 and 2, ABCG2 and aldehyde dehydrogenase (ALDH1) [8]. Following the model of breast cancer stem cells [5], a pancreatic CSC subpopulation was shown to be epithelial-specific antigen (ESA)+/CD44+, but unlike the first, also CD24+[6]. CD44+CD24+ESA+cells represent 0.5% to 1.0% of all pancreatic cancer cells [4] and show self-renewal capacity in vitro, are capable of forming tumor spheres, and can be passaged multiple times without loss of tumor sphere-forming capability [9, 10]. CD133 is a biomarker for putative CSC in several solid tumors [11] and it was used as a marker for flow cytometry to select a subpopulation of tumor cells able to generate tumors in athymic mice [12]; it has been reconfirmed in later studies, by immunohistochemistry, to be present in ductal adenocarcinomas [13]. Furthermore, double positive CD133+/CXCR4+seem to be preferentially located in the migration front of pancreatic tumors [12] and demonstrate increased metastatic abilities [14]. Along with CD133, aldehyde dehydrogenase 1 (ALDH1) is also considered a useful marker of stemness, both of which are currently being used for flow cytometry sorting of stem-enriched side populations [15]. Increased activity of ALDH1 was associated with CSCs and has been correlated with invasion, migration and poor overall survival in patients with pancreatic cancer [16]. Therefore, ALDH (+) cells have stem and mesenchymal cell features and are more tumorigenic than CD44+/CD24+cells [17]. 2.1. Pancreatic cancer stem stells (CSCs) phenotyping and isolation An intriguing and somewhat discouraging observa‐ tion is that only 0.015% of all tumor cells are concomitantly ALDH+and CD44+/CD24+, yet ALDH+cells alone have potent tumorigenic activity, thus, several subsets of tumor-initiating cells might be present within a pancreatic tumor [18]. The majority of CSCs is not positive for cytokeratins (intermediate filament proteins present in differentiated epithelial cells) [12], but for Nestin – an intermediate filament protein and a stem cell marker associated with cell integrity, migration, and differentiation. In pancreatic carcinoma, one third of tumor cells present nestin expression which is correlated with tumor staging and metastasis. Nestin-expressing cells are involved in epithelial-to-mesenchymal transition (EMT) and seem to be the origin of pancreatic intraepithelial neoplasia lesions [19]. Recently, presence of Nestin in various types of malignancy was associated with tumoral angiogenesis and was proposed as an angiogenic marker [20]. Within a recent study, authors comparatively analyzed cancer stem cell markers in normal pancreas and pancreatic ductal adenocarcinoma, yielding surprising results: although Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 31 expression was increased, neither CD133, nor Notch proteins or ALDH1 reached statistical significance; in turn, Jagged 1 was shown to be a robust marker, along with Nestin [8]. Mouse models of ductal pancreatic neoplasia seem to harbor a subpopulation of cells express‐ ing high levels of doublecortin-like kinase 1(DCLK1), alpha tubulin acetyltransferase 1(ATAT1), hairy and enhancer of split-1(HES1), hairy/enhancer-of-split related with YRPW motif 1(HEY1), Insulin-like growth factor 1 receptor (IGF1R), and Abelson murine leukemia viral oncogene homolog 1 (ABL1) with cancer-initiating properties. As this subpopulation is identifiable at very early stages during adenocarcinoma development, it provides new targets for early diagnostic and drug testing [21]. All the studies suggest the importance of CSCs in the prognostic and therapeutic responses of pancreatic cancer patients and underline the necessity of stem cell surface marker characteri‐ zation. In this regard, it is useful to better understand the basic genetic and epigenetic processes of cancer stem cell transformation from highly regulated stem cells and also the interaction between stem cells and the tumor niche [22]. 2.2. Epithelial-to-mesenchymal transition Notch pathways have been shown to increase cellular migration by activating Nuclear factor kappa β (NF-κB), Matrix metalloproteinase 9 and Vascular endothelial growth factor (VEGF) in pancreatic cancer cells [34]. More studies suggest that Notch inhibition can reverse EMT in the Mesenchymal-to-Epithelial Transition (MET) and can be considered a promising therapeutic strategy in cancer treatment [35]. Hedgehog signaling is also involved in embryonic cell growth and organogenesis as well as in regulating genes associated with cell proliferation, differentiation, and cell motility [36]. Some studies showed that the Hedgehog pathway, normally quiescent in adult organs, is very active in cancer where it can increase stromal hyperplasia, myofibroblast differentiation, and production of extracellular matrix, enabling the EMT process in cancer cells [37]. A solid body of literature shows that the EMT process is actively implicated in tumor metastasis and tumor recurrence and that cancer stem cells that have undergone EMT display resistance to therapy [38, 39]. The accepted theory is that CSCs from solid tumors acquire migratory potential together with mesenchymal transition, migrate from the primary tumor, colonize other tissues and form a new metastatic tumor with similar characteristics as the initial one (Figure 1) [40, 41]. In vitro and in vivo studies support EMT involvement in early steps of carcinogenesis, by identifying EMT-associated markers such as mesenchymal-specific markers (i.e. Vimentin and Fibronectin), epithelial specific markers (i.e. E-cadherin and Cytokeratin), and transcription factors (i.e. Snail and Slug) in tumor samples [42]. Moreover, the expression of EMT-specific genes has been identified at the level of the invasive front of primary tumors [32] and reversely, the expression of CSCs markers can be induced by overexpressing Snail or Twist, the most important transcrip‐ tion factors involved in the EMT process [43]. From the other point of view, cancer cells from metastasis after the EMT process can show a CSC phenotype and TGF-β signaling is considered to be a crucial factor involved in these processes [44]. Cellular migratory potential is also increased by up-regulation of Mucin-4 (MUC4) and fibroblast growth factor receptor 1 (FGFR-1) stabilization [45]. Other studies show that the process in pancreatic cancer can also be regulated by Forkhead box protein M1 (FoxM1)- caveolin [46], GLI-Kruppel family member GLI1 (GLI1) [47], hepatocyte growth factor (HGF) or platelet-derived growth factor (PDGF) [48]. 2.2. Epithelial-to-mesenchymal transition Recent studies suggest the involvement of CSCs in the progression, aggressiveness and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer [23, 24]. The epithelial-to-mesenchymal transition concept was first described 40 years ago, in relation to the development of the embryo and germ layer formation [25]. Since then, EMT has been shown to be a key player in several normal biological processes or pathologies, such as: embryogenesis, wound healing or cancer progression. The process is essentially defined by phenotypic changes of epithelial cells towards mesenchymal cells. During embryogenesis, EMT represents the biological process in which cells from the epithelial compartment detach, migrate and acquire a mesenchymal phenotype required for the formation of the mesoderm [26]. EMT also plays a key role upon wounding; the wound healing process is marked by epithelial cell migration to the site following EMT signals from the surrounding tissues and acquisition of the mesenchymal-like phenotype [27]. During this process, changes occur in the expression of specific genes, epithelial cell down-regulation of adherent and tight junction proteins (Claudin1 and 7, Occludin and E-cadherin) and matrix metalloproteinase-increased activity, resulting in increased mobility [28]. The major embryonic signaling pathways Wnt, Notch, Hedgehog and Transforming growth factor beta (TGF-β) are involved in upregulation of EMT-activating transcription factors, including Snail, Twist and Slug families [29].TGF-β signaling, associated with other signaling pathways like Ras/MAPK, is essential for EMT process by repressing junction components like E-cadherin, Claudins, and Occludin via Snail transcription factors. TGF-β is also involved in carcinogenesis, playing dual roles by acting as a tumor suppressor in early tumor development, and paradoxically, by promoting tumor cell invasion in later stages [30]. Wnt signaling is also involved in theEMT program, by stabilizing Snail and β-catenin levels and by blocking Glycogen synthase kinase 3 (GSK-3β) activity, processes also related to Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment 32 cancer metastasis. On the other hand, Snail can interact with β-catenin and it enhances Wnt signaling [31]. Notch signaling is responsible for cell fate, proliferation, differentiation, apoptosis and the maintenance of stem cells and also for hypoxia, which can activate EMT in cancer [32]. It is also considered that Notch can regulate endothelial and mesenchymal markers to sustain mesenchymal transformation [33]. Figure 1. Epithelial-to-mesenchymal transition process Figure 1. Epithelial-to-mesenchymal transition process Figure 1. Epithelial-to-mesenchymal transition process Figure 1. Epithelial-to-mesenchymal transition process 2.2. Epithelial-to-mesenchymal transition Taken into account these observations, EMT- type pancreatic tumor cells represent a highly important research focus for the therapies aiming at reducing or preventing invasion, metastasis and therapeutic resistance in pancreatic cancer. Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 33 2.3. Regulatory pathways in pancreatic cancer stem cells Analysis of expression of CSC-related genes in a purified subpopulation of putative pancreatic CSCs showed that up to 46 canonical pathways are upregulated, including human embryonic stem cell pluripotency, tight junction signaling, NF-kB signaling, Wnt/β-catenin signaling, integrin signaling, and Ephrin signaling networks [49]. In particular, out of most signaling pathways involved in maintaining self-renewal in normal stem cells, pancreatic CSCs are characterized by overexpression of Sonic Hedgehog (Shh), Wnt, Notch, AKT, NF-kB, and BMI1 Polycomb Ring Finger Oncogene(BMI-1). Further, signaling pathways which are not dysregulated in metastatic tumors are overexpressed in the pancreatic CSCs [4, 50]. Hedgehog, Notch, Wnt (Figure 2) are shown to be of particular importance in pancreatic cancer stem cells, due to their role in pancreatic embryonic development and differentiation [51]. These signaling pathways are altered in CSCs and EMT-like cells in pancreatic cancer, being involved in self-renewal of CSCs, tumor growth, invasion, metastasis, and resistance to therapy [52]. Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment 34 34 Figure 2. Factors involved in occurrence of cancer stem cells. The emergence of mutations and aberrant signaling in normal stem cells, progenitors, or differentiated cells triggers the transformation of normal cells into cancer stem cells, losing control of cell division. Figure 2. Factors involved in occurrence of cancer stem cells. The emergence of mutations and aberrant signaling in normal stem cells, progenitors, or differentiated cells triggers the transformation of normal cells into cancer stem cells, losing control of cell division. Notch signaling is involved in the early developmental stages of pancreatic cancer by main‐ taining epithelial cells in a progenitor state. Tumor cells present an overexpression of Notch signaling, high levels of Notch-1 and Notch-2 while normal pancreas shows a weak expression of pathway-related molecules [53, 54]. Notch signaling is involved in cell proliferation, survival, apoptosis and differentiation of pancreatic cells and can promote EMT by controlling some transcription factors and growth factors like Snail, Slug, and TGF-β. Among Notch target genes are found Akt, cyclin D1, c-myc, cyclooxygenase-2 (COX-2), extracellular signal- regulated kinase (ERK), matrix metalloproteinase-9 (MMP-9), mammalian target of rapamycin (mTOR), NF-κB, VEGF, p21cip1, p27kip1, and p53, all involved in development and progres‐ sion of human cancer. Gemcitabine-resistant pancreatic cancer cells present overexpression of Notch-2 and Jagged-1, while Notch1, a key downstream mediator of Kirsten rat sarcoma viral oncogene homolog(KRAS), is responsible for pancreatosphere formation [7, 51, 53]. 2.3. Regulatory pathways in pancreatic cancer stem cells Overex‐ pression of Notch ligand Delta like ligand 4 (Dll-4) in pancreatic cancer cells promotes expression of octamer-binding transcription factor 4(Oct4) and Homeobox Transcription Factor Nanog(Nanog) (transcription factors essential for both early embryonic development and pluripotency maintenance in ES cells) and thus increases the number of CSCs [55, 56]. Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 35 Many studies found that pancreatic cancer stem cell resistance to chemotherapy is linked to activated Notch signaling, but the exact mechanism remains unclear [57, 58]. There is more evidence showing that the Notch signaling pathway is essential in supporting KRAS ability to transform normal cells into tumor stem cells. Notch-1 inhibition with specific siRNA or treatment with γ-secretase inhibitors increases apoptosis and decreases proliferative rates, cell migration and invasive properties of pancreatic cancer cells [53]. In this regard, in pancreatic cancer treatment, Notch signaling inhibition can be quite attractive, as long as there is no data arguing that Notch signaling has a critical role in normal adult pancreatic homeostasis [59]. Targeting Notch signaling as a treatment for metastatic pancreatic cancer could prevent the acquisition of the EMT phenotype and resistance to therapy [60]. Hedgehog signaling is another self-renewal pathway, allowing normal stem cells to become independent of control signals; as a result of mutations in this signaling, transformed cells can use Hedgehog for tumor initiation, progression, and metastasis. In vivo studies showed that compared to normal pancreatic epithelial cells, CD44+CD24+ESA+pancreatic cancer stem cells present with an up-regulation of Sonic Hedgehog (Shh) transcripts (a ligand of Hedgehog signaling) [61]. Moreover, 70% of pancreatic cancer tissue presents overexpression of Shh, suggesting that Hedgehog signaling may be involved in pancreatic carcinogenesis [51]. Many studies showed that Shh signaling can activate pancreatic stellate cells, promotes fibroblast infiltration, and increases secretion of fibronectin, collagen type I, MMPs, and TGF-β [62]. Studies in the pancreatic cancer cell line PANC-1 showed that inhibition of Hedgehog signaling by Smoothened (Smo) suppression can reverse EMT, induce apoptosis via PI3K/AKT inhibi‐ tion, and inhibit the invasion of pancreatic cancer cells [63]. Moreover, combination of focal irradiation with Hedgehog signaling inhibition reduces lymph node metastasis in an ortho‐ topic animal model [64]. Wnt/β-catenin signaling is involved in cell proliferation, migration, apoptosis, differentiation, and stem cell self-renewal in several types of cancer [65]. 2.4. MicroRNAs in pancreatic adenocarcinoma MicroRNAs (miRNAs) are potent regulators of cell function via their role as translational regulators for the synthesis of key proteins. Most often, several miRNAs display different expression profiles in cancer cells, including pancreatic cancers. MiR-21, miR-155 and miR-17−5p appear upregulated in tumoral cells, and these miRs are often called oncogenic miRNAs [60, 74]. Similarly, a series of miRNAs, referred to as tumor sup‐ pressor miRs (miR-34, miR-15a, miR-16−1 and let-7) are downregulated in cancers [54, 75]. Key cell differentiation programs during development are controlled by the members of lethal-7 (Let-7) and miR-200 families. In cancer, loss of Let-7 leads to disease progression and de- differentiation. The EMT process is also regulated by miRNA-dependent mechanisms and the same Let-7 family appears as a regulator of EMT and of stem cell maintenance. According to Hasselman et al [75], inhibition of maturation of Let-7 by nuclear receptor for the cytotoxic ligand TNFSF10/TRAIL (TRAILR2) in pancreatic cancer cell lines, increases their proliferation. This is consistent with high levels of nuclear TRAIL2 in tissue samples from poor outcome patients. Pancreatic neoplasms seem also to exhibit their own pattern of miR overexpression, when compared to normal pancreatic tissue: upregulation of miR-93, miR-95, miR-135b, miR-181c, miR-181d, miR-182, miR-183, miR-190, miR-196b and miR-203, miR-767 and miR-1269 and downregulation of miR-20a and miR-29c [76]. In human pancreatic cancer, DCLK1 regulates EMT by a mechanism dependent on miR-200a [77]. MiRNAs were recently considered to have a role in regulation of CSCs [51]. The population of BxPC-3-LN cells (lymph node metastatic pancreatic cells) contains a 5-fold increased population of CD133+/CXCR4+cells (stem-like cells) compared with the parental (non- metastatic) BxPC-3 cells. Remarkably, a different miRNA pattern is displayed in CSC-like compared with the regular cells: up-regulated miR-572, miR-206, miR-449a, miR-489 and miR-184 were found, as well as downregulated let-7g-3p, let-7i-3p, let-7a-3p, miR-107, miR-128 and miR-141−5p[14]. The miR-200 family members are identified as key regulators of cell maintenance and EMT. It is considered possible that tumor progression is a process resulting in progressive de- differentiation towards a cell type having a stem cell-like phenotype. This process appears to be regulated by miRNA-dependent mechanisms. 2.3. Regulatory pathways in pancreatic cancer stem cells Wnt/β-catenin signaling pathway dysregulation is also associated with chemoresistance in pancreatic cancer and recent studies suggest that nuclear β-catenin is essential for the EMT [66, 67]. In vitro and in vivo studies suggest that activated β-catenin may decrease differentiation of epidermal stem cells, increase self-renewal capacity, and develop epithelial cancers in transgenic mice [68]. Kong D et al. showed that there are some connections between Wnt signaling and Snail, a major regulator of the EMT process. Thus, overexpression of Snail could increase expression of Wnt target genes by interaction with β-catenin [69]. In 2013, Sun L et al. showed that one of the most active signaling pathways in pancreatic cancer stem cells is NF-kB, whose inhibition leads to loss of stem cell properties. This study also showed that aberrant epigenetic processes, like CpG promoter methylation, can be involved in carcinogenesis mediated by cancer stem cells [70]. These results were confirmed by studies conducted on PANC1 and HPAC pancreatic cancer cell lines [51]. Activity of the pro-inflam‐ matory NF-κB induces expression of Shh by pancreatic cancer cells and stromal cells, leading to activation of the Hedgehog pathway [71]. Another possible marker for pancreatic CSCs is Met Proto-Oncogene (c-Met), whose inhibition has been correlated with a decrease of tumor growth and with preventing the development of Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment 36 ncreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment 36 metastases [1, 72]. c-Met is a receptor tyrosine kinases involved in cell survival, growth, angiogenesis and metastasis. c-Met activates many signaling pathways, including Ras-MAPK, PI3K/Akt NF-kB, and Wnt/GSK-3β/β-Catenin and is overexpressed in pancreatic cancer [73]. 2.4. MicroRNAs in pancreatic adenocarcinoma DCLK1 (a putative marker for pancreatic and intestinal cancer stem cells) regulates EMT in human pancreatic cancer cells via a miR-200a-dependent mechanism [77]; it also acts as a regulator of Let-7a in pancreatic and colorectal cancer cells, supporting the concept that these miRNAs may be novel and relevant targets in solid tumor cancers [78]. Sureban et al demonstrated that DCLK1 inhibition results in up-regulation of miRNAs that negatively regulate some key angiogenic and pluripotency Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 3 37 factors [79]. In AsPC1 tumor xenografts, downregulation of c-MYC and KRAS via let-7a was observed by a similar mechanism demonstrated in pancreatic cancer cells. factors [79]. In AsPC1 tumor xenografts, downregulation of c-MYC and KRAS via let-7a was observed by a similar mechanism demonstrated in pancreatic cancer cells. Repression of two tumor-suppressor miRs, miR-143 and miR-145, is reported in pancreatic cancer, as well as in other cancers [80]; moreover, experimental restoration of miR 143/145 levels using nano-vector delivery was demonstrated to inhibit pancreatic cancer cell growth [81]. The miR-143/145 cluster cooperates and inhibits the expression of KRAS2 and ras responsive element binding protein 1 (RREB1), its downstream effector [80]. MiR-145 was demonstrated to inhibit cell proliferation in lung adenocarcinoma, by targeting epidermal growth factor receptor (EGFR). In many cancers, including pancreatic cancer, EGFR is upregulated [82], while inhibition of EGF signaling inhibits cancer initiation and progression [83]. Also a suppressive effect of EGFR on miR-143 and miR-145 was demonstrated on models of colon cancer [84]. These findings are indicators of a negative feedback loop between EGFR and miR-143/145, which is similar to KRAS/RREB1 − miR-143/145. The major role of vascular endothelial growth factor (VEGF) signalling via its receptors, VEGFR1 and VEGFR2, was demonstrated in tumor vascular growth, angiogenesis, and metastasis, while upregulated angiogenic factors in various cancers-colorectal, breast, renal, liver, and ovarian-have been correlated with poor prognosis.Pancreatic ductal adenomacar‐ cinoma (PDAC) exhibits endothelial cell proliferation, a mechanisms that increases angiogenesis. Inhibition of VEGF-A, VEGFR1 and VEGFR2 resulted in inhibition of tumor growth and angiogenesis in mouse models of PDAC. Studies and computational analysis outlined a putative binding site for miR-200 (miR-200a, b and c) in the 3’ UTR of VEGFR1 and VEGFR2 [85]. 2.4. MicroRNAs in pancreatic adenocarcinoma Identification of dysregulated expression of various miRNAs, the existence of regulatory loops between miRNAs and protein regulators of key processes (such as cell growth, angiogenesis, differentiation) suggested the need and potential effectiveness of strategies aiming to restore the ”normal phenotype” expression pattern of miRNAs for cancer treatment. Various ap‐ proaches are developed and investigated, such as the delivery of tumor suppressor miRNAs [86], suppression of expression or action of oncomirs [87], targeting the expression of key regulators (such as DCLK1, adenosine monophosphate activated kinase α1(AMPKα1)[88], leading to miRNAs modulation or even to simultaneous modulation of multiple miRNAs, suggesting that using miRNAs as therapeutic agents or addressing miRNAs as targets represents a potential solution for the therapy of critical cancers. a. Pancreatic stellate cells There is a proven interaction between the CSCs and the tumor stroma, at least in part respon‐ sible for increased metastatic abilities of cancer cells. Tumor-stroma interaction is the new cancer paradigm and in the particular case of pancreatic cancer is supported by the presence of pancreatic stellate cells (PSCs) – a subpopulation of desmin-positive periacinar cells, found as well, but in inactive state, in the normal pancreas [91]. Studied at first in relationship with pancreatic fibrosis [92], they were more recently increasingly investigated in the progression of pancreatic cancer [93-95]. In the activated form, stellate cells secrete an array of pro- inflammatory cytokines and promote an immunosuppresive microenvironment [96], secrete various growth factors (e.g. platelet-derived growth factor, stromal-derived factor 1, epidermal growth factor, insulin-like growth factor 1, fibroblast growth factor) [97], as well as matrix adhesion molecules (collagen type I, secreted protein acidic and rich in cysteine (SPARC), small leucine-rich proteoglycans, periostin) and matrix metalloproteinases (MMP-2 and MMP-9), that have been associated with the invasive phenotype of pancreatic cancer cell lines [41]. This particular pattern of pancreatic cell secretome mediates effects on tumor growth, invasion, metastasis and resistance to chemotherapy and is modulated by CSCs, through release of mitogenic and fibrogenic stimulants, such as Transforming Growth Factor β1 platelet-derived growth factor, sonic hedgehog, galectin 3, endothelin 1 and serine protease inhibitor nexin 2 [97]. Recognition of their importance in tumoral behaviour led efforts to isolate, cultivate and immortalize them for further manipulation with therapeutic purposes [98-100]. Upon activa‐ tion, pancreatic stellate cells suffer a shift of phenotype towards myofibroblast morphology and a subsequent switch of protein expression [101]. Indirect co-culture of pancreatic cancer cells with PSCs seem to favor the stem phenotype of cancer cells, as evaluated by Hamada et al. by the spheroid-forming ability of cancer cells and expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo [90]. The presence of α smooth muscle actin (αSMA) in activated pancreatic stellate cells leads to association with cancer-associated fibroblasts (CAFs) – a cancer modified subpopulation of fibroblasts, identified by the very same marker, that was shown to sustain tumor cells metabolism and favor tumor progression [102]. 2.5. CSCs and tumor environment Although the presence of stromal tissue is described and accepted as a fact in all types of solid cancers, pancreatic adenocarcinoma displays a particularly dense atmosphere of connective tissue, known as “desmoplastic reaction”. Since the new cancer paradigm of “stroma-cancer interaction”, more thorough investigations have focused on the pancreatic tumor environ‐ ment, and it is now accepted that the dense connective tissue surrounding malignant cells is at least partially responsible for hindering drug delivery. The pancreatic cancer stroma is now the focus of a new therapeutic approach called “stroma depletion”, which can be achieved Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment 38 through Hedgehog inhibitors [89]. What stromal cells are responsible for Hedgehog signaling responsiveness is currently under investigation, as it would designate them as new anti-cancer targets. Stromal cells are also of importance when considering the concept of stem cell niche- a unique microenvironment involved in generating hierarchies to maintainself-renewal and to control cell fate. The relationship between CSCs and a putative malignant niche is less well stated than for normal stem cells. CSCs are capable of migrating from the original tumor to distance, behavior that is not common for adult, normal stem cells, but is well documented for the hematopoietic stem cell. Stroma of hematopoietic tissue is a particular one, based on reticular connective tissue, unlike most malignant stromas, rich in dense irregular connective tissue. This would possibly indicate the partial independence of CSC from stem-cell niche [90]. Pro-tumor effect of MSCs Very recent reports have demonstrated that mesenchymal stem cells (MSCs) can function as precursors for CAFs [111, 112]. Interestingly, not all types of MSCs have this particular ability, a recent report from Subramanian et al. arguing that this is not a feature of umbilical-cord derived pluripotent cells[113]. In pancreatic cancer, like in any other type of cancer, these myofibroblast-like cells contribute to inducing EMT in side population cells, maintain tumor- initiating stem cell-like characteristics, including augmenting expression levels of various stemness-associated genes, enhancing sphere-forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs [114]. Bone marrow derived progenitor cells were found to participate to neovascularization of tumors [115], a process that was shown to be dependent on Hedgehog signaling [116]. The recruitment of these progenitors is accomplished by CAFs through stroma-cell derived factor 1(SDF-1) signaling [117]. a. Pancreatic stellate cells CAFs also mediate EMT of tumor cells, possibly through a pro-inflammatory signature [103] – secretome that has also been reported in pancreatic stellate cells, not only in cancer but also in chronic pancreatitis [104] Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 39 39 From tumor-stroma interactions new lessons were learned in diagnostics and therapeutics of pancreatic cancer. Secreted Protein, Acidic, Cysteine-Rich (SPARC) (a member of the family of matricellular glycoproteins that is highly expressed in PSCs and the tumour/stroma interface) is now proposed as marker for accurate diagnostic, as 80% of pancreatic ductal adenocarcinomas seem to express it [105]. Due to its ability to bind to basement membrane collagen IV and ﬁbrillar collagens I, III, V and also to bind albumin [106], it has been used to increase distribution of the chemotherapeutic agent paclitaxel within the tumoral mass [107]. Changes within the stem niche, such as hypoxia, are ”tuning” the behavior of stem cells, inducing the activation of survival, proliferation, differentiation and angiogenesis. b. Mesenchymal stem cells – dual facets in cancer Mesenchymal stem cells (MSCs) are pluripotent cells with homing abilities that are involved in tissue repair, including outside their native niche, that reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues [108]. They also exhibit a natural tendency of homing into tumors – ability that is starting to be exploited in anticancer treatment, using these versatile cells as cargo delivery for cytotoxic drugs or gene therapy [109]. This behavior has been also reported in pancreatic cancer, by the use of genetically engineered labeled MSCs that efficiently accumulatewithin the pancreatic tumor, when injected into tumor-bearing mice [110]. Pro-tumor effect of MSCs 3. CSCs and therapy outcomes In pancreatic cancer, surgery is usually accompanied by other complementary treatments such as multi-chemotherapy regimens and radiotherapy. Despite clear progress in detection and treatment of cancer, current strategies fail to completely remove the tumor and prevent recurrence and metastasis. Existing therapies are toxic and non-specific, being directed towards both normal cells and tumor cells. Most chemotherapeutic regimens are based on gemcitabine, but provided a modest improvement in median survival. The response rate was increased by using more than two chemotherapeutic agents [122]. Human pancreatic cancer tissue contains CSCs defined by CD133 and CXCR4 expression and these cells are highly resistant to standard chemotherapy and are involved in metastasis [12]. Features of CSCs have also been confirmed in brain and colon cancers [9].Therapy failure for other highly malignant tumors has been explained, at least partially, by the chemo-[10, 123] and radio-resistant [124] nature of CSCs. Cancer stem cells therapy resistance is considered to be the result of inappro‐ priate activation of several proliferative signaling pathways, including EGFR, PDGFR(platelet- derived growth factor receptor), stem cell factor (SCF) receptor KIT [125], and activation of Hedgehog and Wnt/β-catenin signaling [50]. Another well sustained argument for chemo‐ therapy resistance is the expression of multidrug resistance-linked genes, out of which most are ATP-binding cassette (ABC) drug transporters [126]. High levels ofABC transporters were documented in pancreatic CSCs and chemotherapeutic agents such as etoposide, doxorubicin, vincristine and paclitaxel are direct substrates of ABC transporters [127]. Gemcitabine uptake, the golden standard for pancreatic adenocarcinoma chemiotherapy, seems to be negatively influenced by expression of ABCG2, though there is no clear evidence that ABC transporters directly efflux gemcitabine or its metabolites in pancreatic cancer cells [90]. Several reports indicate that conventional chemotherapy itself could propagate the CSC population in pancreatic cancer, through exerting a positive selection pressure of CD24/CD44/ESA triple positive CSC fraction [12, 128]. Differential expression of some CSCs biomarkers can be indicative of particular characteristics, such as responsiveness to different therapies or outcomes. Anti-tumor activity An increasing number of reports show that MSCs have the ability of negatively influencing tumor behaviour, in terms of proliferation and invasiveness. Cell cultures co-cultivated or treated with MSCs conditioned media showed inhibited growth [118-120] and co-injection of tumor cells and MSCs in nude animals showed that tumor growth was significantly inhibited [120]. Some authors explain this activity by MSCs to inhibit the expression of Wnt signaling pathway-related factors in tumor cells, consequently unbalancing cellular proliferation and apoptosis [121]. 40 ncreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment To conclude, the presence of MSC within the tumor site is a fact, but its role is still to be determined. To conclude, the presence of MSC within the tumor site is a fact, but its role is still to be determined. 3.1. CSCs as therapeutic targets Different strategies are developed to target specifically CSCs, thus eliminating this particular set of cells. Several key regulatory pathways operating in the stem cells have been proposed and demonstrated to considerably improve the therapy outcomes; relevant examples are Sonic Hedgehog, Notch/Jagged, CD133, TGF beta signaling; specifically addressing such pathways, by small molecule inhibitors, monoclonal antibodies or siRNAs results in increasing the efficacy of therapies, as suggested by in vitro studies, as well as by clinical outcomes. Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 41 Some in vitro studies showed that blocking cis-acting elements, that are common for pluripo‐ tency maintaining Transcription Factor SOX-2 (Sox2), Oct4, and proto-Oncogene C-Myc (c- Myc), dramatically decreased CSCs proliferation and their ability to generate tumors in nude mice [15]. Equally, simultaneous knockdown of OCT4 and its target Nanog led to decreased proliferation, migration, invasiveness and tumorigenesis of putative pancreatic cancer stem cells [129]. Inhibition of the Nodal/Activin receptor Alk4/7 in CSCs decreased almost to zero their self-renewal capacity and tumorigenicity, and reversed the resistance of CSCs to gemci‐ tabine. Concordant with previous reports on stroma-tumor interaction, Lonardo et al. also found the response to gemcitabine was dependent on the amount of stroma which hindered drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor (HHI) en‐ hanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival [130]. The Hedgehog signaling pathway is usually targeted in experimental designs as adjuvant to classic chemotherapy. The combined blockade of Shh and mTOR signaling together with gemcitabine is capable of eliminating pancreatic CSCs [131]. Inhibition of Smoothen (Smo), combined with gemcitabine and mTOR inhibitor rapamycin, led to abrogation of cancer stem cells and the authors reported a long-term disease stabilization or regression and subsequent long-term survival [132]. Notch pathway inhibition by selective γ-secretase inhibitors, such as PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine, inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1 and induced tumor regression in xenograft tumor models. The authors argue that the observed effects are due to PF-03084014 targeting of putative aggressive cancer stem cells [59]. Another potent and selective γ-secretase inhibitor, MRK-003, also led to downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumor- initiating cells capable of extensive self-renewal. 3.1. CSCs as therapeutic targets Pretreatment of a pancreatic adenocarcinoma cell line with MRK-003 significantly inhibited the subsequent engraftment in immunocom‐ promised mice and mixed regimen MRK-003 and gemcitabine of engrafted mice reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis [133].However, some of such pathways are common to normal and CSCs, raising the problem of increasing the selectivity towards cancer stem cells. 3.2. Clinical studies Sunitinib targets multiple receptor tyrosine kinases, including stem cell factor receptor (c-KIT) and it has been shown to have antitumor efficacy in in vivo. The combination of gemcitabine with sunitinib could not surpass the effects of the single agent sunitinib [135]. Cabozantinib – a small kinase inhibitor that targets c-Met and VEGFR2- inhibited viability and spheroid formation and induced apoptosis in pancreatic malignant cells with minor effects in non-malignant cells. In primary, CSC-enriched spheroidal cultures cabozantinib downregulated CSC markers SOX2, c-Met and CD133 and induced apoptosis [73]. Most clinical studies, so far, do not seem to report any significant improvement with various regimens employed [136]. Early clinical data for the Shh inhibitor, GDC-0449 (vismodegib), in combination with either gemcitabine or erlotinib, indicate that these regimens are feasible and well tolerated [137]. However, a phase II trial of gemcitabine plus saridegib versus gemcita‐ bine plus placebo in previously untreated patients with metastatic pancreatic cancer was halted early based on a shorter overall survival rate in the gemcitabine plus saridegib arm [106]. A very interesting new trend in advanced, chemotherapy-resistant cancers, aiming for a different approach, tests personalized peptide vaccination (PPV) – a method to generate an immune response against tumor-associated antigens and so far employed for aggressive cancers such as lung cancer [138] and biliary tract cancer [139]. For advanced pancreatic cancer a phase II clinical trial was also conducted in which vaccine antigens were selected and administered based on the pre-existing IgG responses to 31 different pooled peptides [140]. Other vaccines are aimed at increasing the patient’s immune response against tumor cells – targeting cancer markers with the aid of specialized antigen-presenting cells such as dendritic cells. Currently, there are several vaccines for human pancreatic cancer in clinical trials including: i) whole-cell vaccines, ii) combined dendritic cells with antigen to present to patient leukocytes iii) peptide and DNA vaccines, iv) Ras peptide vaccine; v) vaccine against common cancer mutations, targetable by CD4/8 T cells; vi) Telomerase peptide vaccine; vii) carcinoem‐ brionar antigen (CEA) and Mucin 1; viii) Survivin-targeted vaccine [141]. Also, it was shown that boosting the immune response by additional treatment with dendritic cells (LANEX-DC®) is highly effective and extends the median survival times up to 8.9 months [142]. Lack of response to all of the above mentioned types of therapies led to an investigation of non-conventional therapies. 3.2. Clinical studies Most clinical studies addressing molecular therapies in pancreatic cancer report usage of monoclonal antibodies, for several simple rationales: i) they are already tested as drugs in other types of pathologies, tumoral or not; ii) they block proliferative oversignaling – a characteris‐ tic feature of malignancy; iii) some of them address phenotypic anomalies given by genetic dysregulations, such as EFGR overexpression/ oversignaling. However, these antibodies do not address specifically stem cells, but the larger category of cancer cells. There are some constructs that are, however, effective on the side population of CSCs. A combination of tigatuzumab, a fully humanized death receptor5 (DR5) agonist monoclonal antibody, with gemcitabine proved to be more efficacious in killing both CSCs and adenocarcinoma bulk cells. Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment 42 The combination therapy produced remarkable reduction in pancreatic CSCs, tumor remis‐ sions, and significant improvements in time to tumor progression [134]. Signaling pathways can also be inhibited by small molecule kinase inhibitors that act downstream of the extracellu‐ lar domain of the receptor. Sunitinib targets multiple receptor tyrosine kinases, including stem cell factor receptor (c-KIT) and it has been shown to have antitumor efficacy in in vivo. The combination of gemcitabine with sunitinib could not surpass the effects of the single agent sunitinib [135]. Cabozantinib – a small kinase inhibitor that targets c-Met and VEGFR2- inhibited viability and spheroid formation and induced apoptosis in pancreatic malignant cells with minor effects in non-malignant cells. In primary, CSC-enriched spheroidal cultures cabozantinib downregulated CSC markers SOX2, c-Met and CD133 and induced apoptosis [73]. Most clinical studies, so far, do not seem to report any significant improvement with various regimens employed [136]. Early clinical data for the Shh inhibitor, GDC-0449 (vismodegib), in combination with either gemcitabine or erlotinib, indicate that these regimens are feasible and well tolerated [137]. However, a phase II trial of gemcitabine plus saridegib versus gemcita‐ bine plus placebo in previously untreated patients with metastatic pancreatic cancer was halted early based on a shorter overall survival rate in the gemcitabine plus saridegib arm [106]. The combination therapy produced remarkable reduction in pancreatic CSCs, tumor remis‐ sions, and significant improvements in time to tumor progression [134]. Signaling pathways can also be inhibited by small molecule kinase inhibitors that act downstream of the extracellu‐ lar domain of the receptor. 4. Nanotheragnostics in pancreatic cancer Targeted therapeutic delivery is a way to ensure that drugs reach the designated target at the highest concentration within safety margins, limiting in the same time undesired side effects resulting from unspecific diffusion in well vascularized tissues. This aim is now being resolved with the use of nanomedicine –a multidisciplinary field that aims to utilize nanoscale (up to 100 nm) particles to improve delivery of chemotherapeutics [148]. These constructs fall into several categories – micelles, microemulsions, liposomes, polymers [149] silica and carbon- based nanoparticles [150] and dendrimers [151]. This coating of a nanoparticle can be improved with stabilizing agents (such as polyethylene glycol – PEG) or ligands to direct them to a specific target (such as an antibody towards a cancer cell type). Liposome delivery of active agents has been recently paired with ultrasound technology, by development of ultrasound- responsive stable liposomes. Ultrasound-induced heating triggers phase transition in the phospholipid membrane, leading to drug release in the targeted region [152]. To date, there are at least twelve FDA (Food and Drug Administration) approved liposome-based drugs, most of them being chemotherapeutics for breast, ovarian and pancreatic cancer [153]. Generation of magnetic/metallic nanoparticles was considered a step-forward in magnetic resonance imaging and diagnostics [154], adding a new utility to biomedical nanoscience. Another type of imaging strategy using nanoparticles is optical, through use of carbon nanomaterials that display natural fluorescence emission [155], or use of other infrared light emission agents [156], forming upconversion nanoparticles [157], or incorporated in a wide variety of coating surfaces, such as gold [158] and polymer-based [159]. Photoacoustic imaging is another nanomedical promising technology that combines the benefits of optical imaging methods with the clinically available and cost-effective ultrasound imaging modality [160]. Originally used for investigation of vascularization pattern, based on high endogenous contrast of blood versus surrounding tissues [161] and or/vascular wall/lumen alterations [162], it has been increasingly used in tumor assessment, providing further molecular information on cancer, given by the chemical composition of tissues and by targeted nanoparticles that can interact with extravascular tissues at the receptor level [163]. By incorporating active drugs into imaging nanoparticles, a dual therapeutic and diagnostic agent was generated, thus the emerging field of ”theragnostic”, is widely used especially in cancer research. 3.2. Clinical studies Salinomycin, an anti-protozoa agent that was recently shown to preferentially kill breast CSCs [143], and later investigated in other types of malignancies, was shown to inhibit growth of pancreatic adenocarcinoma CSCs in vitro. In vivo xenografting studies showed that salinomycin combined with gemcitabine could eliminate the engraftment of human pancreatic cancer more effectively than the individual agents [144]. Adamantyl- substituted retinoid-related molecules (ARRs) inhibit growth and induce apoptosis in the pancreatic stem-like cell population, possibly through decreased IGF-1R and β-catenin expression [145]. Isothiocyanate sulforaphane (SF) was used as sensitizer of pancreatic CSCs to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)-induced apoptosis, by quercetin and sorafenib. The combination of SF with a cytotoxic drug efficiently induced Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 43 43 apoptosis along with inhibition of self-renewing potential, ALDH1 activity, clonogenicity, xenograft growth and relapse of gemcitabinetreated tumor cells in nude mice [146]. The flavonoid Quercetin enhances TRAIL-mediated apoptosis, acts as a chemosensitizer for the ABC pump-proteins, and can enhance the effects of sulforaphane in inhibiting the pancreatic CSC characteristics [147]. 4. Nanotheragnostics in pancreatic cancer Most nanoparticles accumulate in tumors due to their intense and leaky neovascularization, but some can be retained there with the use of cancer-specific antigens [164] and stimulated into releasing their chemotherapeutic cargo. Cancer diagnostic and concomitant treatment through nanoparticles benefits from real-time assessment of drug bioavailability and more accurate monitoring of tumor evolution. Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment 44 Pancreatic cancer treatement benefits from development of biomedical nanotechnology, in both clinical practice and fundamental research. A PEGylated polymeric nanoparticle containing a potent antagonist of the Hedgehog transcription factor Gli1 combined with gemcitabine significantly impeded the growth of orthotopic pancreatic cancer xenografts [165]. In in vivo studies, squalene-conjugated gemcitabine nanoparticles decreased tumor growth significantly, prevented tumor cell invasion, and prolonged the survival time of mice bearing orthotopic pancreatic tumors [166]. Liposomal delivery of tissue transglutami‐ nase 2 siRNA effectively blocked the growth of pancreatic adenocarcinoma in nude mice [167]. EGFR monoclonal antibody or peptidylglycine alpha-amidating monooxygenase (PAM4)-conjugated gold nanoparticles induced significant tumor destruction in a murine model of pancreatic carcinoma after radiofrequency radiation [168]. Paclitaxel, one of first- line chemotherapeutic agents before the gemcitabine era, is now available as a positively charged lipid-based complex (known as EndoTAG-1) [169] that in combination with gemcitabine was able to inhibit the incidence of metastasis in pancreatic cancer animal models [170]. A controlled phase II clinical trial for pancreatic cancer showed significant‐ ly increased survival rates of patients treated with EndoTAG®-1 and gemcitabine combina‐ tion therapy [171]. An ongoing phase I study (NCT00968604) of advanced pancreatic cancer is currently investigating the effects of intravenous injection of the liposome nanoparticle BikDD, which contains a pro-apoptotic agent [172]. 4.1. Nanoparticles for cancer stem cell targeted therapy In the same manner that nanoparticles are targeted for the bulk tumor, they can be targeted for CSCs, through the use of antigens against specific CSCs markers (e.g CD-133). Such targeted therapy has already been tested in vitro, against targeting CD133-expressing cancer cells of colon and pancreatic origin, with encouraging results [56]. Breast CSCs-targeted nanoparticle delivery of doxorubicin reduced their mammosphere formation capacity and cancer initiation activity, eliciting tumor growth inhibition in animal models[173]. Apart from cytotoxic drug delivery, nanoparticles can be used to target and modify certain characteristics of CSCs, such as activation of signaling pathways that confer renewal proper‐ ties, targeting metabolism and inhibiting drug efflux transporters in an attempt to sensitize them to therapy [174]. Multi-lamellar vesicle liposomes targeted against CSCs, containing a steroid nucleus, were formulated to disrupt mitochondrial integrity and to facilitate release of cytochrome c to attain programmed cell death [175]. 5. Conclusions CSCs represent key components in the heterogeneous cellular system represented by pancre‐ atic tumors. Their biological features configure them as one of the major players and major targets for investigation; they offer sets of additional and reliable biomarkers for prognosis and stratification. Discovery of target mechanisms and molecules within cancer stem cells is plausible to provide the needed boost for therapy improvement. Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 45 Acknowledgements This work was partly supported by Grants POS CCE 685-152/2010. Author details Cristiana Pistol Tanase1, Ana-Maria Enciu1,2, Maria Linda Cruceru2, Laura Georgiana Necula1,3, Ana Iulia Neagu1,3, Bogdan Calenic1,2 and Radu Albulescu1,4 Laura Georgiana Necula1,3, Ana Iulia Neagu1,3, Bogdan Calenic1,2 and Radu Albulescu Address all correspondence to: bioch@vbabes.ro *Address all correspondence to: bioch@vbabes.ro 1 Victor Babes National Institute of Pathology, Dept. of Biochemistry-Proteomics, Splaiul Independentei, Bucharest, Romania 1 Victor Babes National Institute of Pathology, Dept. of Biochemistry-Proteomics, Splaiul Independentei, Bucharest, Romania 2 Carol Davila University of Medicine and Pharmacy, Eroilor Sanitari, Bucharest, Romania f l I f l h 2 Carol Davila University of Medicine and Pharmacy, Eroilor Sanitari, Bucharest, Romania 3 Stefan S. Nicolau Institute of Virology, Bucharest, Romania 4 National Institute for Chemical Pharmaceutical Research and Development, Bucharest, Romania 4 National Institute for Chemical Pharmaceutical Research and Development, Bucharest, Romania References [1] Herreros-Villanueva M, Zubia-Olascoaga A, Bujanda L. c-Met in pancreatic cancer stem cells: therapeutic implications. World journal of gastroenterology : WJG. 2012;18(38):5321-3. [2] Dima SO TC, Albulescu R, Botezatu A and Popescu I (2012). Novel Biomarkers in Pancreatic Cancer, Pancreatic Cancer-Clinical Management, Prof. Sanjay Srivastava (Ed.), ISBN: 978-953-51-0394-3, InTech, DOI: 10.5772/29001. Available from: http:// www.intechopen.com/books/pancreatic-cancer-clinical-management/novel-biomark‐ ers-in-pancreatic-cancer. [3] Kaur S, Baine MJ, Jain M, Sasson AR, Batra SK. Early diagnosis of pancreatic cancer: challenges and new developments. Biomarkers in medicine. 2012;6(5):597-612. [4] Lee CJ, Dosch J, Simeone DM. 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https://openalex.org/W4323059696	https://publicaciones.unirioja.es/ojs/index.php/brocar/article/download/5223/4049	es		El caso “Sonthonax”: ¿abolicionista o superviviente?	Brocar	2,022	cc-by	13,254	BROCAR, 46 (2022): 11-42. DOI: http://doi.org/10.18172/brocar.5223 Copyright © 2022, Los autores. Artículo en acceso abierto con licencia CC BY (http://creativecommons.org/licenses/by/4.0/) EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS Jonathan Jacobo Bar Shuali Universidad Complutense de Madrid jbar@ucm.es RESUMEN: Contemporáneo de figuras tan icónicas como Jean-Jacques Dessalines y Toussaint Louverture, Léger-Félicité Sonthonax, natural de Oyonnax, ejerció de puente entre la asimilada población esclava del “pueblo de la alta montaña” (Haití) y la República francesa. Misionero de la paz, o representante de los intereses del “abolicionista” Jacques Pierre Brissot, el comisario galo proclamó la primera emancipación de los esclavos en el Caribe francés. Enfrentado a una población realista y mulata, Sonthonax desempeñó un papel crucial en las segunda y tercera comisiones civiles de Saint-Domingue. Retirado de sus funciones por el Consejo de los Quinientos, Léger-Félicité se vio atrapado entre el Consulado y el Imperio. La focalización de diversos ensayos sobre esta figura en su primera visita a la colonia en 1792 ha influido notablemente en un menor análisis de las restantes etapas de su larga travesía. El presente trabajo analiza los aspectos olvidados del diplomático Léger-Félicité Sonthonax en el marco de sus actividades en la isla y en territorio nacional. Asimismo, se pretende atenuar el nulo conocimiento de su obra en el mundo hispanohablante sobre la base de fuentes primarias. Palabras clave: biografía, esclavitud, Revolución haitiana, República francesa, Sonthonax. THE “SONTHONAX” CASE: ABOLITIONIST OR SURVIVOR? ABOUT A FRENCH DIPLOMATIC BETWEEN SAINT-DOMINGUE AND PARIS ABSTRACT: Contemporary with such iconic figures as Jean-Jacques Dessalines and Toussaint Louverture, Léger-Félicité Sonthonax, a native of Oyonnax, acted as a bridge between the assimilated slave population of the “people of the high BROCAR, 46 (2022) 11-42 11 JONATHAN JACOBO BAR SHUALI mountains” (Haiti) and the French Republic. A missionary of peace, or a representative of "abolitionist" Jacques Pierre Brissot’s interests, the Gallic commissioner proclaimed the first emancipation of slaves in the French Caribbean. Facing a royalist and mulatto population, Sonthonax has played a crucial role in the second and third civil commissions in the Saint-Domingue. Having been removed from his duties by the Council of the Five Hundred, Léger-Félicité found himself caught between the Consulate and the Empire. The focus of various essays on this figure on occasion of his first visit to the colony in 1792 significantly influenced a lesser analysis of the remaining stages of his long journey. This paper analyzes the forgotten aspects of the diplomat Léger-Félicité Sonthonax within the framework of his activities on the island and in the national territory with a view to mending the null knowledge of his work in the Spanish-speaking world. Keywords: Biography, slavery, Haitian Revolution, French Republic, Sonthonax. Recibido: 27 de octubre de 2021 Aceptado: 30 de agosto de 2022 1. Introducción Léger-Félicité Sonthonax 1 ha sido objeto de estudio de numerosos trabajos y autores en la Academia franco-haitiana. A pesar de ello, es evidente el desconocimiento parcial sobre este personaje en la península ibérica y gran parte del mundo hispanohablante. Situación que, en gran medida, es consecuencia de la limitada documentación primaria de la que se dispone, además de la mínima traducción a la lengua castellana. El comisario francés es una figura crucial para la historia del actual Estado haitiano. En agosto de 1793 anunció la famosa Proclamation du 29 août 2, otorgando así la emancipación a la totalidad de la El presente trabajo fue pronunciado en el IV Seminario Interdisciplinar de la Asociación Académica “Jóvenes Humanistas” en la Universidad Complutense de Madrid el 14 de abril de 2021. Quiero agradecer a la profesora Dra. María Dolores Domingo Acebrón la confianza que depositó en mi estudio ese año. Asimismo, deseo resaltar la amabilidad de D. Jorge Blanco Mas y la Dra. Alicia Teresa Laspra Rodríguez por su ayuda en la revisión de contenidos. 2 b3903485 John Carter Brown Library (proclamationauno00sont). Proclamation. Au nom de la République.: Nous Léger-Félicité Sonthonax, commissaire civil de la République, délégué aux Iles françaises de l'Amérique sous le vent, pour y rétablir l'ordre & la tranquillité publique (…). Recuperado de (https://jcb.lunaimaging.com/luna/servlet/s/l2fbns). [consultado 05/06/2021] 1 12 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS población negra en la parte occidental de la isla de Santo Domingo: Saint-Domingue o Santo Domingo francés 3. “Todos los negros y mestizos actualmente en la esclavitud, son declarados libres hoy con todos los derechos inherentes a la cualidad del ciudadano francés (…)”. La misión del jacobino Sonthonax tuvo por objeto 4 el desarme de las numerosas revueltas que se daban desde 1791 bajo el mando del sacerdote “vudú” Dutty Boukman (“el hombre del libro” o “Zamba Boukman”) 5. Este último hecho ha ofrecido a los historiadores un amplio debate sobre su figura, discutiendo si la liberación de los esclavos fue fruto de las relaciones del diplomático con el club de “Amigos de los Negros y las Colonias” o una necesidad, con el objetivo de conseguir apoyos entre los jefes rebeldes. Es decir, una “pacificación forzada” para obtener hombres y armas frente a colonos realistas y tropas anglo-españolas en la Guerra del Rosellón. El discurso de 1793 resulta muy significativo ya que, al margen del Decreto del 29 de marzo de 1815 6 por Napoleón I Bonaparte, fue la única emancipación proclamada por un organismo gubernamental francés hasta 1848. No obstante, no debemos olvidar que en 1794 se ratificaron las medidas de Sonthonax en el continente. Creemos que es pertinente aclarar que el presente trabajo no tiene por objeto resumir o explicar el drama histórico que resulta ser la esclavitud negra en el territorio que forma el actual Haití. En este sentido, nos ceñiremos a todo aquello Antonio Jesús PINTO TORTOSA: “Una frontera Definida por el color de la Piel: las controvertidas relaciones entre Haití y la República Dominicana”, Criticae. Revista Científica para el Fomento del Pensamiento Crítico, 1 (2022), pp. 54-62. 4 José LUCIANO FRANCO: Historia de la Revolución de Haití, La Habana, Academia de las Ciencias de Cuba, 1966, pp. 207-208. 5 Antonio Jesús Pinto Tortosa nos recuerda en Una colonia en la encrucijada: Santo Domingo, entre la revolución haitiana y la Reconquista española, 1791-1809 (2012) los orígenes del líder haitiano haciendo uso de las actualizaciones de Buck-Morss, Hegel y Haití. La dialéctica del amo-esclavo: una interpretación revolucionaria (2005). La autora señala el posible origen musulmán de Boukman, así, su apellido pueda hacer referencia al Corán. Por otro lado, se trata de un esclavo fugado de Jamaica que pudo haber trabajado como cochero. Finalmente, formó parte del grupo de los “cimarrones” y lideró el movimiento insurreccional de BoisCaïman hasta noviembre de 1791. Cabe destacar que, según la misma investigadora, un 14 % de la población esclava en Haití era de origen musulmán. 6 Napoleón.Org. La web de historia de la Fundación Napoleón (Fundación Napoleón). Napoléon Bonaparte, la traite des noirs et l’esclavage, 20 mai 1802 et 29 mars 1815. Recuperado de (https://www.napoleon.org/enseignants/documents/video-napoleon-bonaparte-et-le-retablissementde-lesclavage-20-mai-1802-5-min-40/). [consultado 01/06/2021] 3 BROCAR, 46 (2022) 11-42 13 JONATHAN JACOBO BAR SHUALI relevante en las acciones de Sonthonax para así responder a las preguntas que se formulan en este artículo: ¿Qué impulsó a nuestro objeto de estudio a liberar a los esclavos? ¿Se trata de un motivo práctico o político? 2. Estado de la cuestión Las tres comisiones diplomáticas francesas en Saint-Domingue (1791-1797) y la biografía de Sonthonax han originado el interés de sociedades académicas como la Association pour l'étude de la Colonisation Française, la Sociedad de Estudios Robespierristas, el Instituto de Historia de la Revolución francesa, el Souvenir Napoléonien o la propia Fundación Napoleón. Léger-Félicité Sonthonax es un “símbolo” que, tal y como ya indicamos al principio de este trabajo, es transversal en las etapas de la República y el Imperio. Ello da lugar a su análisis desde diversas ópticas y reinterpretaciones. Al igual que sucede con el general Toussaint Louverture, son muchos los movimientos políticos que han tratado de apropiarse de la personalidad y actos de este personaje. Trouillot ya señaló en los 90 el peligro del “silenciamiento” haitiano por parte de los círculos académicos 7, un aspecto que, como ya ha referenciado este autor, se viene desarrollando gradualmente desde mediados del siglo XIX, con la instauración del oficio del “historiador” y la historiografía positivista. No obstante, el caso de estudio de la Revolución haitiana ha sufrido un breve abandono hasta 2021, momento en el que, a raíz de las conmemoraciones por el bicentenario del fallecimiento de Napoleón Bonaparte, se ha puesto bajo revisión su figura 8 (en el caso francés). En otro orden, la bibliografía colonial franco-haitiana se encuentra representada por la historiadora C. Lheureux-Prévot 9, la cual realizó una amplia labor investigadora con el apoyo de la Fundación Napoleón. Trabajó todas las fuentes primarias relativas a las colonias francesas entre 1789 y 1815. En total publicó en su estudio un extenso compendio de 600 referencias, entre las que destacan las referidas a Léger-Félicité Sonthonax, Toussaint Louverture, las tropas polacas y el Ejército francés, Santo Domingo, Guadalupe, Martinica, la situación de los “negros” en Francia, etc. Activistas sociales han denunciado los Decretos 10 del 10 y del 20 de mayo de 1802, leyes para el restablecimiento de la esclavitud, con la esperanza de que el Michel-Rolph TROUILLOT: Silenciando el pasado: el poder y la producción de la Historia, Granada, Comares Historia, 2017, pp. 4-5. 8 Véase https://www.youtube.com/watch?v=Rk73diSlw9o 9 Chantal LHEUREUX-PRÉVOT: “La politique coloniale de la France de 1789 à 1815”, Revue Napoleonica, 1 (2008), pp. 148-194. 10 Jeremy D. POPKIN: A Concise History of the Haitian Revolution, U. S. A., Wiley-Blackwell, 2012, pp. 127-130. 7 14 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS Gobierno de Macron rechazase estas celebraciones. Sin embargo, la cuestión de Napoleón y los esclavos, lejos de ser mitigada, ha suscitado un gran interés dentro de los círculos académicos y divulgativos. En Francia la televisión nacional, TV France, ha producido bajo la dirección de Stéphane Bern el documental Toussaint Louverture: la liberté à tout prix 11. Este hecho ha resultado ser innovador de cara al gran público, desacostumbrado a tratar esta temática en formato audiovisual. Por su parte, los medios, han avivado todo tipo de debates. Es preciso indicar las intervenciones de J. Tulard y T. Lentz en RMC y FRANCE 24 en el mes de mayo (2021) 12. Pero estas acciones no han permanecido únicamente en territorio francés, antiguas colonias como la República de Benín 13, han llevado a cabo exposiciones alrededor de la historia de la esclavitud y Toussaint Louverture. No obstante, Sonthonax no aparece referenciado en ninguno de los proyectos mencionados unas líneas más arriba. Entre los estudios más recientes acerca de la esclavitud y su abolición en el Caribe francés, destacan las dos obras principales de F. Régent: La France et ses esclaves (2007) y Les Maîtres de la Guadeloupe: Propriétaires d'esclaves 1635-1848 (2019). Régent, al contrario, sí analiza el papel de la proclama del 29 de agosto de 1793. Además de ello, precisa las influencias del primer cónsul Bonaparte en el momento de restablecer la esclavitud sobre las islas de Martinica, Guadalupe y Haití. Tal y como se ha señalado con anterioridad, la documentación sobre LégerFélicité Sonthonax es realmente limitada. Los informes y legajos a lo largo de sus actividades como diplomático de la República se encuentran focalizados en el ejercicio de su participación política. Todo ello fomenta las incógnitas acerca de numerosos aspectos personales entre 1789 y 1813; un claro ejemplo es la mínima información que disponemos hoy al respecto de su esposa 14. En cambio, abundan los documentos relativos a su segunda misión, entre 1796 y 1797, en Santo Domingo Secrets d'Histoire (TV France). Toussaint Louverture: la liberté à tout prix. Recuperado de internet (https://www.france.tv/france-3/secrets-d-histoire/2429339-toussaint-louverture-laliberte-a-tout-prix.html). [consultado 05/06/2021] 12 C’est en France (TV FRANCE 24). Bicentenaire de Napoléon: un héritage controversé. Recuperado de internet (https://www.youtube.com/watch?v=Rk73diSlw9o). [consultado 05/06/2021]. RMC (BFM. TV). Jean Tulard, historien: "Napoléon n'était pas raciste". Recuperado de internet (https://www.youtube.com/watch?v=Kl5bLQghjpc). [consultado 05/06/2021] 13 Esclavage, résistance et mémoire à Allada (Musée Toussaint Louverture). EXPOSITION PERMANENTE. Recuperado de internet (https://www.youtube.com/watch?v=Kl5bLQghjpc). [consultado 02/06/2021] 14 Sudhir HAZAREESINGH: Black Spartacus: The Epic Life of Toussaint Louverture, New York, Farrar, Straus and Giroux, 2020, pp. 56-57. La esposa de Léger-Félicité Sonthonax recibió por nombre Marie Eugénie Bléigat, la información sobre esta mujer de color es mínima, hay constancia de su estancia en la plantación Breda en la década de 1780. 11 BROCAR, 46 (2022) 11-42 15 JONATHAN JACOBO BAR SHUALI francés. En esta línea, M. Dorigny, “máximo exponente” en la cuestión de la esclavitud y Sonthonax, afirma la existencia de 1.000 páginas 15 de documentación y correspondencia con la metrópolis. Pese a ello, manuscritos tan importantes como el del 29 de agosto se encuentran en colecciones privadas 16. Es de suma importancia destacar que los testimonios relacionados con el delegado francés también se pueden encontrar en los procesos judiciales que se llevaron en su contra en las etapas del Directorio y del Imperio. En los años 80 R. L. Stein tuvo la oportunidad de profundizar en estos expedientes en su ensayo Léger-Félicité Sonthonax. The Lost Sentinel of the Republic (1985-1986). Señaló un conjunto de 6 volúmenes sobre el caso de Sonthonax y el Comité de Salud Pública. Esta tesis quedó obsoleta ante la aportación de Bénot, autor de La Révolution française et la fin des colonies (19881989), quien ha podido localizar un total de 9 volúmenes 17 y ha llegado a acusar al propio Stein de no proceder a la lectura de estos últimos 18. Asimismo, merece especial atención la única publicación específica de autoría española de los últimos años en torno a la figura de Sonthonax; Léger Félicité Sonthonax en A Coruña: affaire monetario y conflicto diplomático franco-español (1797-1798) (2020). F. Cebreiro analiza el incidente acontecido en A Coruña en diciembre de 1797, por el que la comisión francesa fue acusada por las autoridades locales de “fraude” monetario 19. Un hecho que, además de por su peculiaridad, resalta por la presencia del comisario francés en suelo español. Así, el trabajo de Cebreiro muestra los componentes de la expedición gala en tierras gallegas y algunos aspectos personales de los mismos. También, dentro del mundo hispanohablante, debemos señalar en relación con Sonthonax el trabajo de C. Esteban Deive Los Refugiados franceses en Santo Domingo (1984). Este autor aportó una significativa visión de los colonos sobre las actuaciones del objeto de estudio. Una vez señalada la bibliografía relevante y específica alrededor de las misiones de Sonthonax, es preciso destacar la historiografía tradicional y reciente relacionada con la esclavitud en Saint-Domingue, la República francesa y Napoleón Bonaparte. La nueva patria haitiana edificó su propia historia nacional en base a las obras de Marcel DORIGNY (dir.): Léger-Félicité Sonthonax. La première abolition de l'esclavage. La Révolution française et la Révolution de Saint-Domingue, París, S. F. H. O. M., 2005, pp. 176196. 16 Ibid 17 Débats entre les accusateurs et les accusés dans l’affaire des colonies (1795). 18 Yves BÉNOT: “Le procès Sonthonax ou les débats entre les accusateurs et les accuses dans l’affaire des colonies (an III)”, en Marcel DORIGNY (dir.): Léger-Félicité Sonthonax. La première abolition de l'esclavage. La Révolution française et la Révolution de Saint-Domingue, París, S. F. H. O. M., 2005, pp. 55-64. 19 Francisco CEBREIRO ARES: “Léger Félicité Sonthonax en A Coruña: affaire monetario y conflicto diplomático franco-español (1797-1798)”, Espacio, Tiempo y Forma, 33 (2020), pp. 187-211. 15 16 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS Raban (1825), Malo (1825), Madiou (1843) y Ardouin (1853-1860) entre muchos otros. En su conjunto ofrecían una nueva óptica del conflicto subrayando el papel principal de Louverture. Sin embargo, nuestro objeto de estudio, L. F. Sonthonax, queda desplazado a un segundo plano en un espacio bibliográfico cuyo interés era el desvelar la historia local. En el siglo XX destacan numerosos autores que se han interesado en la reinterpretación de los acontecimientos desde múltiples ópticas: biografías, naciones implicadas, etc. En la década de 1940 Ibarra y Rodríguez coordinó un tomo de la serie Historia del Mundo en la Edad Moderna en el que la temática de Haití apenas recibe unas cuantas páginas, en el capítulo Los estados dependientes de Francia, y Suiza 1800-1814. Quien sí incidió en mayor medida en una serie sobre la historia de América y el Caribe fue el autor de origen cubano J. Luciano Franco entre los 60 y los 70. Este último analizó la correspondencia entre la Corte de Carlos IV y el gobernador de Cuba. Es decir, lo novedoso de Luciano Franco es que profundiza en la visión española sobre la Revolución haitiana. Esto último no significa que no se hubiese hecho ya, sino que se había realizado en menor medida. Uno de los escritos más famosos sobre Dessalines y los rebeldes en castellano fue impreso en México en 1806. Vida de J. J. Dessalines, jefe de los negros de Santo Domingo es un ensayo que trabaja muchos factores de la Guerra de Independencia haitiana, de los cuales, solo unos pocos se han visto seleccionados para este ejercicio. Dubroca o Jean-Louis Dubroca fue un historiador, profesor y autor nativo de la región de Landas. Este erudito publicó más de 30 obras, varias de ellas relacionadas con el Imperio francés, la Guerra de Santo Domingo y la esclavitud. De este modo, entre 1804 y 1805, a raíz de la pérdida de la parte francesa de la isla y la coronación del emperador Dessalines, Dubroca publicó su breve historia y crítica al reciente Imperio haitiano. Anteriormente el autor ya había dedicado parte de sus trabajos a los líderes negros del Caribe francés. Este hecho se materializa con algunos ensayos como La Vie de Toussaint-Louverture, chef des noirs insurgés de Saint-Domingue (1802). En España contamos, además, con Gutiérrez Escudero, quien centró sus estudios en la estructura económica dominicana y haitiana en la etapa previa a la revuelta de 1791. Seguidamente, la tesis de Pinto Una colonia en la encrucijada: Santo Domingo, entre la revolución haitiana y la Reconquista española, 1791-1809 (2012) a pesar de centrar su área de estudio en la parte oriental de la isla, ofrece aspectos realmente interesantes sobre Sonthonax, Napoleón Bonaparte y el problema de la esclavitud. Otra aportación novedosa es el artículo de González-Ripoll (2015) donde se expone una nueva visión de la población negra y los conflictos en la isla a través de una serie de escritos redactados desde Saint-Domingue y Cuba que rozan la nóvela histórica realista de Galdós: la Secret history, or The horrors of St. Domingo de Mary Hassal. En lo referente al mundo hispanoparlante no debemos dejar de mencionar el manual de Gómez, Le spectre de la Révolution noire: l'impact de la Révolution BROCAR, 46 (2022) 11-42 17 JONATHAN JACOBO BAR SHUALI haïtienne dans le Monde atlantique, 1790-1886 (2013). Aquí, tendrá un espacio principal el traslado de información oral, escrita e iconográfica entre los mundos francófono e hispano; el éxodo de los propietarios promonárquicos, la imagen del nuevo estado en Europa, etc. También es pertinente señalar la investigación del dominicano E. Cordero La Revolución haitiana y Santo Domingo (1968), y los sobresalientes resúmenes de la cuestión dominico-haitiana de Moya Pons con su Historia del Caribe (2011) y la imagen que se trató de trasladar sobre los insurgentes en el mundo occidental a través de los resultados expuestos por Jáuregui en su artículo El "Negro Comegente": Terror, colonialismo y etno-política (2009). Para finalizar este apartado, es necesario señalar a los autores más prestigiosos que han estudiado el caso de la Revolución haitiana y la situación de la población de color en Saint-Domingue en lengua inglesa. Así, además de Popkin, a quien haremos mención más abajo, debemos destacar la obra de Buck-Morss Hegel y Haití. La dialéctica del amo-esclavo: una interpretación revolucionaria (2005), el trabajo de C. L. R. James Los jacobinos negros: Toussaint L'Ouverture y la Revolución de Haití (primera edición de 1938), y el no menos importante y más reciente estudio del historiador francés P. Girard, French atrocities during the Haitian War of Independence (2013). Este último distingue tres áreas temáticas principales en su estudio: las mujeres en la Guerra de Independencia haitiana, los crímenes de guerra y la sociedad. A estos estudiosos debemos sumar la labor de Geggus Haitian Revolutionary Studies (2002) y L. Dubois Avengers of the New World: The Story of the Haitian Revolution (2009). En otra línea, aunque se trata de una fuente decimonónica, si deseamos atender a las dificultades de la población esclava es sumamente interesante acudir al relato de M. Rainsford, An Historical Account of the Black Empire of Hayti (1805). Todo este amplio compendio lo podemos localizar en la última publicación de S. Hazareesingh, Black Spartacus (2020). Concluimos así este espacio, con el objeto de ceñirnos a nuestros intereses principales: las acciones de Sonthonax en la colonia y sus consecuencias en la capital francesa. 3. Saint-Domingue, intereses del Gobierno metropolitano El poder económico y los conflictos armados son dos conceptos que van ligados de la mano según los investigadores Gaxotte (1942) y Morazé (1980). SaintDomingue no fue ninguna excepción. En un marco realmente diverso y complejo, mulatos, blancos “realistas”, republicanos, y antiguos esclavos negros se integraron en las diferentes facciones de los enfrentamientos que se darían en la parte occidental de la isla entre 1791 y 1804: la Revolución y Guerra de Independencia haitiana. El resultado de estas contiendas estuvo marcado por el colapso de uno de 18 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS los sistemas económicos de mayor envergadura para el territorio francés y sus colonias, la mano de obra esclava 20. El ya citado historiador y librero francés, Dubroca 21, publicó una extensa narración, y crítica del Imperio haitiano. En su ensayo comprendemos el valor que la población francesa otorgó en los marcos económicos y culturales a su colonia. Así, podremos observar las siguientes descripciones: “Su clima es cálido, pero se templa mucho con los vientos y abundancia de lluvias que la hacen tan húmeda que todo se pudre en poco tiempo. Sin embargo, los europeos acostumbrados a este temperamento gozaban después de buena salud y solían contar, algunos, 80 y 100 años” 22. “Produce mucho azúcar, añil, tabaco, algodón, jengibre, café, té, ámbar y otras drogas medicinales. Riegan y fertilizan esta isla muchos ríos siendo los principales Ozama, Ayña, Nigua, Ville, Gas, Yane, Nissau y Ocoa de modo que apenas se hallará país tan beneficiado de aguas y de diferentes pescados en sus ríos” 23. “Tuvo en labor muchas minas de oro, plata, cobre, cristal y talco (…)”. A través de estos fragmentos se entiende, por lo tanto, la importancia y relevancia que ofrece el territorio haitiano en lo referente a ingenios azucareros, plantaciones de café, tabaco u algodón, además de diversos minerales de gran valor. Asimismo, estas materias suministraban un significativo porcentaje de la Hacienda francesa empleando más de un 40 % de sus puertos. Nantes, Burdeos o Marsella eran destinos asiduos de los productos importados por los grandes terratenientes de SaintDomingue. ¿Pero cómo eran las villas de Santo Domingo francés? ¿Se trataba de una sociedad “con esclavos” o “de esclavos” 24, tal y como señala Popkin? Este último aspecto es realmente innovador, ya que el investigador estadounidense referencia un concepto obvio que numerosos historiadores habían pasado por alto hasta la Antonio GUTIÉRREZ ESCUDERO: “Tabaco y Algodón en Santo Domingo”, en María Justina SARABIA VIEJO, Javier ORTIZ DE LA TABLA DUCASSE, Pablo E. PÉREZ-MALLAÍNA y José J. HERNÁNDEZ PALOMO (eds.): Entre Puebla de los Ángeles y Sevilla. Estudios Americanistas en homenaje al Dr. José Antonio Calderón Quijano, Sevilla, Facultad de Geografía e Historia. Departamento de Historia de América, 1997, pp. 151-169. 21 Véase BNF GALLICA DATA https://data.bnf.fr/fr/12524386/louis_dubroca/ 22 Jean-Louis DUBROCA: Vida de J. J. Dessalines, jefe de los negros de Santo Domingo, México, Oficina de Mariano de Zúñiga y Ontiveros, 1806, pp. 1-4. Todas las transcripciones han sido modernizadas y editadas por el autor de este trabajo a excepción de los topónimos. 23 Ibid 24 Jeremy D. POPKIN: A Concise History…, pp. 14-15. 20 BROCAR, 46 (2022) 11-42 19 JONATHAN JACOBO BAR SHUALI publicación de su trabajo en 2012. En esta línea, merecen especial atención los estudios de Finley sobre sociedades esclavas desde el Mundo Antiguo en los años 80 25. Lo cierto es que a lo largo de la monarquía de Luis XVI y el establecimiento del Consulado, Francia, dispuso de 800.000 esclavos en todo el Caribe. Una cifra considerable teniendo en cuenta que Buck-Morss ha localizado las siguientes cifras en Saint-Domingue: en 1715, 35.451 esclavos; en 1730, 79.545 esclavos; y en 1779, 249.098 esclavos. Esto significa que la población mulata y negra siempre fue mayor que la blanca en las diversas poblaciones de las Antillas y el Caribe francés. En esta línea, es preciso remarcar que el concepto “color”-de couleur era utilizado en referencia a mestizos y mulatos, en cambio “negros” para los que quedaban fuera del grupo anterior. Una de las grandes fuentes para el conocimiento de las cuentas alcanzadas en Saint-Domingue la podemos localizar en los escritos del funcionario del Gobierno colonial P. Albert de Lattre. Este administrador cifra un total de 793 ingenios 26 azucareros en las dos partes de la isla y hasta 200.000 esclavos 27 asentados en la parte occidental. Por otra parte, esta última cantidad contrasta con las indicadas actualmente por la gran mayoría de investigadores; alrededor de 300.000 esclavos 28. 4. Medidas para el control de Santo Domingo francés Los siglos XVI y XVII se vieron marcados por una amplia relación entre los tres grupos mayoritarios de la isla; “grandes” y “pequeños” blancos, población mulata y los esclavos negros. Los matrimonios entre estos espacios, tanto regulados como los que no lo estaban, eran muy frecuentes y la aparición de nuevos propietarios de tierras de color también. En el aspecto cultural 29, la región disponía de católicos, protestantes y judíos (estos últimos expulsados con el Código Negro) que también intervenían en estos vínculos. Sin embargo, en el siglo XVIII se observa un retroceso en los aspectos artísticos y sociales en cuanto a la aceptación de la población negra o de color. Este proceso, que no se dio únicamente en el Caribe francés, originó numerosos conflictos jurídicos y de convivencia que, de igual modo, afectaron a los José Miguel LECAROS SÁNCHEZ: El fenómeno de la esclavitud y del trabajo esclavo. Perspectiva histórico-jurídica e histórica, España, Caligrama, 2019, pp. 70-80. 26 Philippe Albert DE LATTRE: Campagnes des Français à Saint-Domingue et réfutation des reproches faits au capitaine-général Rochambeau, París, Locard, Arthus-Bertrand et Amand Koenig, 1805, pp. 103-214. 27 Ibid 28 El investigador Serge Barcellini indica una población de 30.000 blancos, 35.000 mulatos y negros libres y 450.000 esclavos en Santo Domingo francés en 1790. 29 En este trabajo no se habla del “vudú” como cultura y religión de gran influencia, sin embargo, se ha de tener en cuenta como uno de los múltiples factores que lleva a las revueltas esclavas de 1791. 25 20 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS criollos franceses y españoles. Solamente en la parte occidental se dieron 8 revueltas de esclavos, de las cuales 7 corresponden al período 1700-1787, consecuencia de la arbitrariedad hacia la población mulata libre y a los negros esclavos. Algunos dueños de plantaciones cometían todo tipo de excesos. Mucho antes, hechos similares llevaron al monarca Luis XIV al establecimiento del Code Noir en 1685. Gracias a esta legislación los propietarios no tenían permitido agredir a sus esclavos y estos podían llegar a denunciar cualquier tipo de abuso. Así, se eliminaron las normativas locales y se empezó un “proceso centralizador no armado” que aseguró los ingresos del 20 % de la nobleza francesa. Entre otros aspectos quedaron reguladas diversas cuestiones como el casamiento entre blancos y negros, el “afrancesamiento” y consideración de los derechos de los mestizos, etc. A pesar de ello, las dinámicas implantadas por el nuevo código tardaron en materializarse. Muchas de las normativas se vieron incumplidas por los terratenientes. Un ejemplo evidente de esta etapa en el caso español se da en las obras de Ginés Andrés de Aguirre 30, miembro de la Real Academia de San Fernando y autor de una veintena de “cartones” para tapices en El Pardo y El Escorial. En los óleos al lienzo La Puerta de Alcalá vista desde la Cibeles 31 (1785) y La Puerta de San Vicente32 (1785), hoy en el Museo de Historia de Madrid, destacan dos figuras de tez oscura. La primera de ellas, a la derecha de la Cibeles, entabla una conversación con otros tres individuos. La segunda figura, y posible mulato, se encuentra en el margen izquierdo del conjunto, en este caso con una mujer. En ambas situaciones los tapices desarrollados se encuentran en el Real Monasterio de San Lorenzo de El Escorial, en el sector denominado “Palacio de los Borbones”. El primero de ellos aparece hasta en dos ocasiones en esta ala del edificio, no obstante, el individuo de tez amarronada es representado con piel caucásica, “blanca”. En el tapiz de la Puerta de San Vicente, de la misma forma, ha disminuido el color pardo en la fisionomía del caballero. ¿Cabe la posibilidad de que se trate de dos mestizos o criollos? Al respecto del primer caso, se trata de un oficial de una unidad aún por determinar; en este sentido es interesante señalar que por su uniformidad solo podría pertenecer a un regimiento Escuela española (Museo del Prado). Aguirre, Ginés Andrés de Yecla, Murcia, 1727 - Ciudad de México, 1800. Recuperado de (https://www.museodelprado.es/coleccion/artista/aguirregines-andres-de/ff92169f-5db5-4eac-a195-c17651115576). [consultado 09/06/2021] 31 https://www.museodelprado.es/coleccion/obra-de-arte/la-puerta-de-alcala-vista-desde-lacibeles/fc543a5d-d011-49ac-ae0c-2090cdc93228 32 https://www.museodelprado.es/coleccion/obra-de-arte/la-puerta-de-san-vicente/6243690e63ab-41b6-a6ab-c3a247c4f0de 30 BROCAR, 46 (2022) 11-42 21 JONATHAN JACOBO BAR SHUALI ultramarino 33. En el Estado Militar de 1785 no hay constancia 34 de uniformes marrones con vueltas rojizas, la prenda vestida en la imagen. Finalmente, la única información relativa al representado en el segundo cartón es la siguiente descripción: “(…) y al lado de estos un caballero sentado con una señora en conversación” 35. En conclusión, somos testigos de un claro repudio 36 de la representación pictórica de dos mulatos. Esta propuesta no significa un rechazo total de la población negra o de color, María Antonieta, esposa de Luis XVI, tuvo un “hijo adoptivo” negro 37. Siguiendo el ejemplo del Código Negro, el Gobierno español desarrolló entre 1768 y 1784 un compendio de 3 regímenes jurídicos 38 para su población negra y las provincias ultramarinas; Santo Domingo 1768 39, Luisiana 1769 y “Carolino” 1784. El primer y el tercer código, publicados en la parte oriental de Santo Domingo, fueron elaborados bajo la dirección del Cabildo. El primero de ellos se compuso de 41 títulos bajo la revisión de D. Antonio Dávila y D. J. Campuzano 40. Al igual que en el caso francés, los españoles llevaron a cabo esta legislación para evitar el aumento de “cimarrones” 41, las trifulcas entre blancos y esclavos, el arbitrio de los amos, etc. Se ha valorado la opción de que se trate de un miembro de la Compañía de Guardabosques Reales, unidad creada en 1761. Con la ayuda de diversos “uniformólogos”, se ha llegado a la conclusión de la imposibilidad de dicha afirmación por las prendas y colores que conforman su uniforme. Para profundizar en esta cuestión véase Enrique MARTÍNEZ RUIZ y Magdalena DE PAZZIS PI CORRALES: “Los guardabosques reales: inicio de su funcionamiento y la dotación de equipo”, Espacio, Tiempo y Forma, S. IV-T.7 (1994), pp. 447-466. 34 M. GÓMEZ RUIZ y V. ALONSO JUANOLA: Uniformes militares del Ejército de Carlos III / selección iconográfica, Madrid, Ministerio de Defensa, 1993, pp. 149-252. 35 Las puertas de Madrid (Museo de Historia de Madrid). La Puerta de San Vicente. Recuperado de (https://www.madrid.es/UnidadesDescentralizadas/MuseosMunicipales/MuseoDeHistoriaDe Madrid/EspecialInformativo/La%20pieza%20del%20mes/puerta%20de%20san%20vicente_ WEB.pdf). [consultado 04/06/2021] 36 Antonio Jesús PINTO TORTOSA: “Una frontera Definida por el color de la Piel…”, pp. 5462. Mar GALLEGO-DURÁN: “El racismo científico del siglo XVIII y las estrategias de autorepresentación: La narrativa interesante de Olaudah Equiano”, Estudios Ingleses de la Universidad Complutense, 19 (2011), pp. 71-97. Eduardo BITLLOCH: “Ciencia, Raza y Racismo en el Siglo XVIII”, Ciencia hoy, 33 (1996), pp. 47-58. 37 Jean Amilcar falleció por inanición en el transcurso de la Revolución francesa. 38 Manuel LUCENA SALMORAL: Leyes para esclavos. El ordenamiento jurídico sobre la condición, tratamiento, defensa y represión de los esclavos en las colonias de la América española, España, Fundación Ignacio Larramendi, 2000, pp. 531-1195, 294. 39 Se redactó otro documento en 1783. 40 Manuel LUCENA SALMORAL: Leyes para esclavos…, pp. 531-1195. 41 Entiéndase esclavos fugados de las plantaciones. 33 22 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS El 10 de marzo 42 de 1790 la Asamblea Nacional francesa otorgó el derecho de representación a sus colonias dentro de la Asamblea Constituyente, sin embargo, la política de centralización se intensificó desembocando así en la limitación de los organismos coloniales. Todos los cambios en las instituciones locales debían ser ratificados por el Gobierno central. El 14 de agosto de 1791 los esclavos rebeldes llevaron a cabo la ceremonia de Bois-Caïman. Tras ello, el día 16 43 los colonos tenían noticias de asaltos y ataques masivos a numerosas propiedades. El 13 de septiembre del mismo año la revuelta ya contaba con 10.000 hombres. Entre tanto, para calmar la situación que se daba en la isla, zarpó de la costa francesa la primera comisión diplomática compuesta por Mirbeck, Roume y Saint-Léger. Los esfuerzos “pacificadores” de estos diplomáticos resultaron del todo ineficaces. A pesar de la autoridad de la que disponían, gran parte de la población blanca se había agrupado en torno a la Asamblea General de Cap 44, por ello, todas las medidas elaboradas por los primeros tenían un efecto nulo sobre los colonos. Estas actuaciones por parte de los pobladores blancos son consecuencia de las medidas del 15 45 y del 16 de mayo de 1791. Según estas, los hijos varones, y mayores de edad, de negros y mulatos libres tenían la posibilidad de acceder al voto restringido. Asimismo, los esclavos que dispusieran de una considerable cuantía podían comprar su libertad. El Gobierno francés había promulgado diversos decretos que mejoraban la situación de los antiguos esclavos, no obstante, fue preciso poner en marcha la preparación de una segunda comisión que pudiera controlar los ataques de negros, blancos y mulatos. La “reconquista” o recuperación del poder directo sobre Saint-Domingue se ha solido asociar a una idea originaria de la etapa del Consulado. Pero disponemos de testimonios que nos confirman la concepción de un “imperio colonial” por parte de la población francesa a principios del período republicano: “Sin duda, señores, habrán sido instruidos sobre las vergonzosas maniobras empleadas en esta colonia por los enemigos del Imperio francés, para seducir a las tropas (...)". El testimonio proviene del capitán de artillería destinado en las colonias M. Levavasseur, en el acta tomada del proceso verbal número 498. El oficial jura por su regimiento lealtad a la asamblea, y de igual modo, exige que se solventen los b4042557 John Carter Brown Library (proclamationduro02fran). Proclamation du Roi, sur le décret de l'Assemblée nationale, concernant les colonies (…). Recuperado de (https://jcb.lunaimaging.com/luna/servlet/s/3s98h6). [consultado 1/06/2021] 43 Édouard BRYAND: Histoire de ST.-Domingue depuis 1789 jusqu’en 1794, París, Libraire Pierre Blanchard, 1812, pp. 210-234. 44 Ibid 45 Popkin señala la corta duración del Decreto del 15 de mayo de 1791. El 24 de septiembre del mismo año se derogó. Jeremy D. POPKIN: A Concise History…, pp. 34-36. 42 BROCAR, 46 (2022) 11-42 23 JONATHAN JACOBO BAR SHUALI impagos en las restantes páginas del documento. Este no es el único caso en el que se observa el término “Imperio francés” para hacer referencia a las posesiones ultramarinas. De hecho, en las numerosas actas recopiladas de diferentes asambleas comerciales en puertos tan importantes como Burdeos o Nantes localizamos la utilización de estos términos entre 1790 y 1791: “(…) de ces deux décrets; elle ne pourra pas vouloir la petre de l’Empire par celle de son comerce”. El ciudadano y comerciante de Santo Domingo francés, M. Brard, usó estos mismos términos para indicar el comercio y “extrarradio marino” delante de la Cámara de Comercio de Burdeos el 23 de agosto de 1791. Por ello, la concepción de un proceso centralizador colonial ya existía en la capital francesa desde comienzos de la Revolución, por lo que no resulta extraño que, en 1790, la Asamblea Nacional utilizase el “imperio” 46 para referirse a sus posesiones o que los regimientos de guardias nacionales lo reflejaran así en sus estandartes. Figura 1. Libres bajo un rey ciudadano. Bandera del Batallón Mantos Blancos de la Guardia Nacional de París en 1789. s. a.: Drapeaux de la Garde Nationale de Paris en 1789, París, s. e., 1791-1817. Bajo los adornos superiores figura un navío que conlleva un doble significado: el escudo de la ciudad de París y el poderío comercial, y naval, del Reino de Francia. 46 24 Decreto del 8 de marzo de 1790. BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS 5. Las reformas del comisario Sonthonax, la rebelión de Galbaud y los esclavos Sonthonax es recordado hoy como uno de los grandes precursores de la abolición de la esclavitud. La siguiente, si no se tiene en cuenta la de Napoleón I en 1815, no se daría hasta 1848. Por otra parte, la idea de que el diplomático francés llevase a cabo esta proclama bajo intenciones personales ha sido debatida por numerosos investigadores. Para responder a esta incógnita trataremos en las siguientes líneas la formación de L. F. Sonthonax hasta su nombramiento como comisario de la República francesa en Saint-Domingue. Léger-Félicité Sonthonax nació en Oyonnax en marzo de 1763. La villa en 1786 superaba por poco las 1.000 almas. En la localidad se daba una importante producción artesanal de la que formó parte su propia familia 47. Sonthonax se encontró sujeto a la pequeña burguesía empobrecida que, en la región en la que se ubicaba, era considerada como parte de la élite. Obtuvo su educación elemental en el Colegio de la Orden de Joseph Crétenet en Nantua. En 1781 comenzó sus estudios en derecho por la Universidad de Dijon. En esta etapa tiene su primer contacto “teórico” con el esclavismo. Estudió diversos casos del derecho romano y, a través del Journal de Lyon 48, podrá acceder a los ideales de Franklin. Con el profesorado debate sobre la servidumbre en la antigua Roma. Obtiene su licenciatura el 17 de julio de 1784, tras lo que inicia una estancia en París. Es en este período cuando conoce al abolicionista Brissot, en 1785. Jacques Pierre Brissot de Warville fundó la Sociedad de Amigos de los Negros en febrero 49 de 1788, tres años después de la llegada de Sonthonax a la capital y momento en el que, este último, se encontraba ejerciendo la abogacía en el Parlamento de París. No hay constancia documental entre 1788 y 1790 de una adhesión de L. F. Sonthonax a la sociedad de Brissot 50. De otro modo, podemos observar, la etapa comprendida entre 1791-1799 donde sí disponemos de testimonios que documentan las actividades del objeto de estudio como miembro y “corresponsal” de la misma entidad. Étienne Polverel, futuro miembro de la segunda comisión del Gobierno francés en la colonia, entabla su amistad con Sonthonax en el ejercicio de su cargo como representante de la organización en 1791 51. Los orígenes de la familia se han ubicado en el S. XVII. Louis TRÉNARD: “Oyonnax au temps de Sonthonax”, Revue française d'histoire d'outremer, 316 (1997), pp. 9-21. 49 Refundada en 1796 bajo el nombre de Sociedad de Amigos de los Negros y las Colonias. 50 Robert Louis STEIN: Léger-Félicité Sonthonax. The Lost Sentinel of the Republic, Londres/Toronto, Associated Univ. Presses, 1985, pp. 88-100. 51 Marcel DORIGNY: “Sonthonax et Brissot: Le cheminement d’une filiation politique assume”, en Marcel DORIGNY (dir.): Léger-Félicité Sonthonax. La première abolition de l’esclavage. La Révolution française et la Révolution de Saint-Domingue, París, S. F. H. O. M., 2005, pp. 29-40. 47 48 BROCAR, 46 (2022) 11-42 25 JONATHAN JACOBO BAR SHUALI En septiembre de 1792 desembarcó en Cabo francés una comisión llegada desde Francia. Con una amplia enemistad por parte de los “grandes” y “pequeños” blancos. Tenía la tarea de implantar el Decreto del 4 de abril de 1792 52: limpiar las instituciones y eliminar el clima de cruentos combates y asesinatos. Con Sonthonax alcanzaron la isla 6.000 soldados con su jefe, d’Esparbès, además del ya mencionado Polverel y el comisario Ailhaud 53. Jean-Jacques d’Esparbès ejerció como nuevo gobernador de la parte occidental de la isla a la vez que los comisarios daban lugar al “proceso depurador”. Uno de los hechos de mayor significancia fue el nombramiento de una delegación intermediaria formada por 6 blancos y 6 negros y mulatos que debía sustituir a las asambleas locales previas. En octubre de 1792, Sonthonax trasladó estas medidas a los cuerpos militares. El 11 de octubre 54 el diplomático realizó una proclama a los veteranos de Santo Domingo y a la Guardia Nacional. Las viejas milicias quedaban desintegradas en su totalidad y sus componentes se debían unir a los batallones de la Guardia Nacional. Así, la comisión francesa recalcó la labor patriótica de las guardias y agradecía sus servicios, además de regular las vacantes a la oficialidad y el uso privado del armamento. Unos días después, el Gobierno expedicionario de los comisarios autorizó la entrada de oficiales mulatos en el antiguo Regimiento de Cap. En consecuencia, el 19 de octubre 55 los propietarios blancos y su servidumbre se alzaron en armas, amenazando así la estabilidad de la misión pacificadora. La ciudad quedó devastada, pero gracias al apoyo de los “libertos” se pudo contener la sublevación. Ante la búsqueda de la tranquilidad, a finales de octubre de 1792, los comisionados dieron noticia a “los hombres libres de la patria francesa de Santo Domingo a todos los voluntarios nacionales, soldados de la Guardia Nacional, tropas de línea y empleados en la expedición” del nombramiento del vizconde de Rochambeau 56 como gobernador de Saint-Domingue. D’Esparbès, quien mantenía relaciones con los monárquicos, fue destituido de inmediato. A la inversa de los Jeremy D. POPKIN: A Concise History…, pp. 34-40. Alexis Camille Kimou ATSÈ: La rebelión de Galbaud y la libertad general de los esclavos de Saint-Domingue (1793-1794). Nuevo Mundo Mundos Nuevos [En línea], Debates, (2014). https://doi.org/10.4000/nuevomundo.66356. Ailhaud regresó a Francia a principios de 1793. Su sustituto debía ser M. Delpech. José LUCIANO FRANCO: Historia de la Revolución…, pp. 232-234. 54 Proclamación del 11 de octubre de 1792, se prorroga en Saint-Domingue la misión de salvaguardar el bienestar del Gobierno por parte de la Guardia Nacional. 55 VV. AA.: Libres après les abolitions? Statuts et identités aux Amériques et en Afrique, Bordeaux, Karthala et CIRESC, 2019, pp. 22-23. William S. CORMACK: Patriots, Royalists, and Terrorists in the West Indies: The French Revolution in Martinique and Guadeloupe, 17891802, U. S. A., University of Toronto Press, 2019, p. 313. 56 Ejerció el cargo entre el 21 de octubre de 1792 y el 13 de enero de 1793, Santo Domingo francés nunca fue su destino original. 52 53 26 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS resultados que se esperaban de la comisión en Santo Domingo francés, en 1793, los propietarios de esclavos habían adquirido un considerable potencial militar. En febrero de 1793 se nombró gobernador general de la parte occidental a François-Thomas Galbaud du Fort 57; sin embargo, el militar era favorable al mantenimiento de la esclavitud. El 6 de mayo de 1793 alcanzó la isla con un considerable contingente de tropas. Del 19 al 22 de junio se llevó a cabo en la villa de Cap el conocido proceso como “la traición de Galbaud” 58. Desde finales de mayo los esclavos de la zona “alta” de Cabo se habían alzado pues no confiaban en el general francés. Shonthonax y Polverel debían enfrentarse a tropas regulares procedentes de la parte oriental de Santo Domingo, numerosos grupos de esclavos armados, los grandes propietarios y militares profesionales llegados de la metrópolis con Galbaud du Fort 59. Entre el 20 y el 21 de junio de 1793 60 los esclavistas de Le Cap (Cabo) se unieron a los 2.000 soldados del gobernador. Prácticamente derrotadas, las tropas republicanas solicitaron el auxilio de las bandas de cimarrones 61. Se realizó un llamamiento por parte de los comisarios que fue correspondido por los jefes negros Pierrot y Macaya quienes ayudaron a liberar la ciudad con otros 2.000 hombres 62 en favor del Gobierno de la República. El día 23 la villa se encontraba liberada, a pesar de ello era pasto de las llamas. Al igual que en el comunicado del 11 de junio, la llamada ofreció a los esclavos que lucharan por la República la libertad total. Los hombres de Galbaud y muchos de los colonos de la localidad escaparon de las costas de Haití escogiendo como destino Cuba, la parte española de Santo Domingo, Jamaica o los Estados Unidos. A comienzos de julio los colaboradores realistas eran duramente reprimidos en Cabo. Este aspecto, sumado a la reciente huida, provocó un exilio por parte de la población blanca. En este no participó exclusivamente el “gran propietario”, se trataba, como hemos señalado a lo largo de este trabajo, de grandes y pequeños blancos, muchos acompañados por sus sirvientes de color. Artesanos, nobles, pequeña burguesía, militares, etc. Todos los estratos sociales quedaron afectados por esta fuga “masiva” que se venía dando desde los primeros combates en 1791. A continuación, se adjunta un ejemplo de diferentes casos y condiciones sociales de los exiliados de 1793: Es preciso señalar que él mismo era propietario de diversas plantaciones. Jeremy D. POPKIN: Facing Racial Revolution: Eyewitness Accounts of the Haitian Insurrection, U. S. A., The University of Chicago Press, 2007, p. 180. 59 Édouard BRYAND: Histoire de ST.-Domingue…, pp. 147-168. 60 Ibid 61 Ibid 62 Jeremy D. POPKIN: A Concise History…, pp. 57-58. 57 58 BROCAR, 46 (2022) 11-42 27 JONATHAN JACOBO BAR SHUALI Chitry Barón de Roux Saint-Robert Blandinier Hermanos Duval Conde de Mesme Capitán de infantería Noble Teniente de la gendarmería nativa Propietario de tierras y comerciante Propietarios de tierras Noble y oficial de la Marina Figura 2. Algunos de los exiliados más relevantes en 1793. Elaboración propia en base a los casos recopilados por Carlos Esteban Deive en Los Refugiados Franceses en Santo Domingo (1984). Los individuos aquí reflejados nos indican la disparidad entre los diferentes afectados. Figura 3. (Izda.) François-Thomas Galbaud du Fort. Anónimo, circa 1800. Dominio público. El Museo del Imperio, o Salón de Provenza, dispone de un óleo muy similar de Johann Friedrich Dryander titulado Retrato de un oficial (1795) (Dcha.). ¿Podría tratarse de dos versiones de un mismo trabajo? Así, en el verano de 1793, Sonthonax dedicó las siguientes líneas a lo acontecido: “El pueblo de Santo Domingo, doblegado durante tanto tiempo bajo el yugo de la esclavitud y la tiranía, acaba de recuperar su lugar entre las naciones del mundo y, como el pueblo francés, ha reconquistado sus derechos. Este nuevo orden de las cosas, que debe ser obra del tiempo y efecto de la luz (la verdad) (…) es aquella donde gobierna el frenético 28 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS Galbaud (…), enviado como gobernador a Saint-Domingue, para comenzar destruyendo la ley del 4 de abril” 63. La fuerte presencia de antiguos esclavos y el enfrentamiento con los ejércitos monárquicos, llevó a los diplomáticos a proclamar la libertad de los primeros los días 20, 21 y 22 de junio de 1793. De esta manera, entre el 26 y el 27 de agosto, Polverel ofreció la libertad a los trabajadores que volvieran a sus plantaciones originales en el oeste. Finalmente, ante tales precedentes, Sonthonax se vio en la “obligación” de proclamar la emancipación total el 29 de agosto de 1793. El 24 de septiembre de 1792 64 los tres comisarios publicaron en Cap “en nombre de la nación, de la ley y del rey” un texto indicativo y un conjunto de cinco artículos por los que señalaban los objetivos de la misión diplomática en Saint-Domingue. Entre estas líneas cabe destacar la siguiente aclaración: “Nosotros declaramos que la esclavitud es necesaria para el cultivo y la prosperidad de las colonias, y que no se encuentra dentro de los principios, ni dentro de la voluntad de la Asamblea Nacional y del rey, el tocar en este sentido las prerrogativas de los colonos” 65. Asimismo, todas las proclamas y decretos que se han mostrado a lo largo de este trabajo tuvieron un efecto y visión en la parte española de la isla. Es de especial relevancia mencionar la carta de D. Joaquín García, “mariscal de campo de los Reales Ejércitos” en la parte oriental de Santo Domingo, a “los comandantes generales y demás jefes de nuestras tropas acantonadas en la parte del norte, sur y oeste”: “Los generales de los voluntarios negros libres 66 de nuestros ejércitos me han dirigido diversos ejemplares impresos de dos proclamaciones que se han promulgado a nombre de los que se dicen comisarios civiles de la B3903505 John Carter Brown Library (Sonthonaxcommission00sont). (…) A la société des amis de la liberté (…). Recuperado de (jcb.lunaimaging.com). [consultado 12/06/2021] 64 b4933528 John Carter Brown Library (proclamationauno4158sain). Proclamation.: Au nom de la Nation, de la loi et du Roi. Etienne Polverel, Jean-Antoine Ailhaud & Léger-Félicité Sonthonax, commissaries nationaux-civils, délégués aux Îles françaises (…). Recuperado de (https://jcb.lunaimaging.com/luna/servlet/s/t648lk). [consultado 16/06/2021]. El 21 se decretó la caída de la Monarquía francesa en la capital y, estrictamente, este es el momento en el que da comienzo el gobierno efectivo de la Primera República francesa. Desde agosto de 1792 la familia real se encontraba recluida bajo vigilancia. 65 Ibid. 66 Toussaint Louverture y otros jefes nativos lucharon a favor del Gobierno español en la primera etapa de la Guerra del Rosellón. Jorge VICTORIA OJEDA: “Un líder de la revolución haitiana en Cádiz”, Trocadero, 18 (2006), pp. 191-202. 63 BROCAR, 46 (2022) 11-42 29 JONATHAN JACOBO BAR SHUALI Francia, y han esparcido con ánimo de seducir sus ánimos (principios), y los de sus soldados manifestándome al propio tiempo la justa indignación que ha causado no solo en ellos y en sus oficiales, sino a todos los bravos soldados semejante insolente tentativa, y los falsos, y malvados medios de que estos inicuos se valen; y que sobre todo no pueden oír sin la mayor irritación su mordacidad, y desenvoltura intentando vulnerar, y ofender al mayor, y mejor rey de la tierra, a mi amado bienhechor el señor don Carlos 4.º nuestro rey, y señor a quien todas las naciones deben (…)” 67. 6. Léger-Félicité Sonthonax, el Comité de Salud Pública y la Comisión Colonial El 16 de julio de 1793, al otro lado del Atlántico y totalmente desconocedor de este hecho, Sonthonax fue acusado por los colonos exiliados como cómplice de Brissot y traidor a los objetivos que le habían sido señalados por el Gobierno republicano. Es preciso recordar que la propia comisión se había comprometido a no derogar la esclavitud el día 24 de septiembre de 1792. Muchos han sido los debates y trabajos realizados alrededor de la emancipación del 29 de agosto; aún hoy, los historiadores permanecen divididos al respecto de los intereses de Léger-Félicité Sonthonax. Por un lado, la abolición benefició a las tropas republicanas. Permitió hacer frente al contingente español y más adelante al inglés 68. Así, podemos comprender este proceso como una “pacificación con motivos bélicos” 69. Asimismo, respondía a uno de los objetivos de la Sociedad de Amigos de los Negros: la liberación total de los esclavos. Los pactos del girondino Brissot y el sector jacobino en la Asamblea Legislativa, en 1792, llevaron a Sonthonax a su posición en la comisión francesa destinada en Saint-Domingue. Sin embargo, no alcanzaría su destino sino gracias al mulato Julien Raimond, quien decantaría la balanza a favor del antiguo abogado parisino. Siguiendo esta misma línea, Brissot haría llegar con anterioridad a L. F. Sonthonax unos manuscritos secretos en los que se le indicó los objetivos reales de su misión; la libertad de todos los negros de la isla. Este fue el argumento aportado ante la SGU, LEG. 7151, 18. Archivo General de Simancas (Libertad de esclavos). Dos proclamaciones para los blancos y negros de la vecina colonia para rebatir las proposiciones contenidas en las que mandó publicar el comisario Sonthonax. Recuperado de internet (http://pares.mcu.es/ParesBusquedas20/catalogo/description/1303629). [consultado 10/05/2021] 68 Édouard BRYAND: Histoire de ST.-Domingue…, pp. 150-173. 69 Para profundizar en los beneficios de la “paz” en favor de una autoridad gubernamental o un ejército véase Mark Neocleous (2016). Jean-René Aymes trató un caso similar en la Guerra de Independencia española en su estudio Soult en Andalucía y Suchet en Zaragoza y Valencia: Dos métodos de pacificación diferenciados (2010). 67 30 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS Asamblea y la Convención por gran parte de propietarios de tierras, mulatos y diversos oficiales exiliados 70. En julio de 1793 el diputado conservador Bréard afirmó en la Convención “(…) la tiranía realista ejercida por Sonthonax y Polverel en St. Domingo” 71. Este será seguido por el ciudadano Saint-André que el 3 de septiembre del mismo año pronunciará las siguientes palabras: “(…) ellos han perdido nuestras colonias. Brissot, Pétion, Guadet, Gensonné, Vergniaud, Ducos, Fonfrède, han dirigido las operaciones relativas a nuestras colonias, (y ahora) nuestras colonias se reducen a la situación más espantosa. Los comisarios son culpables de haberlas trastornado de arriba abajo, Sonthonax y Polverel, es obra suya y son cómplices” 72. En conclusión, lo que se exigió al Gobierno francés fue un acta oficial de acusación en contra de los diplomáticos, y su vuelta a territorio nacional para ser juzgados por el Comité de Salud Pública. En 1794 73 los comisarios fueron notificados de sus nuevas obligaciones por un capitán de la Marina; este último les condujo a su nuevo destino. Desembarcaron en Rochefort, de donde fueron llevados a París, lugar en el que serían puestos bajo arresto hasta el 4 de agosto del mismo año. El 30 de enero de 1795 se pusieron en marcha los interrogatorios de la “Comisión de las Colonias” 74 cuyo fin era solventar la incertidumbre alrededor del 29 de agosto de 1793, analizar los citados actos de traición y la mediación entre los colonos y los diplomáticos. Sin embargo, Polverel falleció a consecuencia de las humedades caribeñas en París el 6 de abril de 1795 75. A pesar de la causa, el general francés y exgobernador de Saint-Domingue, Galbaud, consciente de la puesta en libertad de los dos comisarios, hizo llegar una queja formal al “Cuerpo Legislativo” en la que, de igual modo, él mismo exigía los derechos más fundamentales para su familia retenida a la fuerza en su domicilio: “Representantes; Polverel y Sonthonax han sido acusados después de más de un año, y ¡ellos se encuentran en libertad en París! Acusado por Dufay, su criatura y su cómplice, fui arrestado en el momento de mi desembarco, y sin haber Yves BÉNOT: “Le procès Sonthonax ou les débats…”, pp. 55-64. Ibid 72 A. SAINT-ANDRÉ: Acte d’accusation contre plusieurs members de la Convention nationale présenté au nom du Comité de Sûreté Générale par André Amar, membre de ce Comité, París, s. e., 1793, pp. 40-50. 73 Abandonan la isla en junio de 1794. 74 Yves BÉNOT: “Le procès Sonthonax ou les débats…”, pp. 55-64. La comisión y su funcionamiento cesó en agosto de 1795. Se formó con los señores: Garran, Marec, Guyomard, Grégoire, Thibaudeau, Fouché, Mazade, Castillon, Dabray, Lanthenas, Merlino, Grégoire y Mollevaut. Solo los ocho primeros formaban parte del comité original. 75 Véase https://data.bnf.fr/fr/12997859/etienne_de_polverel/ 70 71 BROCAR, 46 (2022) 11-42 31 JONATHAN JACOBO BAR SHUALI sido escuchado; me confundieron con los conspiradores y los traidores. Legisladores, no es el momento de examinar por qué Polverel y Sonthonax no cumplieron con el decreto lanzado en su contra ocho meses después de haber tenido conocimiento de este. (…) Ciudadanos, el legislador (el Cuerpo Legislativo) debe ser impasible al igual que la ley similar a la justicia, debe tener su venda en los ojos y su balanza. Son estos los dos hechos que yo reclamo: I.º La libertad de (…) mi esposa y mis hijos detenidos en Nantes por orden del representante Garroz(s)” 76. Por su parte es preciso destacar la respuesta de Sonthonax, el 8 de enero de 1795, al miembro del Comité de Salud Pública, M. Pelet, quien de nuevo lo había culpado del incendio de Cap. Se ha de tener en cuenta que los ataques fueron continuos desde el propio verano de 1793 y rara vez pudo el comisario ser consciente de estos o contestarlos hasta su regreso a Francia. “Fui comisario civil, los colonos me denuncian como uno de los autores de los movimientos sediciosos que han agitado la colonia de SantoDomingo francés, es este el momento en el que me veo calumniado (por ellos) (…) es cuando lucho contra mis enemigos que la terrible verdad penetrará (…) sin embargo es un miembro del gobierno, Pelet, el que insinúa prejuicios a mis jueces, y engaña a la opinión pública” 77. Podemos ser testigos de cómo el caso de Sonthonax se hizo eco en la ciudadanía parisina y la burguesía francesa, tan dependiente de la mano de obra esclava. Finalmente, Léger-Félicité Sonthonax pudo salir indemne del examen al que fue sometido entre los meses de enero y agosto de 1795. 7. Léger-Félicité Sonthonax y la Francia de Napoleón Bonaparte Siendo el objetivo principal del presente ensayo arrojar luz en la cuestión de la misión de Sonthonax e incidir en su figura, se ha procedido a la traducción de fuentes desconocidas en lengua castellana en torno al diplomático francés en el transcurso de los apartados vistos hasta ahora. Habiendo aclarado aspectos básicos sobre el 29 François-Thomas GALBAUD DU FORT: A la Convention nationale. De la maison d’arrêt de Lazare, l’an deuxième de la République une et indivisible, Francia, s. e., 1793-1795, pp. 2-4. 77 b3903497 John Carter Brown Library (lfsonthonaxaurep00sont). L.F. Sonthonax, au representant du peuple Pelet (de la Lozère), membre du Comité de salut ... Recuperado de (https://jcb.lunaimaging.com/luna/servlet/s/r0kx42). [consultado 17/06/2021] 76 32 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS de agosto de 1793, creemos que no es necesario incidir en mayor medida en el período 1791-1799 78. Así, en las siguientes líneas se procederá, brevemente, a tratar las consecuencias de la primera comisión de Sonthonax en Saint-Domingue, sus efectos en la política francesa y los barrios obreros del París de 1800. En 1797 Sonthonax abandonó por segunda vez Santo Domingo francés. A su marcha, el nuevo eje del poder político giró alrededor del general Toussaint Louverture. El 22 de agosto 79 del mismo año algunos generales al servicio del Gobierno francés como Christophe y Moïse solicitaron al comisario que ejerciese de diputado en favor de sus intereses frente a la República. Asimismo, antes de su marcha, Sonthonax delegó amplios poderes en la personalidad de Toussaint, hecho que disgustó a Napoleón Bonaparte tras el establecimiento del Consulado en 1799 80. En Saint-Domingue el exesclavo y general Breda, Toussaint Louverture, llevó a cabo una serie de políticas en nombre de la República, y en pro de una próspera comunicación administrativa con la capital francesa. Deseaba de este modo mostrar sus dotes como gobernante y la posibilidad de establecer un gobierno autónomo local 81. La isla tenía una sólida estructura basada en una aristocracia dueña de grandes haciendas y numerosos esclavos que, a su vez, se veía obligada a comerciar únicamente con su metrópolis. Napoleón simpatizó con los principios conservadores implantados por Louverture. No obstante, para el primero, el hecho de que las costas de Haití comerciasen con Norteamérica e Inglaterra supuso un problema. En estas transacciones el Ejército local se nutría de mosquetes a la par que ingleses y norteamericanos obtenían considerables beneficios azucareros. Por ello, Napoleón presentó un nuevo modelo de centralización colonial que fue apoyado por oficiales propietarios de tierras, mulatos conservadores, exiliados y miembros del Club Massiac 82. Así, en agosto de 1797, Léger-Félicité Sonthonax, nuevo representante de Santo Domingo francés en el Directorio, partió rumbo a las costas gallegas y, finalmente, alcanzó A Coruña en noviembre 83. Desde su partida de Saint-Domingue tardó 5 meses en llegar a su destino final: París. Ya en la capital en 1798, Sonthonax participó en el Consejo de los Quinientos cumpliendo la promesa que había hecho tiempo atrás a los generales de Louverture bajo los ideales de Brissot. Desde ese Alrededor de este marco cronológico disponemos en castellano de los estudios de Luciano Franco (1966), Buck-Morss (2005) y James (1938). Ninguno de los anteriores dedica el conjunto de sus escritos a Sonthonax, únicamente es mencionado en diferentes apartados. 79 Justo ZARAGOZA: Las insurrecciones en Cuba, apuntes para la historia política de esta isla en el presente siglo, Madrid, Impr. de Manuel G. Hernández, 1872, p. 72. 80 Ibid 81 Susan BUCK-MORSS: Hegel y Haití. La dialéctica del amo-esclavo: una interpretación revolucionaria, Argentina, Grupo Editorial Norma, 2005, pp. 100-300. 82 José LUCIANO FRANCO: Historia de la Revolución…, pp. 200-202. 83 Francisco CEBREIRO ARES: “Léger Félicité Sonthonax en A Coruña…”, pp. 187-211. 78 BROCAR, 46 (2022) 11-42 33 JONATHAN JACOBO BAR SHUALI momento el excomisario francés decide extender los derechos más básicos que defendió en la colonia en territorio nacional. Acude a los barrios obreros, suburbios de San Antonio en París, y narra los hechos acontecidos entre 1792 y 1797 en las posesiones ultramarinas francesas. Encuentra en los trabajadores parisinos a los nuevos “ciudadanos de color” que deben ser rescatados 84. Es preciso recordar que hasta la plena llegada del Primer Imperio francés los obreros habían quedado enmarcados en un segundo plano. Desde la época republicana, militares y hacendados de la talla de Vincent-Marie Viénot de Vaublanc habían mostrado su claro rechazo hacia los trabajadores. Figura 4. Portrait du commissaire Sonthonax. Anónimo, circa 1799-1803. Musée du Peigne et de la Plasturgie, Oyonnax. Dominio público. A su derecha (costado izquierdo del óleo) el comisario civil sostiene la emancipación del 29 de agosto de 1793. Solo en 1794 fue ratificada por el Gobierno francés. Presa de enemigos realistas en la Vendée y terroristas jacobinos en París 85, Napoleón Bonaparte llevó a cabo una ardua tarea policial frente a sus adversarios políticos 86. Consecuencia de esta última fue el arresto e interrogatorio del diputado Yves BÉNOT: “Pourquoi Bonaparte a-t-il exilé Sonthonax? ou: Dix ans après...”, Annales historiques de la Révolution française, 293-294 (1993), pp. 522-525. 85 En este sentido es interesante analizar los diversos atentados organizados para intentar eliminar al futuro emperador francés en la obra de David Chanteranne: Les douze morts de Napoléon (2021). 86 Napoleón aprendió a controlar a la policía a raíz de los levantamientos de abril, mayo y octubre de 1795. El 3 de octubre se da la sublevación realista del barrio de Le Peletier (París). 84 34 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS francés en 1799. Sonthonax permaneció temporalmente aislado y arrestado en la Rochelle; de hecho, el Gobierno consular pretendía exiliarlo a la Guayana. Esta situación se mantuvo así hasta que Joseph Fouché, antiguo miembro de la Comisión de las Colonias, abogó por su antiguo compañero de militancia. Veterano entre los jacobinos, Fouché se había posicionado a favor de Sonthonax en los juicios de 1795. A partir de este momento el futuro ministro de la policía siempre veló por el excomisario. Los atentados del 24 de diciembre de 1800 y de enero de 1801 hicieron pensar 87 al primer cónsul que se trataba de una estratagema jacobina. Es por ello por lo que se procedió al arresto de numerosos representantes de corte jacobino entre los que se encontraba el propio Sonthonax 88. A pesar de ser liberados muchos de ellos, otros, como se da en el caso de nuestro objeto de estudio, permanecieron en el punto de mira de Napoleón. En 1803 el Gobierno francés forzó el exilio de Léger-Félicité Sonthonax de la capital. Se trataba de una herramienta muy usada por Napoleón Bonaparte que, de hecho, aplica de igual modo a la conocida Mme. Staël o Anne-Louise Germaine Necker 89. A pesar de ello, el diputado trató de evitar el mencionado castigo a toda costa. De este proceso destacan cinco escritos dirigidos a los Bonaparte. Según Y. Bénot, de estos documentos dos se encuentran sin fechar y uno de ellos, el referente al 11 de agosto de 1803, puede aportar información poco veraz por parte del propio Sonthonax 90. Así, el antiguo comisario optó por contactar a una autoridad política y miembro del “clan”, además de académico de su mismo “gremio”: José Bonaparte. Quizás se podría aplacar la indiferencia del cónsul francés ante su situación. “Al senador José Bonaparte. Ciudadano senador, Me apresuro a denunciar a su conocimiento un error por parte de la autoridad del que soy víctima en este momento, y cuyas consecuencias serían desastrosas para el Gobierno (francés) si no se reparara. El 6 de este mes el prefecto de la policía me hizo notificar verbalmente a través del jefe de división Bertrand, la orden del gran juez de salir de París Sin embargo, Bonaparte destaca el día 5 junto a la iglesia de Saint-Roch. Para mantener un orden centralizado disuelve la Guardia Nacional de París y crea la Legión Policial. Jean TULARD: Napoleón, Barcelona, Crítica, 2014, pp. 75-95. 87 Xavier ROCA-FERRER: Madame de Rémusat. Las guerras privadas del clan Bonaparte. Memorias de una dama de palacio en la corte de Josefina y Napoleón Bonaparte, Barcelona, Arpa, 2019, pp. 302-303. 88 Ibid 89 Ibid. Solo el 5 de enero de 1801 exilia a 130 antiguos miembros del partido de la “Montaña”, los jacobinos. 90 Yves BÉNOT: “Pourquoi Bonaparte…”, pp. 522-525. BROCAR, 46 (2022) 11-42 35 JONATHAN JACOBO BAR SHUALI inmediatamente para retirarme a la comuna en la que nací. Una justa deferencia por las decisiones del poder ministerial me hizo obedecer inmediatamente la parte de la orden que me era ejecutable. Salí de los límites, y exigí, tanto contra la medida en sí, como sobre la designación de un municipio, donde no tengo fuego, lugar ni establecimiento. Hace 20 años que me encuentro domiciliado en París, primero como abogado en el Parlamento y luego en el Primer tribunal (…). Desde la Revolución, las diversas misiones que he cumplido, las múltiples magistraturas que me han sido investidas, no hubo interrupción (sobre) mi domicilio. (…) Buscando los motivos de un acto injusto, descubrí que se me acusaba de unas declaraciones hechas por mí mismo, o, dijeron, que testifiqué mi alegría por la guerra que acaba de comenzar entre Francia e Inglaterra. (…) Perdóneme, ciudadano senador, este tipo de digresión que me desgarra la gravedad de las circunstancias (…). En cuanto a lo que me concierne, desapruebo absolutamente todos estos propósitos absurdos (…). Le ruego con mucha urgencia que me defienda de la injusta persecución que sufro, que invite al gran juez a la revisión de los hechos, así como a revocar la orden que se ha dictado contra la libertad de mi persona. Salud y respeto. Sonthonax” 91. Al analizar la carta del 30 de junio de 1803 no es difícil deducir el efectivo manejo de la publicidad y los medios por parte del Gobierno consular, tanto es así que el propio caso de Sonthonax llegó al público como posible agente del régimen inglés 92. A pesar de los innumerables esfuerzos por parte del antiguo comisario, y sus intentos de acercamiento a la capital, Léger-Félicité Sonthonax falleció en julio de 1813 en su villa de origen, Oyonnax. NAF 1301-1309. Bibliothèque Nationale de Paris (Archives et manuscrits NAF 1308 VIII SZ. 265 feuillets). Collection de lettres originales de différents personnages du XVIIIe et surtout du XIXe siècle, provenant de la succession du libraire Lefèvre. Recuperado de (https://archivesetmanuscrits.bnf.fr/ark:/12148/cc38988p/cd0e2290). [consultado 24/06/2021] 92 Yves BÉNOT: “Pourquoi Bonaparte…”, pp. 522-525. 91 36 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS 8. Conclusiones Sin la figura de Léger-Félicité Sonthonax el Estado francés no hubiera podido comenzar su larga trayectoria hacia la emancipación total de la población esclava en las colonias francesas en 1848. A pesar de la existencia en territorio nacional de la Sociedad de Amigos de los Negros y su posterior refundación bajo el nombre de “Amigos de los Negros y las Colonias”, fue Sonthonax quien tuvo la decisión final sobre el campo de operaciones que se daba en agosto de 1793 en la colonia de Saint-Domingue. Actualmente se puede afirmar una colaboración espontánea con el club mencionado más arriba y, en una segunda etapa, la adhesión de Sonthonax al mismo es indudable. De este modo, los historiadores todavía analizan si a largo plazo se siguió por parte de los diplomáticos algún tipo de indicaciones “secretas” en las diferentes medidas que se llevaron a cabo en la isla. Sin embargo, es más probable, debido a la situación estratégica en la que se encontraron Polverel y Sonthonax, que la libertad total de los esclavos se diera por una necesidad concreta de tropas y armamento, elementos de los que careció el Ejército republicano en el Caribe. Por otro lado, es evidente que desde junio de 1793 se había prometido la liberación de numerosos grupos de cimarrones que, asimismo, se habían integrado en las fuerzas de los comisarios. La presión ante tales precedentes desembocó en el día 29 de agosto de 1793 con la proclama de Sonthonax. De no haber sido así, los antiguos esclavos se habrían podio levantar ante sus “generales”, algo a lo que ya se encontraban acostumbrados. Los nuevos “libertos” y el Gobierno republicano fueron conscientes del potencial económico que suponía la parte occidental de Santo Domingo, el actual Haití, es por ello por lo que la nueva aristocracia negra solicitó a Sonthonax su representación en el Directorio, misión que llevó a cabo defendiendo los intereses de los subordinados de Toussaint Louverture. Así, Sonthonax permaneció en París hasta la etapa consular, momento en el que centró todos sus esfuerzos en los suburbios obreros. Fue esta última decisión la que le condenó a su clasificación de “jacobino radical” y condujo a su expulsión de la ciudad en el verano de 1803, como a tantos otros jacobinos y detractores desde enero de 1801. A pesar de todo ello, Sonthonax no es una figura conocida por el público general y menos aún en los ámbitos hispanoparlantes. En los territorios de influencia francófona, como es el caso del propio Estado haitiano, se ha procedido a la elaboración y documentación del personaje. Hasta ahora, solo se han publicado dos biografías concisas sobre la figura del comisario francés, siendo una de estas una obra conjunta. Por el contrario de lo que puedan aparentar los acontecimientos históricos, Léger-Félicité Sonthonax nunca fue obviado en la memoria del floreciente estado. Así, durante el Gobierno de Toussaint Louverture se edificó un panteón en BROCAR, 46 (2022) 11-42 37 JONATHAN JACOBO BAR SHUALI memoria de la Proclamation du 29 août y la persona de Sonthonax en el que rezaba el siguiente extracto: “Amigos míos, venimos a haceros libres. Los franceses dan libertad al mundo. Sois libres. Cuidad vuestra libertad. Viva la libertad. Viva la República. Viva Robespierre” 93. Bibliografía BÉNOT, Yves: “Pourquoi Bonaparte a-t-il exilé Sonthonax? ou: Dix ans après...”, Annales historiques de la Révolution française, 293-294 (1993), pp. 522-525. BÉNOT, Yves: “Le procès Sonthonax ou les débats entre les accusateurs et les accuses dans l’affaire des colonies (an III)”, en Marcel DORIGNY (dir.): LégerFélicité Sonthonax. La première abolition de l'esclavage. La Révolution française et la Révolution de Saint-Domingue, París, S. F. H. O. M., 2005, pp. 55-64. BITLLOCH, Eduardo: “Ciencia, Raza y Racismo en el Siglo XVIII”, Ciencia hoy, 33 (1996), pp. 47-58. BRYAND, Édouard: Histoire de ST.-Domingue depuis 1789 jusqu’en 1794, París, Libraire Pierre Blanchard, 1812. BUCK-MORSS, Susan: Hegel y Haití. La dialéctica del amo-esclavo: una interpretación revolucionaria, Argentina, Grupo Editorial Norma, 2005. CEBREIRO ARES, Francisco: “Léger Félicité Sonthonax en A Coruña: affaire monetario y conflicto diplomático franco-español (1797-1798)”, Espacio, Tiempo y Forma, 33 (2020), pp. 187-211. CHANTERANNE, David: Les douze morts de Napoléon, París, Passés Composés, 2021. CORMACK, William S.: Patriots, Royalists, and Terrorists in the West Indies: The French Revolution in Martinique and Guadeloupe, 1789-1802, U. S. A., University of Toronto Press, 2019. DORIGNY, Marcel: “Sonthonax et Brissot: Le cheminement d’une filiation politique assume”, en Marcel DORIGNY (dir.): Léger-Félicité Sonthonax. La première abolition de l’esclavage. La Révolution française et la Révolution de SaintDomingue, París, S. F. H. O. M., 2005, pp. 29-40. Este extracto ha llegado al presente gracias al oficial inglés Marcus Rainsford. Marcus RAINSFORD: An Historical Account of the Black Empire of Hayti, U. S. A., Duke University Press Durham and London, 2013, p. 135. 93 38 BROCAR, 46 (2022) 11-42 EL CASO “SONTHONAX”: ¿ABOLICIONISTA O SUPERVIVIENTE? ACERCA DE UN DIPLOMÁTICO FRANCÉS ENTRE SAINT-DOMINGUE Y PARÍS DUBOIS, Laurent: Avengers of the New World: The Story of the Haitian Revolution, U. S. A., Harvard University Press, 2009. DUBROCA, Jean-Louis: Vida de J. J. 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ZARAGOZA, Justo: Las insurrecciones en Cuba, apuntes para la historia política de esta isla en el presente siglo, Madrid, Impr. de Manuel G. Hernández, 1872. Recursos tomados de internet ATSÈ Alexis-Camille Kimou, «La rebelión de Galbaud y la libertad general de los esclavos de Saint-Domingue (1793-1794)», Nuevo Mundo Mundos Nuevos [En línea], Debates (2014). https://doi.org/10.4000/nuevomundo.66356 b3903485 John Carter Brown Library (proclamationauno00sont). Proclamation. Au nom de la République.: Nous Léger-Félicité Sonthonax, commissaire civil de la République, délégué aux Iles françaises de l'Amérique sous le vent, pour y rétablir l'ordre & la tranquillité publique (…). Recuperado de internet (https://jcb.lunaimaging.com/luna/servlet/s/l2fbns). [consultado 05/06/2021] b4042557 John Carter Brown Library (proclamationduro02fran). Proclamation du Roi, sur le décret de l'Assemblée nationale, concernant les colonies (…). 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Napoléon Bonaparte, la traite des noirs et l’esclavage, 20 mai 1802 et 29 mars 1815. Recuperado de internet (https://www.napoleon.org/enseignants/documents/videonapoleon-bonaparte-et-le-retablissement-de-lesclavage-20-mai-1802-5-min-40/). [consultado 01/06/2021] BROCAR, 46 (2022) 11-42 41 JONATHAN JACOBO BAR SHUALI NAF 1301-1309. Bibliothèque Nationale de Paris (Archives et manuscrits NAF 1308 VIII S-Z. 265 feuillets). Collection de lettres originales de différents personnages du XVIIIe et surtout du XIXe siècle, provenant de la succession du libraire Lefèvre. Recuperado de internet (https://archivesetmanuscrits.bnf.fr/ark:/12148/cc38988p/cd0e2290). [consultado 24/06/2021] RMC (BFM. TV). Jean Tulard, historien: "Napoléon n'était pas raciste". Recuperado de internet (https://www.youtube.com/watch?v=Kl5bLQghjpc). [consultado 05/06/2021] Secrets d'Histoire (TV France). Toussaint Louverture: la liberté à tout prix. 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https://openalex.org/W3040258418	https://revista.univap.br/index.php/revistaunivap/article/download/1977/1566	Portuguese	null	ANÁLISE DA CONCENTRAÇÃO ENERGÉTICA BRASILEIRA DAS INDÚSTRIAS DE CAL E GESSO, VIDREIRO E NÃO ENERGO-INTENSIVOS	Revista UniVap	2,020	cc-by	5,630	ANÁLISE DA CONCENTRAÇÃO ENERGÉTICA BRASILEIRA DAS INDÚSTRIAS DE CAL E GESSO, VIDREIRO E NÃO ENERGO-INTENSIVOS ANALYSIS OF THE BRAZILIAN ENERGY CONCENTRATION OF LIME AND PLASTER, GLASS AND NON-ENERGY-INTENSIVE INDUSTRIES Luiz Moreira Coelho Junior1 Filipe Vanderlei Alencar2 Edvaldo Pereira Santos Júnior3 Resumo: A Empresa de Pesquisa Energética (EPE) constitui o segmento de “Outras Indústrias” na matriz energética brasileira com os setores de cal e gesso, vidreiro e não energo-intensivos. Este artigo analisou a concentração energética do setor brasileiro de outras indústrias (SBOI), de 1970 a 2016. As informações foram extraídas do Balanço Energético Nacional (BEN) da EPE. A concentração energética foi determinada pelos indicadores: Razão de concentração [CR(p)], Índice de Hirschman - Herfindahl (HHI), Índice de Theil (E) e índice de Gini (G). Os resultados apresentaram que o consumo energético do SBOI teve um crescimento médio de 3,59% a.a. de 1970 até 1982, o Óleo Combustível teve como principal fonte a energética e foi substituída pela eletricidade a partir de 1983, devido ao tamanho das firmas e a disponibilidade de outras fontes de energia. A demanda por gás natural (1995 a 2016) cresceu 5,66% a.a., principalmente, em função dos incentivos no segmento vidreiro. O CR(p) mostrou-se altamente concentrado por meio dos valores médios de CR(1) de 47,69%, CR(2) de 70,54% e CR(4) de 91,51%.O HHI inferiu como muito concentrado e o E corroborou com essas análises. O G mostrou tendência de concentração com desigualdade médio a forte para muito forte. Conclui-se que o SBOI é concentrado na sua demanda energética. Palavras-chave: Economia da energia; matriz energética; indicadores de concentração. Abstract: The Energy Research Company (EPE) is the "Other Industries" segment in the Brazilian energy matrix with lime and gypsum, glass and non-energy-intensive sectors. This article analyzed the energy concentration of the Brazilian sector of other industries (SBOI) from 1970 to 2016. The information was extracted from the National Energy Balance (BEN) of the EPE. The energy concentration was determined by the following indicators: Concentration ratio [CR(p)], Hirschman-Herfindahl index (HHI), Theil index (E) and Gini index (G). The results showed that SBOI energy consumption had an average growth of 3.59% per year. From 1970 to 1982, Fuel Oil was the main energy source, being replaced by electricity since 1983, due to the size of the firms and the availability of other energy sources. The demand for natural gas (1995 to 2016) increased by 5.66% pa, mainly due to the incentives in the glass segment. 11 11 Recebido em 02/2018. Aceito para publicação em 10/2019. ANÁLISE DA CONCENTRAÇÃO ENERGÉTICA BRASILEIRA DAS INDÚSTRIAS DE CAL E GESSO, VIDREIRO E NÃO ENERGO-INTENSIVOS ANALYSIS OF THE BRAZILIAN ENERGY CONCENTRATION OF LIME AND PLASTER, GLASS AND NON-ENERGY-INTENSIVE INDUSTRIES CR(p) was highly concentrated by means of the mean values of CR(1) of 47.69%, CR(2) of 70.54% and CR(4) of 91.51% .HHI inferred as very concentrated and E corroborated these analyses. G showed a tendency of concentration from medium to strong to a very strong inequality. It is concluded that SBOI is concentrated in its energy demand. Keywords: Energy economy; energy matrix; concentration indicators. 1 Economista, Dr., Professor do Departamento de Engenharia de Energias Renováveis, Universidade Federal da Paraíba - UFPB, Brasil. E-mail: luiz@cear.ufpb.br. 2 Eng. de Energias Renováveis, Universidade Federal da Paraíba - UFPB, Brasil. E-mail: alencarfv@gmail.com. 3 Eng. de Energias Renováveis, Mestrando em Energias Renováveis, Universidade Federal da Paraíba - UFPB, Brasil. E-mail: edvaldo.junior@cear.ufpb.br. Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 1 INTRODUÇÂO De acordo com a Empresa de Pesquisa Energética - EPE (2014), o consumo De acordo com a Empresa de Pesquisa Energética - EPE (2014), o consumo 1 Economista, Dr., Professor do Departamento de Engenharia de Energias Renováveis, Universidade Federal da Paraíba - UFPB, Brasil. E-mail: luiz@cear.ufpb.br. 2 Eng. de Energias Renováveis, Universidade Federal da Paraíba - UFPB, Brasil. E-mail: alencarfv@gmail.com. 3 Eng. de Energias Renováveis, Mestrando em Energias Renováveis, Universidade Federal da Paraíba - UFPB, Brasil. E-mail: edvaldo.junior@cear.ufpb.br. Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 12 energético das indústrias de Cal, Gesso e Vidro são classificados na matriz energética brasileira como “Outras Indústrias”. Esses setores são bastantes diversificados nos seus processos produtivos e em relação à demanda de fontes de energia. No Brasil, em 2014, o setor vidreiro apresentou déficit na balança comercial de US$ 547,3 milhões e importações de US$ 800,4 milhões sendo o vidro, o principal produto importado. Do faturamento mundial de 2011 para o segmento de vidros, o Brasil representou 5,3% (US$ 33,4 bilhões). O setor brasileiro de Cal, em 2013, produziu 543 milhões de toneladas e foi o 5º no ranking mundial, gerando R$ 2,6 bilhões, que atendeu basicamente o mercado interno (BAJAY; LEITE; GORLA, 2010a). O setor brasileiro de outras indústrias (SBOI) tem sua importância no PIB, refletindo também na demanda energética. Segundo o Balanço Energético Nacional (BEN) de 1970 a 2016, o consumo no SBOI ocupou entre a 3ª e a 6ª do ranking industrial. Em 2016, O SBOI ficou em 4º lugar e consumiu 7,9 milhões tep (9,02%), sendo a Eletricidade (50,72%), o Gás Natural (28,76%) e a Lenha (10,78%) responsáveis com cerca de 90% da oferta interna de energia deste segmento (EPE, 2017). A eletricidade se tornou a principal fonte no SBOI devido aos ganhos produtividade. Santana e Bajay (2016) mostraram que a eficiência energética diminuiu o consumo de energia com a mesma produção, devido a evolução tecnológica, melhoria de processos e substituição das fontes de energia. Foram identificadas as potencialidades e previu-se uma queda de 20% da demanda energética para 2020. A substituição foi uma marca inovadora no âmbito energético, porém, atualmente, a palavra de ordem é aproveitamento energético (EPE, 2014). Para a identificação dos ganhos e que seja possível averiguar essas mudanças graduais do consumo energético (eficiência e intensidade) é necessária a utilização de índices de desempenho. Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 2 MATERIAIS E MÉTODOS Os dados usados para medir a concentração energética do Setor Brasileiro de Outras Indústrias (SBOI) foram obtidos do Balanço Energético Nacional (BEN). A unidade de medida utilizada foi em tonelada equivalente de petróleo (tep), entre 1970 e 2016. Analisou-se a conjuntura do consumo energético do SBOI nos anos de 1970, 1975, 1980, 1985, 1990, 1995, 2000, 2005, 2010 e 2016. Avaliaram-se os ganhos e perdas das fontes energéticas por meio da Taxa Geométrica de Crescimento (TGC), Equação 1 (CUENCA; DOMPIERI, 2017).  % 1 *100 F O V t TGC V    = −       (1) (1) 𝑻 em que, VF é o consumo energético referente ao ano final; VO é o consumo energético referente ao ano inicial; ∆t é a diferença entre o ano final e o inicial. 𝑻 em que, VF é o consumo energético referente ao ano final; VO é o consumo energético referente ao ano inicial; ∆t é a diferença entre o ano final e o inicial. A concentração energética foi mensurada por meio de índices parciais (parte dos dados) e sumárias (totalidade de informações). O índice parcial usado foi a razão de concentração e os índices sumários utilizados foram o Índice de Hirschman-Herfindahl, o Índice de Entropia de Theil e o Índice de Gini. A Razão de concentração [CR(p)], Equação2, foi proposto Bain (1959). Este indicador avaliou a parcela do consumo energético p (i = 1, 2, 3,..., p) fontes da matriz energética do SBOI. ( ) 1 i p CR p S i = = (2) ( ) 1 i p CR p S i = = (2) em que, Si é o percentual consumido da fonte de energia i em relação ao total. Foram utilizados o principal [CR(1)], os dois [CR(2)] e os 4 [CR(4)] maiores fontes energéticas. em que, Si é o percentual consumido da fonte de energia i em relação ao total. Foram utilizados o principal [CR(1)], os dois [CR(2)] e os 4 [CR(4)] maiores fontes energéticas. O Índice de Herfindahl – Hirschman (HHI), Equação 3, foi reconhecida por Hirschman (1964) em que foi calculado por meio do somatório do quadrado do Si de i até p fontes energéticas do SBOI. 1 INTRODUÇÂO Os indicadores de concentração são componentes importantes para medir estrutura de mercado, oferta e demanda em apenas um indicador (POSSAS, 1999, COELHO JUNIOR, 2013). Os indicadores de concentração podem ser utilizados em diversas áreas do conhecimento, como na engenharia florestal com os ensaios de Coelho Junior, Reszende e Oliveira (2013), Coelho Junior (2016) e Simioni et al. (2017), na agropecuária com Costa e Santana (2014), Cuenca e Dompieri (2017) e Coelho Junior (2017), no bancário com Luft e Zilli (2013), na siderurgia com Oliveira e Sperb (2016). No setor energético com Beiral, Moraes e Bacchi (2013), Coelho Junior et al. (2017), Coelho Junior et al. (2018) e Coelho Junior, Santos Junior e Alencar (2019). Em busca de compreender melhor o comportamento da demanda energética industrial, este artigo analisou a concentração energética do setor brasileiro de outras indústrias (SBOI), de 1970 a 2016. Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 13 p p São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 Revista Univap - revista.univap.br 2 MATERIAIS E MÉTODOS 2 1 i p HHI S i = = (3) 2 1 i p HHI S i = = (3) em que, p é o número de fontes de energia; Si é a participação da fonte energética i do consumo total da SBOI. O HHI encontra-se entre 1/n (limite inferior) e 1. Porém, quando as quantidades de fontes participantes variam ao longo do tempo, é necessário ajusta-lo para melhor 14 comparação. Resende e Boff (2002) ajustaram o HHI conforme a Equação 4. comparação. Resende e Boff (2002) ajustaram o HHI conforme a Equação 4. ( ) 1 '' 1 1 HHI p HHI p =  − − (4) (4) A utilização dessa equação implica em um intervalo de variação entre 0 e 1 para o HHI. Feito este ajuste, o HHI’’ será avaliado entre 0 e 1, sendo que 0 implica em condições de desconcentração e 1 implica em monopólio. A classificação é descrita pela tabela 1. Tabela 1 - Classificação da concentração pelo HHI’’. Tabela 1 - Classificação da concentração pelo HHI’’. Tabela 1 - Classificação da concentração pelo HHI’’. Classificação Intervalo Desconcentrado 0 ≤ HHI’’ ≤ 0,1 Pouco concentrado 0,1 ≤ HHI’’ ≤ 0,18 Muito concentrado HHI’’ > 0,18 Fonte: Resende e Boff (2002) Tabela 1 - Classificação da concentração pelo HHI’’. Classificação Intervalo Desconcentrado 0 ≤ HHI’’ ≤ 0,1 Pouco concentrado 0,1 ≤ HHI’’ ≤ 0,18 Muito concentrado HHI’’ > 0,18 Fonte: Resende e Boff (2002) O Índice de Entropia de Theil (E), Equação 5, foi proposto Theil (1967). ( ) ln 1 i i p E S S i =  = (5) ( ) ln 1 i i p E S S i =  = (5) em que, p é a quantidade de fontes de energia; Si é o matrix share, percentual, da fonte energética i do consumo total da SBOI. em que, p é a quantidade de fontes de energia; Si é o matrix share, percentual, da fonte energética i do consumo total da SBOI. Valores pequenos inferem em concentrações de energia mais elevadas. A quantidade de fontes de energia participantes é proporcional ao valor do E que varia entre 0, condição de monopólio, e ln(p), infere em participações iguais entre todas as fontes de energia (RESENDE; BOFF, 2002). Analogamente ao HHI, é sugerido por Resende e Boff (2002) que para análises interanuais, a Entropia seja ajustada de acordo com Equação 6. Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 2 MATERIAIS E MÉTODOS E’’ apresenta como concentração máxima (E’’ = 0) e mínima concentração (E’’ = 1). ( ) ( ) 1 '' ln ln 1 i i p E S S p i = −  = (6) ( ) ( ) 1 '' ln ln 1 i i p E S S p i = −  = (6) O Coeficiente de Gini (G), Equação7, foi desenvolvido Gini (1955) em “Variabilità e mutabilità”. Trata-se de uma medida de desigualdade, primariamente usada para avaliar desigualdade de renda, mas pode ser usada para mensurar a desigualdade do consumo de energia na SBOI. 15 ( ) 1 1 ij i p S S i G p     +   =   = −  (7) em que, Sij é a participação acumulada entre as fontes energéticas i até j; Si é participação percentual, da fonte energética i em relação ao consumo total do SBOI. Este índice está contido 0 e 1. O G classifica a desigualdade: nula a fraca (0 < G ≤ 0,25), fraca a média (0,25 < G ≤ 0,5), média a forte (0,5 < G ≤ 0,7), forte muito forte (0,7 < G ≤ 0,9) e muito forte a absoluta (0,9 < G ≤ 1). e sta U ap e sta u ap b São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 Revista Univap - revista.univap.br 3 RESULTADOS E DISCUSSÃO Em 1995, o SBOI consumiu 343 mil tep de Gás Natural enquanto que, em 2016, foram 2,18 milhões tep, chegando com 28,75% do total do segmento. A TGC neste período (1984 a 2016) foi 18,81% a.a. O setor vidreiro foi um dos precursores no consumo de Gás Natural do SBOI e é o principal consumidor quando comparado aos demais segmentos de outras Indústrias. Este combustível substituiu o óleo combustível, oferecendo vantagens técnicas para o melhor controle da temperatura na produção do vidro. Houve redução nos custos no sistema produtivo fabril pela logística por não possuírem contaminantes, aumentando o tempo entre as manutenções dos equipamentos (BAJAY; LEITE; GORLA, 2010a; MELO; MEDINA, 2005). O consumo do Óleo Combustível caiu de 644 mil tep (1970) para 56 mil tep (2014). Teve crescimento de 1970 até 1980, onde atingiu o pico de demanda com 2,2 milhões tep, correspondendo a 56,8% do consumo total do SBOI, e depois caiu até 2014 chegando a 59 mil tep. Esta queda foi resultado da Crise do Petróleo de 1979 que, de acordo com Pedrosa Junior e Corrêa (2016), elevou o preço do barril de petróleo e afetou severamente o consumo de Óleo Combustível por parte do SBOI. A partir de 1983, a Eletricidade foi a principal fonte do SBOI. Apresentou um consumo estável, em que saiu de 1970 (468 mil tep) para 2016 (3,85 milhões tep, 50,72% do consumo total), com um crescimento médio de 5,66% a.a. Bajay, Leite e Gorla (2010b) afirmaram que o principal segmento consumidor de Eletricidade do SBOI é o não energo-intensivo e as principais finalidades para o uso da Eletricidade se distribui em 20% para aquecimento direto e 60% para execução de força eletromotriz. A Lenha foi outra fonte de destaque no SBOI, que em 1970 teve consumo de 541 mil tep e 1981 caiu para 486 mil tep. Porém, a Crise de 1979 estimulou a demanda por lenha como reflexo de substituição do Óleo combustível. De 1982 até 1986, saiu de 605 mil tep para 961 mil tep, respectivamente. De 1987 (914 mil tep) até 2016 (819 mil tep) houve queda no consumo da Lenha, mas teve sua importância, pois contemplou em 10,78% da matriz energética do SBOI. As demais fontes não apresentaram boa representatividade. A Figura 1 apresenta a evolução dos indicadores de concentração energética do setor de outras indústrias brasileira (SBOI), período de 1970 a 2016. 3 RESULTADOS E DISCUSSÃO A Tabela 2 apresenta a evolução do consumo energético do Setor Brasileiro de Outras Indústrias (SBOI), em 10³ tonelada equivalente de petróleo (tep), de 1970 a 2016. Este segmento foi composto por 12 fontes na matriz energética. Em 2016, o SBOI representou 9,02% da demanda interna da energia industrial, e 3,15% do consumo energético nacional. O SBOI consumiu, em 1970, 1,67 milhões tep e, em 2016, este valor chegou a 7,59 milhões tep, apresentando um crescimento médio de 3,35% a.a. no período estudado. Este aumento do consumo de energia acompanhou o crescimento econômico das empresas que compõem o segmento. Tabela 2 - Evolução do consumo energético do Setor Brasileiro de Outras Indústrias (SBOI), em 10³ tonelada equivalente de petróleo (tep), de 1970 a 2016. Fontes 1970 1975 1980 1985 1990 1995 2000 2005 2010 2016 Gás natural - - - 27 262 343 567 984 1.901 2.183 Carvão vapor - - 8 25 9 6 152 99 87 11 Lenha 541 480 444 837 697 594 576 703 874 819 Bagaço de cana - - - 2 4 9 - - - - Óleo diesel 127 127 114 58 120 159 150 113 144 120 Óleo combustível 644 1.636 2.211 799 835 1.031 803 358 177 56 Gás liq. de petr. 31 77 103 71 54 94 179 148 153 166 Querosene 16 56 68 43 25 7 5 5 1 - Gás canalizado 4 9 27 40 22 2 - - - - Eletricidade 306 561 1.161 1.520 1.674 1.939 2.534 3.024 3.380 3.851 Carvão vegetal - - 8 29 16 13 8 10 12 12 Outras secundarias de petróleo - 3 227 4 2 15 374 380 481 376 Total 1.670 2.948 4.371 3.455 3.720 4.213 5.347 5.823 7.211 7.592 Fonte: EPE (2017). Tabela 2 - Evolução do consumo energético do Setor Brasileiro de Outras Indústrias (SBOI), em 10³ tonelada equivalente de petróleo (tep), de 1970 a 2016. Em 1984 ocorreram os primeiros registros do Gás Natural na matriz energética do SBOI com 9 mil tep (0,3%). O estímulo do consumo do gás natural no setor industrial Revista Univap revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 16 ocorreu com a implantação do gasoduto Brasil-Bolívia, na década de 90 (EPE, 2014). Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 3 RESULTADOS E DISCUSSÃO ISSN 2237-1753 18 De 1970 até 2016, a razão de concentração das quatro principais fontes [CR(4)] variou entre 96,0% (1970) e 83,56% (2000), chegando 2016 com 95,21%, proporcionando uma média de 91,51%. Segundo a classificação de Bain (1959), a matriz energética da SBOI foi altamente concentrada nas quatro principais fontes, no período analisado. A terceira fonte energética do SBOI foi lenha (1973 – 1983,1987 – 2000 e 2003 – 2016), eletricidade (1970 – 1972), óleo combustível (1984 – 1986 e 2002) e gás natural (2001). E a quarta fonte foi a lenha (2001 e 2002), óleo diesel (1970 – 1975, 1981, 1982), querosene (1982), gás natural (1987 – 2000), gás liquefeito de petróleo (1984 – 1986) e outras secundárias do petróleo (1976 – 1980 e 2003 – 2016). A Figura 1.b apresenta a evolução do Índice de Herfindahl-Hirshman (HHI) do consumo energético do SBOI, no período de 1970 a 2016. O HHI’’ e as diferenças entre o HHI e o limite inferior mostraram que durante todo período, o consumo ficou classificado como muito concentrado. Observando evolução do HHI’’ em destaque de 3 períodos: 1970-1977; 1978-2001e 2002-2016. De 1970 até 1977, o HHI’’ teve tendências de crescimento com oscilações que saiu de 0,1760 para 0,2638, em que sua classificação foi pouco concentrada para muito concentrada. De 1978 a 2001, o valor do HHI’’ caiu de 0,2431 para 0,2043 permanecendo seu grau de concentração. De 2002 a 2016, o HHI’’ teve tendências de crescimento saindo de 0,2178 para 0,2831, devido ao alto consumo de Eletricidade. A diferença entre HHI e o limite superior, entre 1970 a 1980 aumentou, elevando a concentração na matriz energética do SBOI. Após 1981 até 2000, a linha do HHI e do limite superior diminuiu, e diminuiu a concentração do consumo. De 2001 até 2014, voltou a crescer, sendo coerente com o comportamento do HHI’’. A Figura 1.c apresenta a evolução da Entropia de Theil da SBOI, no período de 1970 a 2016. O índice de Entropia de Theil ajustada (E’’), no período analisado, apresentou valores mínimo de 0,5714 (1984) e máximo de 0,7116 (2000), classificando com uma concentração alta. De 1970 até 1984 o E’’ decresceu, com oscilações, em que saiu de 0,6613 (1970) para o mínimo de 0,5714 (1984). A diferença entre o limite superior e E teve um aumento de 0,5708 (1970) para 1,0651 (1984), confirmado pelo E’’. 3 RESULTADOS E DISCUSSÃO A Figura 1.a mostra o comportamento da razão de concentração [CR(q)] do consumo energético do SBOI, no período de 1970 a 2016. De 1970 até 2016, a razão de concentração da principal fonte [CR(1)] ficou entre 56,78% (1976) e 38,47% (1981) com média de 47,59%, inferindo que é altamente concentrado. As fontes dominantes no processo de obtenção energética no setor outras indústrias foram óleo combustível (1970 – 1982) e a eletricidade (1983 – 2016). O CR(1) cresceu de 38,58% (1970) para 56,78% (1976) e com a Crise de 79, o CR(1) caiu, atingindo seu valor mais baixo em 1981 e a partir de 1982 cresceu gradativamente de 38,60% para 50,72% (2016) da matriz energética do Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 17 SBOI, mostrando-se assim altamente dependente da principal fonte energética. De 1970 até 2016, a razão de concentração das duas principais fontes [CR(2)] saiu de 70,98% chegando ao valor máximo de 79,48% e teve valor mínimo de 61,25% (2001), proporcionando média de 70,53%. A segunda principal fonte do SBOI foi a lenha (1970 – 1972 e 1984 – 1986), eletricidade (1973 – 1982), óleo combustível (1983 e 1987 – 2001) e gás natural (2002 – 2016). De 1970 a 1976, o CR(2) apresentou tendências de crescimento chegando 74,72%. Entre 1977 e 1981 não houve muitas variações e a partir de 1982 teve crescimento gradativo saindo de 73,15% para 79,48%, em 2016. Figura 1 - Evolução dos indicadores de concentração energética do setor de outras indústrias brasileira (SBOI), no período de 1970 a 2016. Sendo que (a) Razão concentração; (b) Índice Herfindahl-Hirschman; (c) Índice de entropia de Theil e; (d) Índice de Gini. Figura 1 - Evolução dos indicadores de concentração energética do setor de outras indústrias brasileira (SBOI), no período de 1970 a 2016. Sendo que (a) Razão concentração; (b) Índice Herfindahl-Hirschman; (c) Índice de entropia de Theil e; (d) Índice de Gini. Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 a) b) d) c) a) b) d c) d) c) Revista Univap - revista.univap.br e sta U ap e sta u ap b São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 p p São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 3 RESULTADOS E DISCUSSÃO De 1985 até 2000, houve um aumento expressivo, o E’’ saiu de 0,5739 para o pico, respectivamente. A diferença entre o limite superior e E cresceu de 1,0589 (1985) para 0,6641 (2000). Esta diminuição da concentração foi consequência da forte inserção do consumo do Gás Natural na matriz energética do SBOI. De 2001 até 2016, ocorreu uma diminuição no E’’, saiu de 0,6993 para 0,6449, respectivamente. Enquanto a diferença entre o limite superior e E aumentou de 0,6924 (2001) para 0,8177 (2016). Ocorreu um aumento da concentração, devido à queda do consumo do Gás Natural e aumento do consumo de Eletricidade, enquanto as demais fontes mantiveram o consumo absoluto quase constante. A Figura 1.d representa a evolução do Coeficiente de Gini (G) da SIOB, no período de 1970 a 2016. Ao longo do período estudado o G variou entre os intervalos: 0,5632 (1970) e 0,7393 (1995). A classificação da concentração ficou entre média a forte (1970 Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 19 – 1979, 1998 – 2015) e muito forte (1980 – 1997 e 2016). De 1970 até 1995 o G tendeu a crescer, saindo do mínimo até o máximo, mudando de classificação da desigualdade média a forte para forte a muito forte, entre 1979 e 1980. Este crescimento do G, bem como a mudança da desigualdade foi decorrente do aumento de fontes participantes, passando de 7 fontes (1970) para 12 (1995). De 1996 até 2016 o G decresceu, com oscilações, saiu de 0,7225 para 0,7116, respectivamente. Variou a classificação da desigualdade de forte a muito forte para médio a forte entre os anos de 1997/1998 e 2015/2016. A queda na desigualdade é condizente com a queda do número de fontes participantes da matriz energética, em 1996 tinham-se 11 fontes participantes, já em 2016 10 fontes compuseram a matriz energética. 4 CONCLUSÃO O consumo energético do SBOI teve um crescimento médio anual de 3,59% a.a.. De 1970 até 1982, o Óleo Combustível teve como principal fonte a energética e foi substituída pela eletricidade a partir de 1983, devido ao tamanho das firmas e a disponibilidade de outras fontes de energia. A demanda por gás natural (1995 a 2016) cresceu 5,66% a.a., principalmente, em função dos incentivos no segmento vidreiro. O CR(p) mostrou-se altamente concentrado por meio dos valores médios de CR(1) de 47,69%, CR(2) de 70,54% e CR(4) de 91,51%.O HHI inferiu como muito concentrado e o E corroborou com essas análises. O G mostrou tendência de concentração com desigualdade médio a forte para muito forte. O SBOI é concentrado na sua demanda energética. Revista Univap - revista.univap.br São José dos Campos-SP-Brasil, v. 26, n. 50, jul. 2020. ISSN 2237-1753 5 REFERÊNCIAS BAIN, J. Industrial Organization. New York: J. Wiley, 1959. 274 p. AIN, J. Industrial Organization. New York: J. Wiley, 1959. 274 p. BAIN, J. Industrial Organization. New York: J. Wiley, 1959. 274 BAJAY, S. V.; LEITE, A. A. F.; GORLA, F. D. Oportunidades de Eficiência para Indústria: Relatório Setorial: Setor Vidreiro. Brasília: CNI, 2010a. 56 p. (Relatório 16). 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https://openalex.org/W4237801735	http://jurnal.fk.unand.ac.id/index.php/jka/article/download/975/851	Indonesian	null	Gambaran Karakteristik Tingkat Kontrol Penderita Asma Berdasarkan Indeks Massa Tubuh (IMT) di Poli Paru RSUP. Dr. M. Djamil Padang pada Tahun 2016	Jurnal kesehatan Andalas/Jurnal Kesehatan Andalas	2,019	cc-by-sa	3,709	http://jurnal.fk.unand.ac.id http://jurnal.fk.unand.ac.id 89 Artikel Penelitian Artikel Penelitian Abstrak Asma adalah penyakit heterogen, yang ditandai dengan terjadinya inflamasi kronik saluran pernapasan. Salah satu faktor risiko asma yang berkaitan erat dengan kontrol asma adalah obesitas. Selain itu underweight juga terkait dengan fungsi paru yang menurun dan asma. Tujuan penelitian ini adalah menentukan gambaran karakteristik tingkat kontrol penderita asma berdasarkan indeks massa tubuh (IMT) di Poli Paru RSUP Dr. M. Djamil Padang pada tahun 2016. Jenis penelitian ini adalah deskriptif observasional restrospektif dengan menggunakan data rekam medis pasien asma rawat jalan di Poli Paru RSUP Dr. M. Djamil Padang antara 1 Januari sampai 31 Desember 2016 dan didapatkan sebanyak 63 data yang memenuhi kriteria sampel. Data diolah dengan menggunakan program komputer dan disajikan dalam bentuk tabel distribusi dan frekuensi. Hasil penelitian menunjukkan bahwa pasien asma laki-laki (49,2%) dan perempuan (50,8%) hampir merata, sebagian besar berusia 40 – 60 tahun (47,6%), bekerja sebagai PNS (31,7%), memiliki IMT ≥ 23,0 (49,2%), dengan tingkat kontrol asma berupa asma terkontrol sebagian (61,9%), asma terkontrol penuh terbanyak ditemukan pada IMT normal (3,2%), asma tidak terkontrol terbanyak pada IMT normal (17,5%), dan asma terkontrol sebagian terbanyak pada IMT berat badan lebih & obes (31,7%). Kata kunci: asma, indeks massa tubuh, tingkat kontrol asma Abstract Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. One of the asthma risk factors that is closely related to asthma control is obesity. Other than that underweight is also associated with decreased lung function and asthma. The objective of this study was to describe the characteristic of asthma control level based on body mass index in the pulmonary polyclinic RSUP Dr. M. Djamil Padang in 2016. This observational descriprive study used medical records of patients with asthma who were the outpatient in the pulmonary polyclinic RSUP Dr. M. Djamil Padang from 1 january until 31 December 2016 and obtained as many as 63 data that met the criterias. Data were processed using computer program and presented in the form of distribution tables and frequency. This study showed that male (49,2%) and female (50,8%) asthma patients are almost evenly distributed, mostly aged 40 – 60 years (47,6%), work as civil servant (31,7%), has a body mass index ≥ 23,0 (49,2%), with the level of asthma control is partially controlled asthma (61,9%),fully controlled asthma mostly on normal IMT (3,2%), uncontrolled asthma mostly on normal IMT (17,5%), and partially controlled asthma most commonly found on overweight and obese (31,7%). ( ) Keywords: asthma, body mass index, level of asthma control Affiliasi penulis: 1. Prodi Kedokteran Fakultas Kedokteran Universitas Andalas Padang (FK Unand), 2. Bagian Paru FK Unand. 3. Bagian Paru FK UNAND Korespondensi: Fanny Permata Andriani, Email : fannypermataa@gmail.com, Telp : 0823-9068-7429 PENDAHULUAN Asma merupakan gangguan inflamasi kron pada saluran napas yang melibatkan banyak sel d elemennya yang menjadi masalah kesehat masyarakat yang serius diberbagai negara di selur Keywords: asthma, body mass index, level of asthma control Gambaran Karakteristik Tingkat Kontrol Penderita Asma Berdasarkan Indeks Massa Tubuh (IMT) di Poli Paru RSUP. Dr. M. Djamil Padang pada Tahun 2016 Fanny Permata Andriani1, Yessy Susanty Sabri2, Fenty Anggrainy3 Fanny Permata Andriani1, Yessy Susanty Sabri2, Fenty Anggrainy3 Jurnal Kesehatan Andalas. 2019; 8(1) PENDAHULUAN Asma merupakan gangguan inflamasi kronik pada saluran napas yang melibatkan banyak sel dan elemennya yang menjadi masalah kesehatan masyarakat yang serius diberbagai negara di seluruh http://jurnal.fk.unand.ac.id 90 dunia. Asma dapat menyebabkan penurunan produktivitas, serta menurunkan kualitas hidup bagi penderitanya.1 Di Amerika Serikat, asma tidak terkontrol dengan baik hingga mencapai angka 41-55%.6 Di Indonesia berdasarkan data di Poliklinik Alergi Ilmu Penyakit Dalam Rumah Sakit Cipto Mangunkusumo didapatkan, 64% pasien tidak terkontrol, 28% terkontrol sebagian, dan 8% terkontrol penuh.7 Faktor- faktor yang mempengaruhi kontrol asma diantaranya adalah usia, jenis kelamin, merokok, genetik, infeksi saluran pernapasan, dan berat badan yang berlebih. Diantara faktor tersebut yang berkaitan erat dengan kontrol asma adalah obesitas. Banyak penelitian yang mengungkapkan bahwa obesitas adalah faktor risiko asma dan memiliki hubungan yang signifikan antara peningkatan indeks massa tubuh dengan perkembangan asma.6 Obesitas adalah faktor risiko utama untuk gejala respirasi dan penyakit dengan kondisi kronik seperti penyakit kardiovaskular, asma, dan penyakit paru obstruktif kronis. Sebagai tambahan, beberapa penelitian melaporkan bahwa individu yang underweight juga terkait dengan fungsi paru yang menurun dan asma.8 Asma menjadi salah satu masalah kesehatan utama baik di negara maju maupun di negara berkembang. Menurut data dari laporan Global Initiatif for Asthma (GINA) tahun 2017 dinyatakan bahwa angka kejadian asma dari berbagai negara adalah 1- 18% dan diperkirakan terdapat 300 juta penduduk di dunia menderita asma.1 Prevalensi asma menurut World Health Organization (WHO) tahun 2016 memperkirakan 235 juta penduduk dunia saat ini menderita penyakit asma dan kurang terdiagnosis dengan angka kematian lebih dari 80% di negara berkembang.2 Di Amerika Serikat menurut National Center Health Statistic (NCHS) tahun 2016 prevalensi asma berdasarkan umur, jenis kelamin, dan ras berturut-turut adalah 7,4% pada dewasa, 8,6% pada anak-anak, 6,3% laki-laki, 9,0% perempuan, 7,6% ras kulit putih, dan 9,9% ras kulit hitam.3 Angka kejadian asma di Indonesia berdasarkan data Riset Kesehatan Dasar (Riskesdas) tahun 2013 mencapai 4,5%. Menurut Kementrian Kesehatan RI tahun 2011 Penyakit asma masuk dalam sepuluh besar penyebab kesakitan dan kematian di Indonesia dengan angka kematian yang disebabkan oleh penyakit asma diperkirakan akan meningkat sebesar 20% pada 10 tahun mendatang, jika tidak terkontrol dengan baik.4 Pasien asma dengan obesitas memiliki keparahan asma yang lebih, rendahnya kontrol asma, dan rendahnya respon terhadap pengobatan asma yang dinilai dari gejala, sesak napas dan mengi, terbangun malam hari, dan keterbatasan aktivitas sehari-hari yang lebih sering dan menetap pada individu dengan obesitas yang menderita asma. Jurnal Kesehatan Andalas. 2019; 8(1) PENDAHULUAN Selain itu, penderita lebih sering menggunakan obat pelega, membutuhkan rawatan ketika terjadi asma akut dan kunjungan ke dokter yang tak terduga dibandingkan dengan pasien asma non obes.9 Asma lebih sulit dikontrol pada pasien yang obes, maka dalam terapi asma upaya untuk menurunkan berat badan harus disertakan dalam rencana terapi asma dengan obes. Penurunan 5-10% berat badan dapat meningkatkan kontrol asma.1 Riskesdas nasional tahun 2013 menyatakan bahwa angka kejadian asma di Sumatera Barat adalah 2,7%.4 Data dari Profil Kesehatan Dinas Kesehatan Kota Padang (DKK) penyakit asma termasuk ke dalam daftar 10 penyebab kematian terbanyak di kota Padang tahun 2013, dengan angka kematian 11 orang perempuan dan 5 orang laki-laki, terlihat bahwa angka kejadian asma di Indonesia khususnya di Sumatera Barat masih tinggi dan perlu mendapatkan penanganan yang signifikan agar penderita asma mampu memiliki kualitas hidup yang baik.5 Asma tergolong kepada 10 penyakit penyebab kematian di Sumatera Barat namun, data mengenai jumlah penderita asma yang obes dan pola sebaran tingkat kontrol penderita asma belum diketahui, sementara prevalensi obesitas terus meningkat. Obesitas merupakan salah satu faktor komorbiditas yang memperburuk kondisi asma, sehingga dengan diketahuinya jumlah penderita asma yang obes dan tingkat kontrolnya, tujuan jangka panjang manajemen Tujuan jangka panjang manajemen asma adalah untuk mencapai kontrol gejala yang baik, memelihara tingkat aktivitas normal dan untuk meminimalkan risiko eksaserbasi, memperbaiki keterbatasan aliran udara dan efek samping pengobatan.1 http://jurnal.fk.unand.ac.id 91 tidak memiliki data yang lengkap seperti berat badan, tinggi badan dan tidak adanya data asthma control test (ACT) selama 2 bulan berturut-turut pada periode tersebut. asma dapat dicapai karena pengobatan tidak hanya terfokus pada obat-obatan medis melainkan melibatkan penanganan terhadap faktor yang memperburuk asma sehingga dapat meminimalisasi risiko eksaserbasi dan diharapkan dapat menurunkan angka kematian akibat asma di Sumatera Barat. Tabel 1. Distribusi pasien asma berdasarkan jenis kelamin Jenis Kelamin n % Laki-laki 31 49,2 Perempuan 32 50,8 Total 63 100 Tabel 1. Distribusi pasien asma berdasarkan jenis kelamin METODE Jenis penelitian ini adalah deskriptif observasional retrospektif dengan populasi seluruh pasien yang terdiagnosis asma dengan pemeriksaan spirometri di Poli Paru RSUP Dr. M. Djamil Padang pada periode 1 Januari hingga 31 Desember 2016. Sampel penelitian ini adalah seluruh pasien asma di Poli Paru RSUP Dr. M. Djamil Padang, yang memenuhi kriteria inklusi dan tidak memiliki kriteria eksklusi. Kriteria inklusi sampel adalah pasien asma yang datang berobat ke poli paru RSUP. Dr. M.Djamil Padang dari 1 januari sampai 31 Desember 2016, dan pasien asma yang memiliki data ACT 2 bulan berturut- turut tanpa riwayat kunjungan ke Instalasi Gawat Darurat (IGD) dan rawatan dalam 1 bulan terakhir. Kriteria eksklusi adalah pasien asma dalam keadaan hamil. Penelitian dilaksanakan dari September 2017 hingga Februari 2018. Berdasarkan data di atas dapat diketahui bahwa jumlah pasien asma laki-laki dan perempuan hampir sama besar, namun persentase tertinggi ditemukan pada perempuan yaitu sebesar 50,8%. Tabel 2. Distribusi pasien asma berdasarkan umur Usia (tahun) n % 18 – 40 9 14,3 40 – 60 30 47,6 >60 24 38,1 Total 63 100 Tabel 2. Distribusi pasien asma berdasarkan umur Data di atas menunjukkan bahwa pasien asma terbanyak pada kelompok umur dewasa madya 40 – 60 tahun (47,6%). Pengolahan data dilakukan dengan cara memeriksa kelengkapan data, menyusun data yang berhasil dikumpulkan dengan mengelompokkan data sesuai kriteria yang diinginkan, kemudian dilakukan penghitungan jumlah serta persentase setiap kelompoknya. Data disajikan dalam bentuk tabel dan grafik dengan menggunakan program komputer. Tabel 3. Distribusi pasien asma berdasarkan status pekerjaan Pekerjaan n % Pedagang 0 0 Buruh/Tani 6 9,5 PNS 20 31,7 TNI/Polri 0 0 Pensiunan 6 9,5 Wiraswasta 5 7,9 IRT 13 20,6 Tidak Bekerja 4 6,3 Dll 9 14,3 Total 63 100 Tabel 3. Distribusi pasien asma berdasarkan status pekerjaan Jurnal Kesehatan Andalas. 2019; 8(1) PEMBAHASAN PEMBAHASAN Penelitian ini bertujuan untuk mengetahui gambaran karakteristik tingkat kontrol penderita asma berdasarkan Indeks Massa Tubuh (IMT) di Poli Paru RSUP Dr. M. Djamil Padang. Data diambil dari bagian instalasi rekam medis di RSUP Dr. M. Djamil Padang dan dipilih berdasarkan kriteria yang telah ditetapkan. Dari 166 pasien asma yang memenuhi kriteria inklusi hanya 63 pasien. Tabel 4 menunjukkan bahwa IMT pasien asma terbanyak berada pada klasifikasi berat badan berlebih & obes (49,2%) sementara IMT berat badan kurang/underweight (6,3%). Jenis kelamin pasien asma antara laki-laki dan perempuan didapatkan jumlah yang hampir sama besar, yaitu 49,2% laki-laki dan 50,8% perempuan. Hal ini sama dengan penelitian yang dilakukan oleh Shaheen et al tentang pengaruh indeks massa tubuh (IMT) pada penderita asma dewasa, didapatkan perempuan sebesar 55% dan laki-laki 45%.10 Tabel 5. Distribusi pasien asma berdasarkan tingkat kontrol asma Klasifikasi ACT n % Tidak Terkontrol 21 33,3 Terkontrol Sebagian 39 61,9 Terkontrol Penuh 3 4,8 Total 63 100 Tabel 5. Distribusi pasien asma berdasarkan tingkat kontrol asma Penelitian tersebut menunjukkan antara laki- laki dan perempuan didapatkan jumlah yang hampir sama besar, namun secara umum dari didapatkan perempuan cenderung menderita asma dibandingkan dengan laki-laki. Hal ini disebabkan diameter saluran napas dan fungsi paru pada laki-laki lebih besar daripada perempuan. Resistensi saluran napas berbanding terbalik sebanyak 4 kali lipat dibandingkan dengan diameter saluran napas, sehingga resistensi saluran napas dengan mudah meningkat ketika diameter saluran napas kecil. Faktor lainnya adalah terdapatnya polimorfisme genetik pada perempuan tetapi tidak ditemukan pada laki-laki seperti cyclooxygenase-2-765C. Polimorfisme genetik ini meningkatkan kapasitas monosit untuk memproduksi prostaglandin yang dapat meningkatkan inflamasi pada saluran napas. Selain pengaruh dari anatomi dan genetik, hormon pada wanita juga memiliki peranan penting dalam menyebabkan asma. Progesteron meningkatkan sekresi IL-4 dan estrogen meningkatkan tingkat IgE total.11 Berdasarkan data di atas diketahui bahwa tingkat kontrol asma paling banyak didapatkan pada klasifikasi kelompok asma terkontrol sebagian yaitu sebesar 61,9%), sedangkan yang terkontrol penuh hanya 4,8%. Tabel 6. Persentase pasien asma berdasarkan tingkat kontrol asma dan IMT Klasifikasi IMT ACT Terkontrol Penuh Terkontrol Sebagian Tidak Terkontrol Berat Badan Kurang 0% 4,8% 1,6% Normal 3,2% 23,8% 17,5% Berat Badan Lebih & Obes 1,6% 31,7% 15,9% Total 4,8 61,9 33,3 Tabel 6. HASIL Telah dilakukan penelitian tentang gambaran karakteristik tingkat kontrol penderita asma berdasarkan indeks massa tubuh di Poli Paru RSUP Dr. M. Djamil Padang pada tahun 2016 yang dilakukan di bagian rekam medis RSUP Dr. M. Djamil Padang periode 1 Januari sampai 31 Desember 2016. Jumlah pasien yang terdiagnosis asma selama periode tersebut sebesar 166 orang namun, sampel yang terkumpul sesuai dengan kriteria inklusi berjumlah 63 orang. Hal ini dikarenakan sebagian besar sampel Tabel di atas menunjukkan bahwa pada umumnya (31,7%) pasien asma bekerja sebagai Pegawai Negri Sipil (PNS) dan tidak ada pasien asma yang bekerja sebagai pedagang maupun TNI/Polri. Jurnal Kesehatan Andalas. 2019; 8(1) http://jurnal.fk.unand.ac.id 92 92 Tabel 4. Distribusi pasien asma berdasarkan indeks massa tubuh (IMT) Klasifikasi IMT n % Berat badan kurang 4 6,3 Normal 28 44,4 Berat badan lebih & Obes 31 49,2 Total 63 100 PEMBAHASAN Persentase pasien asma berdasarkan tingkat kontrol asma dan IMT Usia pasien asma terbanyak adalah pasien usia dewasa madya (40 – 60 tahun) yaitu 30 orang (47,6%), diikuti oleh usia dewasa lanjut (>60 tahun) 24 orang (38,1%) dan usia dewasa awal (20 – 40 tahun) 9 orang (14,3%). Distribusi serupa ditemukan pada penelitian yang dilakukan oleh Fuad Akbar (2014) mengenai hubungan IMT dengan tingkat kontrol asma Berdasarkan data di atas diketahui bahwa pasien asma yang mencapai asma terkontrol penuh terbanyak ditemukan pada klasifikasi IMT normal (3,2%), asma terkontrol sebagian terbanyak ditemukan pada IMT berat badan lebih & obes (31,7%), dan asma tidak terkontrol terbanyak ditemukan pada IMT normal (17,5%). Jurnal Kesehatan Andalas. 2019; 8(1) http://jurnal.fk.unand.ac.id 93 sebesar 2,8%.12 Hasil penelitian ini sama dengan yang dilakukan oleh Sastre et al yang meneliti hubungan antara indeks massa tubuh dan tingkat kontrol asma di Spanyol didapatkan jumlah pasien yang underweight 5%, normal 33,8%, overweight 36,3% dan obesitas lebih dari 24,6%.15 pada pasien asma, didapatkan usia pasien dewasa madya 40 orang (56,3%), dewasa lanjut 19 orang (26,8%), dan usia dewasa awal 12 orang (16,9%).12 Penelitian tersebut menunjukkan bahwa asma dapat terjadi pada semua usia. Usia pasien terbanyak pada rentang 40 – 60 tahun karena pada usia tersebut pasien asma dapat menderita asma sejak masa anak atau remaja yang berlangsung terus atau timbul setelah periode remisi tetapi dapat juga terjadi pada dewasa tua atau lebih dari 65 tahun. Asma usia lanjut (ageing asthmatic) secara klinis terdiri dari 2 kelompok yaitu; kelompok yang menderita asma sejak masa anak atau remaja dan asma onset lambat. Di samping itu, insiden asma meningkat seiring dengan bertambahnya usia. Pada penelitian ini pasien dengan usia diatas 60 tahun mengalami penurunan yaitu sebanyak 24 orang (38,1%). Hal ini dapat terjadi karena pada populasi usia lanjut sering terjadi underdiagnosed yang disebabkan oleh penurunan sensitivitas terhadap gejala, gejala klinis yang tidak spesifik dan sering menjadi efek perancu dari komorbid.13 Penelitian ini mendapatkan IMT pasien asma paling banyak berada pada klasifikasi berat badan berlebih. PEMBAHASAN Teori yang menjelaskan obesitas dapat menyebabkan asma melibatkan beberapa faktor seperti genetik, hormonal, diet, dan mekanik.15 Terdapat satu teori yang mendukung pandangan kurang umum mengenai asma dapat menyebabkan obesitas, yaitu individu dengan asma membatasi tingkat aktivitas mereka karena takut aktivitas dapat memicu eksaserbasi asma, yang kemudian mengarah ke gaya hidup yang santai dan peningkatan risiko obesitas.16 Tingkat kontrol asma terbanyak ditemukan pada kelompok asma terkontrol sebagian (61,9%), asma tidak terkontrol (33,3%), sementara hanya 4,8% pasien asma yang dapat mencapai asma terkontrol penuh. Hal ini berbeda dengan penelitian yang dilakukan oleh Cendrawati dan Amin (2016) mengenai faktor yang berpengaruh pada tingkat kontrol asma, didapatkan tingkat kontrol asma pada pasien asma lebih banyak berada pada tingkat asma tidak terkontrol yaitu sebesar 48,7%, diikuti dengan asma terkontrol sebagian sebesar 35,9%, dan 15,4% asma terkontrol penuh.17 Penelitian lain yang dilakukan oleh Fuad Akbar mendapatkan asma tidak terkontrol 88,7%, asma terkontrol sebagian 11,3%, dan tidak ada pasien yang terkontrol penuh.12 Berdasarkan data di Poliklinik Alergi Ilmu Penyakit Dalam Rumah Sakit Cipto Mangunkusumo didapatkan, 64% pasien tidak terkontrol, 28% terkontrol sebagian, dan 8% terkontrol penuh.7 Berdasarkan status pekerjaan dapat dilihat bahwa sebagian besar (31,7%) pasien asma bekerja sebagai Pegawai Negri Sipil (PNS), diikuti dengan pekerjaan Ibu Rumah Tangga (IRT) sebesar 20,6%, Dll 14,3%, Buruh/Tani dan Pensiunan sama-sama berjumlah 9,5%, Tidak bekerja sebesar 6,3% dan 0% untuk pedagang dan TNI/Polri. Pada penelitian ini tidak dilakukan pengelompokan tingkat kontrol asma berdasarkan status pekerjaan sehingga tidak dapat diperoleh bagaimana gambaran kontrol asma berdasarkan pekerjaan. IMT pasien asma terbanyak berada pada klasifikasi berat badan lebih & obes (49,2%), diikuti dengan berat badan normal (44,4%), dan berat badan kurang (6,3%). Penelitian ini berbeda dengan penelitian yang dilakukan oleh Sri Khodijah mengenai hubungan antara IMT dengan tingkat kontrol asma pada penderita asma umur lebih dari atau sama dengan 18 tahun, didapatkan IMT pasien asma paling banyak berada pada klasifikasi IMT normal 52,3%, dan hanya 47,7% IMT berlebih.14 Penelitian oleh Fuad Akbar mendapatkan IMT sebagian besar pasien asma adalah IMT normal sebesar 43,7%, sementara obesitas sebesar 18,3%, dan berat badan kurang Hasil yang berbeda antara penelitian ini dengan penelitian di atas yang mendapatkan tingkat kontrol pasien asma terbanyak berada pada level asma terkontrol sebagian kemungkinan disebabkan penelitian ini mengambil nilai rata-rata dari data ACT pasien asma 2 bulan berturut-turut yang tidak mengalami eksaserbasi dalam 1 bulan terakhir sehingga data ACT yang diambil cenderung dalam rentang terkontrol. Jurnal Kesehatan Andalas. PEMBAHASAN 2019; 8(1) Jurnal Kesehatan Andalas. 2019; 8(1) http://jurnal.fk.unand.ac.id 94 mempengaruhi hasil penelitian seperti riwayat merokok, pengetahuan terhadap asma, kepatuhan dalam menjalani pengobatan, dan penyakit komorbid. Mengenai faktor perancu berupa riwayat merokok, penelitian yang dilakukan oleh Boulet et al (2008) menunjukkan bahwa pasien asma yang merokok mempunyai tingkat kontrol asma yang lebih buruk dibandingkan pasien dengan asma yang telah berhenti merokok atau yang tidak pernah merokok.20 Braido (2013) mengungkapkan bahwa adanya penyakit komorbid pada pasien asma seperti penyakit refluks gastroesofagus, gangguan tidur, obstructive sleep apnea dan rinitis mempengaruhi saluran napas, memperberat manajemen penyakit, dan pencapaian asma kontrol.21 Berdasarkan tingkat kontrol asma dan IMT dapat dilihat bahwa tingkat asma terkontrol penuh terbanyak didapatkan pada mereka dengan IMT normal diikuti dengan IMT berat badan lebih & obes dan untuk asma tidak terkontrol paling banyak didapatkan pada IMT normal kemudian diikuti dengan IMT berat badan lebih & obes dan IMT berat badan kurang/underweight. Hasil penelitian ini sama dengan penelitian yang dilakukan oleh Fuad Akbar mendapatkan distribusi yang hampir serupa yaitu, IMT normal cenderung memiliki tingkat kontrol asma berupa asma tidak terkontrol yaitu sebesar 39,5%, asma terkontrol sebagian paling banyak didapatkan pada IMT berat badan lebih & obes sebesar 7,1%, dan asma terkontrol penuh terbanyak pada IMT normal yaitu 43,7%.12 Namun hasil penelitian ini berbeda dengan penelitian yang dilakukan oleh Atmoko et al (2009) mengenai pengaruh obesitas terhadap tingkat kontrol asma yang mendapatkan asma tidak terkontrol pada pasien asma dengan IMT ≥25 kg/m2 sebesar 52,3%, dan 47,7% pada IMT <25 kg/m2.18 SIMPULAN Sebagian besar pasien asma berada pada kelompok umur dewasa madya 40 – 60 tahun dengan jenis kelamin terbanyak pada perempuan. IMT pasien asma sebagian besar berada pada rentang berat badan berlebih & obes Asma terkontrol penuh terbanyak ditemukan pada IMT normal, asma tidak terkontrol terbanyak pada IMT normal, dan asma terkontrol sebagian paling banyak didapatkan pada IMT berat badan lebih & obes. Studi epidemiologi yang mengevaluasi sejumlah faktor risiko dan hubungannya dengan kontrol asma telah menunjukkan hubungan yang bermakna antara obesitas dengan kontrol asma yang buruk. Demoly et al (2009), menggunakan kuesioner Asthma Control Test (ACT) untuk mengukur kontrol asma. Tiga puluh persen dari mereka yang memiliki kontrol asma yang buruk memiliki indeks massa tubuh yang lebih tinggi atau sama dengan 30 kg/m2 dibandingkan dengan 22,7% pasien dengan kontrol asma yang baik.19 Pasien asma dengan IMT yang tinggi memiliki skor Asthma Control Questionnaire (ACQ) yang tinggi setelah dilakukan penyesuaian terhadap usia, jenis kelamin dan keparahan asma yang ditentukan berdasarkan pedoman GINA. Pada penelitian tersebut menunjukkan pasien dengan IMT yang lebih tinggi memiliki gejala pagi hari yang lebih sering, keterbatasan aktivitas sehari-hari yang lebih besar, sesak napas, mengi, dan membutuhkan pengobatan yang lebih sering.19 DAFTAR PUSTAKA 1. National Health Lung and Blood Institute. Global initiatif for asthma. Global strategy for asthma management and Prevention; 2017. 2. World Health Organization (WHO). Asthma 2016 (diakses 15 Desember 2017). Tersedia dari: http://www.who.int/mediacentre/factsheets.html 3. National Center Health Statistic. Asthma. 2016. (diakses tanggal 15 Desember 2017). Tersedia dari: http://www.cdc.gov/nchs/fastats/asthma.htm 4. Badan Penelitian dan Pengembangan Kesehatan Departemen Kesehatan RI. Riset Kesehatan Dasar. Jakarta: Departemen Kesehatan RI; 2013. 5. Dinas Kesehatan Kota Padang. Profil Kesehatan Kota Padang. 2013. (diakses 13 September 2017). Tersedia dari: http://dinkeskotapadang1.files. wordpress.com/2014/narasi+profil+2013+edisi+ter bit+2014.pdf 5. Dinas Kesehatan Kota Padang. Profil Kesehatan Kota Padang. 2013. (diakses 13 September 2017). Tersedia dari: http://dinkeskotapadang1.files. wordpress.com/2014/narasi+profil+2013+edisi+ter bit+2014.pdf Hasil yang berbeda antara penelitian ini dengan penelitian di atas kemungkinan disebabkan adanya variabel-variabel perancu yang tidak dimasukkan dalam penelitian ini sehingga mungkin saja dapat Jurnal Kesehatan Andalas. 2019; 8(1) http://jurnal.fk.unand.ac.id 95 umur lebih dari atau sama dengan 18 tahun di bbkpm Surakarta [naskah publikasi]. Surakarta: 2014. 6. Novosad S, Khan S, Bruce W. Role of obesity in asthma control, the obesity-asthma phenotype. J Allergy 2013;58(5):1-5. 7. Yunus F. The Asthma Control Test, A new tool to improve the quality of asthma management. Dalam: Suryanto E, Suradi, Reviono, Rima A, editor (penyunting). Preceeding book perhimpunan dokter paru Indonesia. Edisi ke-1. Surakarta: Indah Comp 2005.hlm.361. 15. Sastre J, Olagube JM, Vina AL, Vega JM. Increased body mass index does not lead to a worsening of asthma control in a large adult asthmatic population in spain. J Investig Allergol and Clin Immunol. 2010;20(7):551–5. 16. Chinn S, Rona RJ, Can the increase in body mass index explain the rising trend in asthma in children?. Thorax. 2001;56(11):845-50. 8. Liu Y, Pleasants RA, Croft JB. Body mass index, respiratory conditions, asthma, and chronic obstructive pulmonary disease. J Respir Med. 2015;5(109):851-9. 17. Cendrawati NW, Amin M. Faktor yang berpengaruh pada tingkat kontrol asma di RSUD Dr. Soetomo Surabaya. J Respir Indo. 2016;36(1):41-6. 9. Trunk C, Ulrik CS. Obesity and asthma: impact on severity, asthma control, and response to the therapy. Respir Care. 2013;58(5):867-72. 18. Atmoko W, Yunus F, Wiyono WH. Prevalence of controlled asthma in asthma clinic Persahabatan Hospital Jakarta 2009 (abstract). Respir. 2009;14:247. 10. Shaheen SO, Sterne JA, Montgomery SM. Birth weight, body mass index, and asthma in young adults. Thorax. 1999;54:396-402. 19. Demoly P, Paggiaro P, Plaza V. Prevalence of asthma control among adults in france, Germany, Italy, Spain, and the UK. Europ Resp Rev. 2009;44(112):105-12. 11. Choi IS. DAFTAR PUSTAKA Gender-specific asthma treatment. Allergy Asthma Immunol. 2011; 3(2):74-80. 12. Akbar F. Hubungan indeks massa tubuh dengan tingkat kontrol asma pada pasien asma di RSU Dr. Soedarso Pontianak [skripsi]. Pontianak: Fakultas Kedokteran Universitas Tanjungpura; 2014. 20. Boulet LP, Gerald MF, Melvor RA, Simmerman S, Chapman KR. Influence of current or former smoking on asthma management and control. Can respir J. 2008;15:275-9. 13. Marlen FS, Yunus F. Asma pada usia lanjut. J Respir Indo. 2008;28(3):11. 21. Braido. Failure in asthma control: reasons and consequences. Genoa Italy: Hindawi publishing corp; 2013. 14. Khodijah S. Hubungan antara indeks massa tubuh dengan tingkat kontrol asma pada penderita asma Jurnal Kesehatan Andalas. 2019; 8(1) Jurnal Kesehatan Andalas. 2019; 8(1)
https://openalex.org/W4385222163	https://www.qeios.com/read/4M83EK/pdf	English	null	Review of: "Cloud-based geospatial services for building capacity and safeguarding heritage in climatically marginal landscapes"	null	2,023	cc-by	400	Qeios, CC-BY 4.0 · Review, July 25, 2023 Review of: "Cloud-based geospatial services for building capacity and safeguarding heritage in climatically marginal landscapes" Zhiyuan Zhu1 1 Northwest A&F University Zhiyuan Zhu1 1 Northwest A&F University Potential competing interests: No potential competing interests to declare. Qeios ID: 4M83EK · https://doi.org/10.32388/4M83EK Potential competing interests: No potential competing interests to declare. This is a meaningful study. The author introduces five tools written in GEE, demonstrating the universality of spatial cloud computing and remote sensing - they provide a way to communicate the threats posed by floods, erosion, and sand dune movement to heritage, as well as rapid urbanization, making mitigation strategies more intelligent. Let us see the possibility of using technology to visualize and mitigate heritage threats in some of the most remote regions of the world and extreme climate conditions on Earth. There are several issues that still need further discussion: 1. Who are the 5 tools written by GEE introduced by the author for? Farmers or the government? In most impoverished and underdeveloped regions of the world, local residents (especially farmers) have not received education and face difficulties in living. They make every effort to maintain their livelihoods and have no ability or willingness to explore the issue of heritage protection. For local governments, they are more concerned with how to purchase and adopt this technology, that is, who comes and uses it, how to use it, and how to introduce it for effective management, rather than government officials learning this technology themselves. So, this article provides a detailed introduction to the application prospects of GEE in the most remote areas and extreme climate conditions on Earth, without discussing the fundamental question of how these technologies can truly be applied in these areas, whether through the market behavior of enterprises or the public welfare activities of public welfare organizations? GEE is a great tool. Currently, it relies on many academic papers published by GEE, but it is essentially a commercial platform. How to apply it to real life requires the government, Google, other enterprises, scientists, ordinary residents, and other parties to jointly negotiate, and how to promote technology and enterprise development, achieving the unity of social and economic benefits. Based on the above, I also hope to see a richer, broader, and deeper level of thinking in this article, not just at the technical level. Qeios ID: 4M83EK · https://doi.org/10.32388/4M83EK 1/1
https://openalex.org/W3180878429	https://hal.archives-ouvertes.fr/hal-03275521v2/document	English	null	Initiation of deep convection through deepening of a well‐mixed boundary layer	Quarterly journal of the Royal Meteorological Society	2,021	cc-by	8,813	R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Abstract The present study examines mechanisms by which deep moist convection may be initiated through deepening of a well-mixed convective boundary layer under large-scale low-level convergence. The process is examined using a standard formulation for the well-mixed boundary layer, in which the depth increases exponentially with time under a constant convergence with height. Considera- tion is also given to a case with a more realistic mean vertical velocity profile which has a maximum in the middle troposphere and attenuates to zero at the tropopause. In the latter case, the unstable well-mixed layer under convergence grows to a troposphere-deep mixed layer. Importantly, under these scenarios, the so-called convective inhibition (CIN) does not inhibit deepening of a well-mixed convective boundary layer, nor does deep moist convection need to overcome CIN to initiate. Correspondence J.-I. Yano, CNRM, Météo-France, 31057 Toulouse Cedex, France. Email: jiy.gfder@gmail.com Correspondence J.-I. Yano, CNRM, Météo-France, 31057 Toulouse Cedex, France. Email: jiy.gfder@gmail.com Initiation of deep convection through deepening of a well-mixed boundary layer Jun-Ichi Yano CNRM UMR3589, CNRS and Météo-France, Toulouse, France CNRM UMR3589, CNRS and Météo-France, Toulouse, France Received: 8 February 2021 Revised: 14 June 2021 Accepted: 27 June 2021 Received: 8 February 2021 Revised: 14 June 2021 Accepted: 27 June 2021 Received: 8 February 2021 Revised: 14 June 2021 Accepted: 27 June 2021 DOI: 10.1002/qj.4117 DOI: 10.1002/qj.4117 © 2021 The Authors. Quarterly Journal of the Royal Meteorological Society published by John Wiley & Sons Ltd on behalf of the Royal 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 original work is properly cited. wileyonlinelibrary.com/journal/qj 1 1 INTRODUCTION has been gradually discarded based on a criticism that moisture convergence is better considered to be feedback of large-scale circulations to convection in response to growing convection, rather than as a prerequisite for ini- tiating convection (cf., Emanuel et al., 1994). However, the attempt to statistically predict thunderstorms by mois- ture convergence has hardly died out even today (e.g., van Zomeren and van Delden, 2007). It is phenomenologically well established that low-level convergence tends to induce deep moist convection (e.g., Byers and Braham, 1949; Weaver, 1979; Wilson and Schreiber, 1986). A very simple explanation for this ten- dency is to interpret it as a mechanism to gather mois- ture to the lower atmosphere, which is necessary to ini- tiate convection. The idea has naturally led to the use of moisture convergence as a condition for closing a con- vection parametrization, as originally proposed by Kuo (1965, 1974). This idea has been popular during earlier developments of convection parametrization (e.g., Anthes, 1977; Bougeault, 1985; Tiedtke, 1989). However, the idea Processes leading to the initiation of deep moist con- vection are somehow yet not well understood. Our lack of understanding further leads to technical difficulties in properly timing initiation of deep convection in numer- ical modelling at large scales both for weather forecasts and global change projections. In the context of convection wileyonlinelibrary.com/journal/qj Q J R Meteorol Soc. 2021;1–11. YANO YANO 2 2 well-mixed layer would simply keep growing even in the presence of CIN. Intuitively, a deeper well-mixed layer pro- vides a more favourable condition for initiation of deep moist convection. Although the present study does not address the question of the initiation explicitly, it naturally follows that initiation of moist deep convection should not be hindered by the presence of CIN either, because the well-mixed layer is already growing at its expense. parametrization, this is a ‘trigger’ problem: a certain trig- ger condition must be satisfied for turning on a convection parametrization (cf. review at Yano et al., 2013). A standard approach for addressing convective initia- tion in terms of parcel lifting (e.g., Roff and Yano, 2002; Manzato and Morgan, 2003) may be summarized as fol- lows. A small quantity of air mass is lifted from the bound- ary layer (or surface) in isolation, maintaining its pressure as the same as the environment. 1 INTRODUCTION Under a standard sound- ing, a lower layer of the atmosphere is stably stratified, thus a parcel lifted from the surface first loses its buoy- ancy with ascent until it reaches a lifting condensation level (LCL). At that level, the parcel is saturated. Beyond that level, latent heating by condensation of water vapour curbs the decrease of buoyancy with height. If the par- cel is moist enough, the increase of buoyancy by latent heat overcompensates the decrease of buoyancy by adi- abatic cooling, and finally, the parcel become positively buoyant, and convection may be initiated as a result of a buoyancy-induced instability. The level at which the par- cel buoyancy turns from negative to positive is called the level of free convection (LFC) for this reason. The originality of the present article is to focus on the dynamics of the well-mixed boundary layer in the pres- ence of large-scale convergence. Studies of the well-mixed boundary layer are usually focused on an environment with large-scale divergence, because it is an ideal envi- ronment for study in isolation. Once large-scale conver- gence kicks in, the well-mixed boundary layer is destroyed and replaced by deep moist convection, which would be a less interesting situation from a point of view of boundary-layer meteorology. In turn, the deep-convection studies are focused on a state with low-level convergence, but with fewer considerations on the processes within a pre-existing well-mixed boundary layer. The present article points out an important link of the well-mixed boundary-layer dynamics to the deep moist-convective dynamics. Under this parcel-based description of convective initi- ation, an air parcel must somehow be lifted from a surface layer to LFC for moist convection to be initiated. A cer- tain external force is required to lift a parcel to this level, because this layer is negatively buoyant, and thus a par- cel cannot be lifted spontaneously. A vertical integral of the buoyancy over this layer is called the convective inhi- bition (CIN), thus convection must be initiated by over- coming this barrier of CIN, according to the parcel-based description. Variations to this problem with alternative approaches are found in reviews by, for example, Nugent and Smith (2014). Nevertheless, all the existing approaches take a similar line of argument in which a barrier of CIN must somehow be overcome to initiate convection. 1 INTRODUCTION Furthermore, this very intuitive parcel-based argument provides a basis for some convective initiation condi- tions in parametrizations (e.g., Mapes, 2000; Bretherton et al., 2004). The present article emphasizes a simple (even trivial) fact that, under the presence of large-scale convergence, the well-mixed boundary layer simply continues to deepen with time, so long as standard mixed-layer assumptions remain valid. Especially when a standard assumption of constant convergence with height is taken, it can be shown that the well-mixed layer height deepens with time expo- nentially, a type of instability. I propose it as an important precondition for transformation of the well-mixed bound- ary layer into deep moist convection. Considering that there is less familiarity in the deep-convection community with the problems of the well-mixed boundary layer, the article proceeds rather ped- agogically; the tendency of the well-mixed boundary layer to keep deepening with low-level convergence is demon- strated in two different ways. In the next section, a sim- ple geometrical consideration of heat (entropy) budget is employed, thus the analysis can be understood without prior knowledge of the well-mixed boundary-layer dynam- ics. In Section 3, the same analysis is repeated with a standard formulation for the well-mixed boundary layer. In the latter case, a solution of exponentially growing mixed-layer depth naturally arises under a condition of low-level convergence. The basic claim of the present arti- cle is better supported by presenting these two different simple boundary-layer models, with substantially different assumptions in detail, which lead essentially to the same conclusion. At the same time, the overall equivalence of these two models is established by deriving the latter from The present article will present a different perspec- tive based on the dynamics of a convectively well-mixed boundary layer. More precisely, the article presents initi- ation of deep moist convection as a natural consequence of continuous deepening of a well-mixed boundary layer under a presence of large-scale convergence. From a point of view of the dynamics of the well-mixed boundary layer, the inversion at the top, as a major source of CIN, does not hinder a growth of the well-mixed boundary layer, but rather it is a consequence of a growth of the well-mixed boundary layer; the convective boundary-layer air is continuously penetrating into a stable layer so that an inversion is created. 1 INTRODUCTION Thus, if nothing else happens, the 3 (a) (b) F I G U R E 1 Schematics describing the well-mixed boundary layer heat budget. (a) When F+ > 0, the shaded area indicates accumulated entropy, Ht, over time, t, by the surface flux, H. (b) When F+ < 0, the gain of entropy (red shading) due to the surface flux, H, for the growth of mixed layer is equal to the sum of the surface-flux supply, Ht, and an excess entropy (blue shading) due to the forcing, F+, to the free atmosphere [Colour figure can be viewed at wileyonlinelibrary.com] YANO (a) the former in the Appendix A. The article is concluded with further discussion. (a) Throughout the article, a horizontally homogeneous atmosphere is considered for the sake of a lucid and sim- ple demonstration. Thus, all the variables depend only on the height, z. To actually address the question of trans- formation of the well-mixed boundary layer into cumulus convection, the horizontal dependence of a growing per- turbation must be considered explicitly by removing this assumption. However, as it turns out, deriving a solu- tion becomes much more involved. For this reason, this generalization is left for a future study. (b) (b) Furthermore, a dry atmosphere is assumed for the sake of simplicity throughout the article. A basic extension of the analysis to the moist case is straightforward, and essen- tially a matter of replacing the potential temperature by the equivalent potential temperature. Otherwise the iden- tical formulation is still applicable, which leads to identical conclusions. However, for more careful analysis, the actual formulation becomes much more involved; Section 4.2 gives further remarks. 2 A SIMPLE GEOMETRICAL ANALYSIS F I G U R E 1 Schematics describing the well-mixed boundary F I G U R E 1 Schematics describing the well-mixed boundary layer heat budget. (a) When F+ > 0, the shaded area indicates accumulated entropy, Ht, over time, t, by the surface flux, H. (b) When F+ < 0, the gain of entropy (red shading) due to the surface flux, H, for the growth of mixed layer is equal to the sum of the surface-flux supply, Ht, and an excess entropy (blue shading) due to the forcing, F+, to the free atmosphere [Colour figure can be viewed at wileyonlinelibrary.com] F I G U R E 1 Schematics describing the well-mixed boundary layer heat budget. (a) When F+ > 0, the shaded area indicates accumulated entropy, Ht, over time, t, by the surface flux, H. (b) When F+ < 0, the gain of entropy (red shading) due to the surface flux, H, for the growth of mixed layer is equal to the sum of the surface-flux supply, Ht, and an excess entropy (blue shading) due to the forcing, F+, to the free atmosphere [Colour figure can be viewed at wileyonlinelibrary.com] As an initial condition, we consider an atmosphere with a constant lapse rate, d𝜃∕dz, given in terms of the potential temperature, 𝜃. Thus 𝜃(z, t = 0) = 𝜃0 + ( d𝜃 dz ) z. (1) (1) We assume a constant heat flux, H (with units K⋅m⋅s−1) from the surface at z = 0. As a result, a well-mixed bound- ary layer is formed and grows from the surface. As is phenomenologically known, we assume that the potential temperature becomes a constant value, 𝜃m, with height, z, over this well-mixed layer extending to a height, zm. Above the well-mixed layer (z > zm), a free atmosphere is consid- ered (designated by a subscript + throughout), which is assumed to linearly evolve with time, t, under a constant forcing, F+: The well-mixed layer deepens in response to the sur- face flux, H. The supplied heat flux, Ht, over time t, must be equal to an area swept on a (𝜃, z)-plane from the ini- tial condition to a state at time t as indicated by shading in Figure 1a, thus Ht = Δ0𝜃+ Δm𝜃 2 zm, (4) (4) where Δ0𝜃= 𝜃m(t) −𝜃m(t = 0), and Δm𝜃= 𝜃(zm, t) −𝜃+ (zm, t = 0) = F+t. 2 A SIMPLE GEOMETRICAL ANALYSIS For now, we neglect an inversion and set 𝜃m = 𝜃+(zm, t). Note that when F+ < 0, Equation (4) reduces to: where Δ0𝜃= 𝜃m(t) −𝜃m(t = 0), and Δm𝜃= 𝜃(zm, t) −𝜃+ (zm, t = 0) = F+t. For now, we neglect an inversion and set 𝜃m = 𝜃+(zm, t). Note that when F+ < 0, Equation (4) d t 𝜃+(z, t) = 𝜃(z, t = 0) + F+t, (2) (2) and we may assume reduces to: Ht = zmΔ0𝜃 2 −zm(−F+t) 2 . F+ = − [ w ( d𝜃 dz ) + QR ] . (3) (3) Thus, the gain of entropy (red shading to the left) for the growth of the mixed layer is equal to the sum of the surface flux supply, Ht, and an excess entropy (blue shading to the right) provided Here, w is a prescribed large-scale vertical velocity, which is assumed to be constant in this section, and QR is a constant radiative cooling rate. YANO F I G U R E 2 Plots of Equation (10) with sgn(F+) = 1 (dashed) and sgn(F+) = −1 (solid) by evolution of the free atmosphere, as shown in Figure 1b. by evolution of the free atmosphere, as shown in Figure 1b. By referring to Figure 1, we also find that the well-mixed layer depth, zm, and the change, Δ0𝜃, of the surface potential temperature over time t are related by By referring to Figure 1, we also find that the well-mixed layer depth, zm, and the change, Δ0𝜃, of the surface potential temperature over time t are related by Δ0𝜃−F+ t = ( d𝜃 dz ) zm, or Δ0𝜃= ( d𝜃 dz ) zm + F+t. (5) (5) Substitution of Equation (5) into Equation (4) leads to: H t = 1 2 ( d𝜃 dz ) z2 m + (F+ t)zm. (6) F I G U R E 2 Plots of Equation (10) with sgn(F+) = 1 (dashed) and sgn(F+) = −1 (solid) (6) By solving the above for zm, we obtain over half a day under a modest large-scale low-level con- vergence. The evolved mixed layer is deep enough to reach the level of free convection (LFC) in common situations and, under conventional wisdom, deep moist convection will be spontaneously initiated from there. 2 A SIMPLE GEOMETRICAL ANALYSIS zm = ( d𝜃 dz )−1 { −F+t + [ (F+t)2 + 2 ( d𝜃 dz ) Ht ]1∕2} (7) by choosing a solution with positive zm. When the envi- ronmental air is descending (i.e., w < 0), the forcing, F+, is positive, and we find that the mixed-layer depth asymp- totically evolves as zm →H∕F+. On the other hand, under large-scale ascent, the forcing, F+, may become negative, and the system never reaches equilibrium, but the mixed layer keeps deepening with time. This is the situation expected under large-scale low-level convergence. A finite inversion jump, Δi𝜃= 𝜃+(zm) −𝜃m (≠0), can be included in the above analysis by simply replacing F+t by F+t −Δi𝜃. Furthermore, when a finite downward entrainment flux at the top of the mixed layer (given by rH with r a constant, taken downward positive) is further con- sidered, we just have to replace H by (1 + r)H. It transpires that the basic conclusion of Equation (8) does not change by these generalizations. The solution (7) can be presented in a more com- pact manner after non-dimensionalization by setting zm = zm̃zm and t = t∗̃t. Here, the non-dimensionalization scales for the mixed-layer depth and the time, respectively, are 3 ANALYSIS BASED ON A STANDARD MIXED-LAYER FORMULATION Furthermore, the right-hand side of Equation (9) may be expressed by z′ = z′ 0e−Dt, (15) (15) with z′ 0 an initial perturbation. Thus, when the system is divergent, associated with a mean descent, and D > 0, the equilibrium state is stable. On the other hand, when the system is convergent, associated with a mean ascent, and D < 0, the unphysical equilibrium state becomes unstable: a large-scale low-level convergence makes the well-mixed convective boundary layer grow exponentially with time. Note that the characteristic growth time-scale, D−1 ∼105 s ∼1 day is comparable to that obtained in the last section. −w′𝜃′\|\|\|z=zm- = weΔi𝜃 (10) (10) in terms of the entrainment rate, we. We re-write the verti- cal eddy flux in this manner so that the inversion strength, Δi𝜃no longer appears in the equation explicitly: dzm dt = w + we. (11) (11) Here, the result is presented in terms of a perturbation analysis. However, recall that the original full system itself (13) is linear, thus zm = zm + z′ m constitutes a full solution of Equation (13). In other words, we can set an initial per- turbation, z′ 0, large enough so that we can make zm(t = 0) = zm + z′ 0 positive, although zm is negative under con- vergence. Thus, the exponential growth of the well-mixed layer depth also becomes physically meaningful in this respect. In the following, the entrainment rate, we, is consid- ered to be a constant for the ease of analysis. It is further shown in Appendix A that Equation (9) can be derived from the geometrical formulation of the last section. As also suggested therein, Equation (11) is valid also in the limit of Δi𝜃→0. Furthermore, as in many studies of the well-mixed layer (e.g., Schubert et al., 1979), a background divergence, D, is assumed to be constant for now, and However, another unphysical aspect of this solution is that the mixed-layer depth keeps growing exponentially without reaching any equilibrium. We should realize that this behaviour is due to a rather artificial assumption of a constant divergence, D, with height. Thus, when D < 0, the large-scale vertical velocity, given by w = −Dz, simply increases linearly with height without limit. Clearly, this is unrealistic. w = −Dz. 3 ANALYSIS BASED ON A STANDARD MIXED-LAYER FORMULATION zm = H \|F+\| ∼103 m and t∗= zm* ( d𝜃 dz ) 1 \|F+\| = ( d𝜃 dz ) H F2 + ∼105s ∼1 day. zm* = H \|F+\| ∼103 m In this section, an analysis equivalent to the last section is performed based on a standard formulation for the well-mixed boundary layer (cf. Stevens, 2006), with slightly different mixed-layer-top assumptions. As seen immediately below, in this case, an equation for the depth, zm, of the well-mixed layer can be considered in a stand-alone manner, thus no other variable needs to be considered. This formulation can also be directly extended to the moist atmosphere without modifications, including a possibility of clouds at the top of a mixed layer. Evaluat- ing the associated evolution of the potential temperature (and other thermodynamic variables) is straightforward, but without providing any further insight to the given problem. Section 4.2 gives further discussion. These typical scales are obtained by setting \|F+\| ∼10−5K ⋅s−1 ∼1 K ⋅day−1, H ∼10−2K ⋅m ⋅s−1, d𝜃∕dz ∼10−3K ⋅m−1. The actual typical heat flux is 𝜌CpH ∼102 W⋅m−2, and large-scale vertical velocity is \|w\| ∼10−2 m⋅s−1. After non-dimensionalization, the solution (7) reduces to: ̃zm = −sgn(F+)̃t + (̃t2 + 2̃t)1∕2. (8) (8) We assume an inversion, Δi𝜃= 𝜃+(zm) −𝜃m, of the potential temperature at the top of the well-mixed The non-dimensional solution (8) is plotted in Figure 2: a well-mixed boundary layer can grow to a depth of 2 km YANO 5 boundary layer. The mixed layer deepens relative to a back- ground vertical motion, w, at a rate that excess entropy is transported downwards by vertical eddy flux, −w′𝜃′\|z=zm-. Thus, perturbation analysis can be automatically extended glob- ally, because Equation (13) is linear. boundary layer. The mixed layer deepens relative to a back- ground vertical motion, w, at a rate that excess entropy is transported downwards by vertical eddy flux, −w′𝜃′\|z=zm-. Thus, The perturbation problem around the equilibrium solution is given by (d dt + D ) z′ m = 0 (dzm dt −w ) Δi𝜃= −w′𝜃′\|\|\|z=zm-. (9) (9) with a prime designating a perturbation. The solution is immediately obtained as Here, zm−refers to a level immediately below the mixed-layer height, and a possible contribution of radia- tion is neglected. Alternatively, this contribution can be included as a part of vertical eddy flux. 4.1 Summary and implications (18) The present study suggests that deep moist convection is initiated through the deepening of a well-mixed convec- tive boundary layer under a large-scale low-level conver- gence. This tendency of the well-mixed boundary layer is demonstrated by two similar, simple theoretical models, but which are substantially different in detail. The first, considered in Section 2, is based on a simple geometrical argument of the heat budget of the well-mixed boundary layer, assuming a constant large-scale vertical velocity with height. In this case, the well-mixed layer depth increases in quasi-linear manner under a large-scale convergence over a half day period up to a depth of a few kilometres. The second, considered in Section 3, is based on a stan- dard formulation for the depth of a well-mixed layer. When the large-scale divergence (or convergence) is assumed to be constant with height, as is commonly assumed in the- oretical analyses, the well-mixed layer simply grows expo- nentially with time under convergence. A more realistic solution is obtained by assuming a profile of the large-scale vertical velocity, which reaches the maximum at the mid- dle troposphere, and vanishes at the top of the troposphere. In this case, the unstable standard well-mixed layer grows into a troposphere-deep mixed layer. This final state could even be very crudely interpreted as a state of deep moist convection. These two equilibria are approximately given by zm ≃ we∕D and h, when the top zero-velocity level is high enough and we∕hD ≪1. Thus we obtain two equilib- rium mixed-layer depths: the first corresponds to a usual boundary-layer depth, and the second extends to a full troposphere. By setting the lower and the upper equilibrium solutions, respectively, to be z1 and z2 (thus z1 < z2), Equation (17) is rewritten as dzm dt = D h (zm −z1)(zm −z2). (19) (19) This form is much easier to solve analytically. The solu- tion is more conveniently presented in two equivalent but different forms, depending on the sign of D, so that expo- nential contributions decay with time in both cases: when a background state is diverging, zm = z1 + 𝜀z2e−Dt 1 + 𝜀e−Dt , (20) (20) and when a background state is converging, zm = z2 + (z1∕𝜀)eDt 1 + eDt∕𝜀 , (21) (21) Various alternative interpretations of deep-convection initiation under convergence are already available in lit- erature. 3 ANALYSIS BASED ON A STANDARD MIXED-LAYER FORMULATION (12) (12) By substituting Equation (12), Equation (11) reduces to By substituting Equation (12), Equation (11) reduces to (d dt + D ) zm = we. (13) (13) An equilibrium solution is identified from Equation (13) as To amend this problem, we modify the large-scale ver- tical velocity to q Equation (13) as zm = we∕D. (14) (14) w(z) = −Dz ( 1 −z h ) , (16) (16) Note that, when the background large scale is con- vergent, D < 0, then the equilibrium depth, zm, becomes negative, thus unphysical. In other words, a well-mixed layer may reach an equilibrium state only under a diver- gent large-scale background state. Nevertheless, even an unphysical equilibrium solution provides a good refer- ence point for performing a perturbation analysis. In the present case, the issue is inconsequential, because the thus w no longer grows linearly with height, but it turns back to zero at a height z = h. This height may be taken at the level of the tropopause, thus the existence of the top height, h, does not affect an initial exponential growth of zm, but ultimately influences it, when zm becomes deep enough. Here, D is a constant as before, corresponding to the divergence at z = 0. YANO YANO As a result, Equation (11) for the well-mixed layer depth turns into: to that stage. However, to describe this transformation process, the horizontal dependence of the perturbation growth must be explicitly taken into account by removing the assumption of horizontal homogeneity. dzm dt = −Dzm ( 1 −zm h ) + we. (17) (17) Equilibrium solutions are, in this case, given by Equilibrium solutions are, in this case, given by 4 DISCUSSION zm = h 2 [ 1 ± ( 1 −4we hD )1∕2] . (18) 4.1 Summary and implications At the most technical level, analyses have been per- formed under rather simple treatments at the top of the well-mixed boundary layer, assuming a constant heat flux and a constant entrainment rate in Sections 2 and 3 r ti l V lidit f th i lifi ti b YANO YANO 7 F I G U R E 3 Explosive evolution of an Arctic well-mixed boundary layer under a constant convergence, simulated by Dimitrelos et al. (2020). Shading denotes the domain-averaged droplet mixing ratio, ranging from 0.0125 g⋅kg−1 (green) to 0.35 g⋅kg−1 (red) on a logarithmic scale. Contours are the ice-crystal mixing ratio (g⋅kg−1). Reproduced from Figure 3(h) of Dimitrelos et al. (2020), but using a logarithmic scale in the vertical [Colour figure can be viewed at wileyonlinelibrary.com] YANO continues to grow by penetrating its air into an inversion layer, thus the existence of CIN does not block its growth. Another common approach is to interpret the convec- tive initiation process in term of a lifting parcel from the boundary layer. Under this picture, the main obstacle is convective inhibition (CIN), which is re-interpreted as an inversion strength, Δ𝜃, under the well-mixed layer for- mulation adopted herein. Importantly, the inversion does not hinder the deepening of the well-mixed layer at all under the standard description presented in Section 3. Phenomenologically, the well-mixed layer deepens by a downward vertical eddy flux, −w′𝜃′\|z=zm-, at the top, which is known to be approximately proportional to the sur- face flux. Although a stronger inversion may reduce the entrainment rate proportionally (cf. Equation 12), the inversion itself does not hinder the deepening of the well-mixed layer. It is rather other way round: the down- ward flux at the mixed-layer top generates the inversion. A stronger downward flux creates a stronger inversion, as a simple extension of a geometrical argument in Section 2 can show. In this manner, CIN does not play a role of con- vection inhibition in the context of a well-mixed convective boundary layer. If deep moist convection can be inter- preted as a transformation of a continuously deepening well-mixed convective boundary layer, this transforma- tion process should not be hindered by a presence of CIN either, because the well-mixed layer is already growing at its expense. 4.1 Summary and implications Under large-scale descent, a standard environment considered for studies of a well-mixed boundary layer, it reaches an equilibrium state due to the presence of a large-scale descent which tends to suppress the growth driven by entrainment. However, once the large-scale movement turns from descent to ascent, there is no longer a mechanism to restrict the well-mixed boundary layer to an equilibrium. Thus, it keeps growing, leading to deep convection. Based on this very simple picture, the deep-convection community should not be concerned with the existence of the so-called CIN. Unfortunately, this is not the case. In fact, for many of the parametrizations in current operational models, the initiation of convection is controlled by CIN (e.g., Zhao et al., 2009, 2016; Walters et al., 2019) 4.1 Summary and implications A historically important one is to interpret it in terms of moisture budget. Superficially, the present inter- pretation may somehow appear to be very akin to this historical interpretation, but is actually fundamentally dif- ferent. This historical interpretation is based on an analysis of the budget of the column-integrated moisture in the atmosphere. Convergence is simply a mechanism of pro- viding more moisture to an atmospheric column. There is no role of the boundary layer in this interpretation. In contrast, the moisture does not play any active role in the present analysis. The argument is solely based on a ten- dency of the boundary layer to deepen with time under a converging flow. The top of boundary layer may ulti- mately reach a LCL, and further to the LFC, thus leading to conditional instability. However, these aspects are not considered explicitly, but only implied as what follows. with 𝜀= z0 −z1 z2 −z0 , (22) with (22) and z0 is an initial depth. Thus, the depth, zm, asymptoti- cally approaches the lower and the upper equilibria, z1 and z2, respectively, when D > 0 and D < 0. So long as the stability of the standard mixed-layer with a depth zm = z1 ≃we∕D is concerned, we recover the same conclusion as previously: it is stable and unstable under divergence and convergence, respectively. However, by introducing a more realistic large-scale vertical veloc- ity profile, we can infer the subsequent evolution of an unstable boundary layer; it no longer keeps growing, but it asymptotically approaches a state of a troposphere-deep well-mixed layer. In reality, the well-mixed layer would be transformed into deep moist convection before getting 7 g c YANO 7 F I G U R E 3 Explosive evolution of an Arctic well-mixed boundary layer under a constant convergence, simulated by Dimitrelos et al. (2020). Shading denotes the domain-averaged droplet mixing ratio, ranging from 0.0125 g⋅kg−1 (green) to 0.35 g⋅kg−1 (red) on a logarithmic scale. Contours are the ice-crystal mixing ratio (g⋅kg−1). Reproduced from Figure 3(h) of Dimitrelos et al. (2020), but using a logarithmic scale in the vertical [Colour figure can be viewed at wileyonlinelibrary.com] Another common approach is to interpret the convec- tive initiation process in term of a lifting parcel from the boundary layer. 4.1 Summary and implications Under this picture, the main obstacle is convective inhibition (CIN), which is re-interpreted as an inversion strength, Δ𝜃, under the well-mixed layer for- mulation adopted herein. Importantly, the inversion does not hinder the deepening of the well-mixed layer at all under the standard description presented in Section 3. Phenomenologically, the well-mixed layer deepens by a downward vertical eddy flux, −w′𝜃′\|z=zm-, at the top, which is known to be approximately proportional to the sur- face flux. Although a stronger inversion may reduce the entrainment rate proportionally (cf. Equation 12), the inversion itself does not hinder the deepening of the well-mixed layer. It is rather other way round: the down- ward flux at the mixed-layer top generates the inversion. A stronger downward flux creates a stronger inversion, as a simple extension of a geometrical argument in Section 2 can show. In this manner, CIN does not play a role of con- vection inhibition in the context of a well-mixed convective boundary layer. If deep moist convection can be inter- preted as a transformation of a continuously deepening well-mixed convective boundary layer, this transforma- tion process should not be hindered by a presence of CIN either, because the well-mixed layer is already growing at its expense. Here, growth of the convective well-mixed layer is t l h i f r r ti f i r i d continues to grow by penetrating its air into an inversion layer, thus the existence of CIN does not block its growth. Under large-scale descent, a standard environment considered for studies of a well-mixed boundary layer, it reaches an equilibrium state due to the presence of a large-scale descent which tends to suppress the growth driven by entrainment. However, once the large-scale movement turns from descent to ascent, there is no longer a mechanism to restrict the well-mixed boundary layer to an equilibrium. Thus, it keeps growing, leading to deep convection. Based on this very simple picture, the deep-convection community should not be concerned with the existence of the so-called CIN. Unfortunately, this is not the case. In fact, for many of the parametrizations in current operational models, the initiation of convection is controlled by CIN (e.g., Zhao et al., 2009, 2016; Walters et al., 2019) 4.2 Limits of the present study The present study is based on very simple theoretical anal- yses, and it hardly presents a full picture of the problem. 4.2 The present study is based on very simple theoretical anal- yses, and it hardly presents a full picture of the problem. At the most technical level, analyses have been per- formed under rather simple treatments at the top of the well-mixed boundary layer, assuming a constant heat flux and a constant entrainment rate in Sections 2 and 3, respectively. Validity of these simplifications can be questioned. However, a large-eddy simulation (Dimitrelos et al., 2020) indeed demonstrates that such an explosive growth of a well-mixed layer actually is realized. The case here assumes a large-scale divergence of 1.5 × 10−6s−1 con- stant with height under Arctic conditions. An exponential tendency of the growth of the mixed-layer depth from Here, growth of the convective well-mixed layer is not a sole mechanism for generation of an inversion and CIN. A notable example is the so-called elevated mixed layer, which is a consequence of advection of warm moist air originating from the Gulf of Mexico to higher lati- tudes (e.g., Lanicci and Warner, 1997). However, the main point remains the same: regardless of being self-generated or externally generated, the convective well-mixed layer YANO 8 8 its transformation into deep moist convection, because the latter is characterized by localized updraughts sur- rounded by environmental descent. Instead, a well-mixed layer deepens all the way through the troposphere, as a solution from the present analysis shows. Mathemati- cally, it is straightforward to add horizontal dependence to a standard mixed-layer formulation. As a result, the formulation must also be modified in such manner that resulting local circulations arising from horizontal inho- mogeneity are also taken into account. This turns out not to be a difficulty, either. However, my own preliminary investigation reveals that the resulting analysis is rather involved and lengthy. Thus, it will be reported in a separate article. 100 to 1,500 m over a period of t = 6–30 hr is convinc- ingly shown in Figure 3 (reformatting their figure 3(h)) by making the vertical axis logarithmic. Note that in this sim- ulation, by assuming an Arctic situation, the surface-flux values are by an order of magnitude smaller than those typ- ical over the tropical oceans. Thus, if a similar simulation were performed in a tropical environment, the growth of the well-mixed layer would also be about ten times faster. 4.2 The most obvious limit in the physical formulation is the use of dry dynamics, and the contribution of mois- ture remains implicit. However, extension to the moist case is conceptually rather straightforward. Recall that the basic premise of the well-mixed layer is simply to assume that the main role of the convective dynamics is to mix the conserved variables of a system homoge- neously in the vertical. Such a variable is the potential temperature in a dry well-mixed layer. When moisture is added to a system, the main modification is to replace it by the equivalent potential temperature, because the latter is conserved along with the total water when the precip- itation can be neglected. Otherwise, the procedure of the analysis overall remains the same, although it becomes more involved in the detail; full discussions appear in Lilly (1968); Carson (1973); Deardorff (1976); Schubert et al. (1979). For the geometrical formulation in Section 2, the development of the well-mixed layer of moisture must be considered separately under this generalization, because of decreasing moisture with height rather than increasing. Appenedix B shows that an identical result (Equation 8) as for the potential temperature is obtained in the end. So long as an equation for the evolution of the well-mixed layer height, zm, is concerned, its form remains identi- cal to Equation (13), as already emphasized in Section 3. The only necessary modification is to replace the potential temperature, 𝜃, in Equations (11) and (12) by the virtual potential temperature, 𝜃v. Thus, as far as our very crude description of the evolution of well-mixed layer height is concerned, the given formulations can be extended to the moist case without any fundamental modifications. Probably, the most serious limit of the present analy- sis is in its perturbation approach. It would be relatively easy to envisage a fully nonlinear analysis under a certain numerical procedure, but with the latter detail still left to be sorted out. Inclusion of precipitation and the associated microphysics would be another aspect to be incorporated into this nonlinear formulation. Under such an approach, transformation of a convectively well-mixed boundary layer into deep moist convection may be described in full, still assuming a local vertical mixing for homogenization of variables. REFERENCES Anthes, R.A. (1977) A cumulus parameterization scheme utilizing a one-dimensional cloud model. Monthly Weather Review, 105, 270–286. Tiedtke, M. (1989) A comprehensive mass flux scheme for cumulus parameterization in large-scale models. Monthly Weather Review, 177, 1779–1800. van Zomeren, J. and van Delden, A. (2007) Vertically integrated moisture flux convergence as a predictor of thunderstorms. Atmo- spheric Research, 83, 435–445. Arakawa, A. and Schubert, W.H. (1974) Interaction of a cumulus cloud ensemble with the large-scale environment, Part I. Journal of the Atmospheric Sciences, 31, 674–701. Walters, D., Baran, A.J., Boutle, I., Brooks, M., Earnshaw, P., Edwards, J., Furtado, K., Hill, P., Lock, A.P., Manners, J., Morcrette, C.J., Mulcahy, J., Sanchez, C., Smith, C., Stratton, R.A., Tennant, W., Tomassini, L., Van Weverberg, K., Vosper, S.B., Willett, M., Browse, J., Bushell, A., Carslaw, K., Dalvi, M., Essery, R., Gedney, N., Hardiman, S., Johnson, B., Johnson, C., Jones, A., Jones, C., Mann, G., Milton, S.B., Rumbold, H., Sellar, A., Ujiie, M., Whitall, M., Williams, K. and Zerroukat, M. (2019) The Met Office Unified Model Global Atmosphere 7.0/7.1 and JULES Global Land 7.0 configurations. Geoscientific Model Development, 12, 1909–1963. https://doi.org/10.5194/gmd-12-1909-2019. Bougeault, P. (1985) A simple parameterization of the large-scale effects of cumulus convection. Monthly Weather Review, 100, 683–689. Bretherton, C.S., McCaa, J.R. and Grenier, H. (2004) A new parameterization for shallow cumulus convection and its appli- cation to marine subtropical cloud-topped boundary layers. Part I: description and 1D results. Monthly Weather Review, 132, 864–882. Browse, J., Bushell, A., Carslaw, K., Dalvi, M., Essery, R., Gedney, N., Hardiman, S., Johnson, B., Johnson, C., Jones, A., Jones, C., Byers, H.R. and Braham, R.R.Jr. (1949) The Thunderstorm. Washing- ton, DC: U.S. Govt. Printing Office. Carson, D.J. (1973) The development of a dry inversion-capped con- vectively unstable boundary layer. Quarterly Journal of the Royal Meteorological Society, 99, 450–467. Weaver, J.F. (1979) Storm motion as related to boundary-layer con- vergence. Monthly Weather Review, 107, 612–619. Wilson, J.W. and Schreiber, W.E. (1986) Initiation of convective storm at radar-observed boundary-layer convergence lines. Monthly Weather Review, 114, 2516–2536. Deardorff, J.W. (1976) On the entrainment rate of a stratocumulus-topped mixed layer. Quarterly Journal of the Royal Meteorological Society, 102, 563–583. Yano, J.-I., Bister, M., Fuchs, Z., Gerard, L., Phillips, V., Barkidija, S. and Piriou, J.M. (2013) Phenomenology of convection-parameterization closure. Atmospheric Chemistry and Physics, 13, 4111–4131. Dimitrelos, A., Ekman, A.M.L., Caballero, R. and Savre, J. ORCID Jun-Ichi Yano https://orcid.org/0000-0002-2643-7572 ORCID Jun-Ichi Yano https://orcid.org/0000-0002-2643-7572 Stevens, B. (2006) Bulk boundary-layer concepts for simplified mod- els of tropical dynamics. Theoretical and Computational Fluid Dynamics, 20, 279–304. 4.3 Implications for the convection parametrization problem The present study may be considered a first step for con- structing a convective initiation condition in a more for- mal and robust manner. A main problem to note, in this respect, is the fact that the majority of current convection parametrizations are constructed without any explicit cou- pling with the boundary layer. The present study suggests the crucial importance of such a coupling, especially for initiating deep moist convection as a result of large-scale low-level convergence. No doubt many operational fore- cast centres are already working towards this goal. How- ever, importantly, this issue should not be reduced to a narrow question of identifying, say, a coupling parame- ter. In this respect, it is worthwhile revisiting the original formulation of mass-flux convection parametrization by Arakawa and Schubert (1974); they introduced a specific well-mixed layer model, similar in design to the present study, for the purpose of coupling convection with the boundary layer. Likewise, any renewed efforts for coupling between deep convection and the boundary layer should also involve modifications in the treatment of both compo- nents. It is hoped that the present theoretical investigation suggests a general direction towards this goal. From a more formal point of view, all those extra fac- tors can be taken into account by including the possible time dependencies of various parameters (H, Ft, D, we) in both formulations. As it turns out, analytical solutions are still available in both cases even under those circum- stances, and it can also be easily shown that the main conclusions do not change so long as these parameters evolve smoothly with time. The most crucial limit of the present study is in assum- ing a horizontal homogeneity of the well-mixed layer. As long as we consider only a proper convective well-mixed boundary layer, this assumption would not be serious. However, this very assumption hinders us from describing YANO YANO 9 ACKNOWLEDGEMENTS Mapes, B.E. (2000) Convective inhibition, subgrid-scale triggering energy, and stratiform instability in a toy tropical wave model. Journal of the Atmospheric Sciences, 57, 1515–1535. An earlier conversation with Anton Beljaars motivated me to pursue this idea. Discussions with Annica Ekman and Rodorigo Caballero are acknowledged. Antonio Dim- itrelos has kindly provided Figure 3 for the present manuscript. Thanks are also due to Michael Whitall and Adrian Lock for providing a reference. Thanks are also due to the QJ chief editor, Andrew Ross, for his exceptional patience with this editorial process. Nugent, A.D. and Smith, R.B. (2014) Initiating moist convection in an inhomogeneous layer by uniform ascent. Journal of the Atmospheric Sciences, 71, 4597–4610. Roff, G.L. and Yano, J.-I. (2002) Tropical convective variability in the CAPE phase space. Quarterly Journal of the Royal Meteorological Society, 128, 2317–2333. Schubert, W.H., Wakefield, J.S., Steiner, E.J. and Cox, S.K. (1979) Marine stratocumulus convection. Part I: governing equations and horizontally homogeneous solutions. Journal of the Atmo- spheric Sciences, 36, 1286–1307. REFERENCES (2020) A sensitivity study of Arctic air-mass transformation using large-eddy simulation. Journal of Geophysical Research, 125. https://doi.org/10.1029/2019/JD031738. Emanuel, K.A., Neelin, J.D. and Bretherton, C.S. (1994) On large-scale circulation in convective atmospheres. Quarterly Jour- nal of the Royal Meteorological Society, 120, 1111–1143. Zhao, M., Held, I.M., Lin, S.-J. and Vecchi, G.A. (2009) Simula- tions of global hurricane climatology, interannual variability, and response to global warning using a 50 km resolution GCM. Jour- nal of Climate, 22, 6653–6678. Kuo, H.-L. (1965) On formation and intensification of tropical cyclones through latent heat released by cumulus convection. Journal of the Atmospheric Sciences, 22, 40–63. Zhao, M., Golaz, J.-C., Held, I.M., Ramaswamy, V., Lin, S.-J., Ming, Y., Ginoux, P., Wyman, B., Donner, L.J., Paynter, D. and Guo, H. (2016) Uncertainty in model climate sensitivity traced to rep- resentations of cumulus precipitation microphysics. Journal of Climate, 29, 543–560. Kuo, H.-L. (1974) Further studies of the parameterization of the influ- ence of cumulus convection on large-scale flow. Journal of the Atmospheric Sciences, 31, 1232–1240. Lanicci, J.M. and Warner, T.T. (1997) A case study of lid evolu- tion using analyses of observational data and a numerical model simulation. Weather and Forecasting, 12, 228–252. How to cite this article: Yano, J.-I. (2021) Initiation of deep convection through deepening of a well-mixed boundary layer. Quarterly Journal of the Royal Meteorological Society, 1–11. Available from: https://doi.org/10.1002/qj.4117 How to cite this article: Yano, J.-I. (2021) Initiation of deep convection through deepening of a well-mixed boundary layer. Quarterly Journal of the Royal Meteorological Society, 1–11. Available from: https://doi.org/10.1002/qj.4117 Lilly, D.K. (1968) Models of cloud-topped mixed layers under a strong inversion. Quarterly Journal of the Royal Meteorological Society, 94, 292–309. Manzato, A. and Morgan, G.Jr. (2003) Evaluating the sounding insta- bility with the lifted parcel theory. Atmospheric Research, 67–68, 455–473. YANO YANO 10 APPENDIX A. DERIVATION OF EQUATION (11) FROM THE SYSTEM OF SECTION 2 The derivation here suggests that the existence of a small, but finite, inversion is essential for the growth of a well-mixed layer: it grows by generating an inversion by penetrating convective plumes into a free atmosphere. In this Appendix, Equation (11) for the evolution of the mixed-layer height, zm, is derived from the system of Section 2. Two derivations are presented. The first is heuristic, which elucidates an implicit role of an inversion in the system of Section 2. The second is a full derivation. A.2. Full derivation When a full system of Section 2 is considered, reduction to Equation (11) appears only under approximations, most likely due to minor inconsistencies in the formulation of Section 2, but not of any serious nature. A.1. Heuristic derivation For a heuristic derivation, we begin with the simplest case with F+ = 0 and Δi𝜃= 0. In this case, Equations (4) and (7) reduce to Under the presence of an inversion, Equations (4) and (7) are modified into: (7) reduce to (A1) (A2) (H −H(−))t = Δ0𝜃+ Δm𝜃−Δi𝜃 2 zm, (A6) Δ0𝜃= ( d𝜃 dz ) zm + Δm𝜃−Δi𝜃. (A7) Ht = Δ0𝜃 2 zm, (A1) Δ0𝜃= ( d𝜃 dz ) zm. (A2) (H −H(−))t = Δ0𝜃+ Δm𝜃−Δi𝜃 2 zm, (A6) Δ0𝜃= ( d𝜃 dz ) zm + Δm𝜃−Δi𝜃. (A7) Ht = Δ0𝜃 2 zm, (A1) Δ0𝜃= ( d𝜃 dz ) zm. (A2) (A6) (A1) (A7) (A2) By substituting Equation (A2) into Equation (A1), we obtain: From Equation (A7), we find: By substituting Equation (A2) into Equation (A1), we obtain: 1 From Equation (A7), we find: z2 m 2 = ( d𝜃 dz )−1 Ht. z2 m 2 = ( d𝜃 dz )−1 Ht. Δ0𝜃+ Δm𝜃−Δi𝜃 2 = 1 2 ( d𝜃 dz ) zm + Δm𝜃−Δi𝜃. By taking a time derivative of the above, we obtain: By taking a time derivative of the above, we obtain: Substituting this expression into the right-hand side of Equation (A6), and taking a time derivative of the result: dzm dt = H Δ0𝜃, (A3) (A3) Δ0𝜃dzm dt + zm d dt(Δm𝜃−Δi𝜃) = H −H(−). (A8) (A8) also using Equation (A2). In a final step, we need to recover a small, but non-vanishing inversion, Δi𝜃(≠0). Note that the heat flux must decreases linearly with height, thus a simple geometrical consideration suggests also using Equation (A2). In a final step, we need to recover a small, but non-vanishing inversion, Δi𝜃(≠0). Note that the heat flux must decreases linearly with height, thus a simple geometrical consideration suggests In writing the above result, Equation (A7) has already been applied to the first term. In writing the above result, Equation (A7) has already been applied to the first term. In deriving the final result, we need to introduce sev- eral approximations. First, the temporal change of Δi𝜃is expected to be much smaller than that of Δm𝜃, thus H∕(zm −Δiz) = −H(−)∕Δiz. Here, H(−) = w′𝜃′\|z=zm- is the heat flux at z = zm- just below the inversion. By taking a limit of Δiz →0, we obtain Here, H(−) = w′𝜃′\|z=zm- is the heat flux at z = zm- just below the inversion. A.1. Heuristic derivation By taking a limit of Δiz →0, we obtain d dt(Δm𝜃−Δi𝜃) ≃d dtΔm𝜃. (A9) (A9) H∕zm →−H(−)∕Δiz. Furthermore, radiation is neglected in Equation (3), and it should be realized that it is always a good approxi- mation to set: ( ) Further recalling Equation (A2) and Δi𝜃= (d𝜃∕dz)Δiz, thus H∕Δ0𝜃→−H(−)∕Δi𝜃. (A4) (A4) ( d𝜃 dz ) zm ≃Δ0𝜃. ( d𝜃 dz ) zm ≃Δ0𝜃. Finally, by substituting Equation (A4) into Equation (A3), we obtain Using these two approximations, Using these two approximations, APPENDIX B. DEVELOPMENT OF A WELL-MIXED MOISTURE LAYER F I G U R E B1 Schematic describing the well-mixed moisture layer Development of a well-mixed layer with any conserved variable can be formulated as for the potential tempera- ture, as presented in Section 2. However, when a back- ground value decreases with height, a special consider- ation becomes necessary. This Appendix considers this issue by taking the moisture as an example. In extend- ing the formulation of Section 2 to the moist case, this consideration becomes important, because the equivalent potential temperature also decreases with height in the lower troposphere. layer Evolution of moisture above the well-mixed layer (z > zm) may be given by q+(z, t) = q(z, t = 0) + F+t, (B3) (B3) with with F+ = −w (dq dz ) . (B4) (B4) The derivation proceeds in parallel to Section 2. For simplicity, some notations for the moisture are kept the same as for the potential temperature in Section 2. This should not cause any confusion, because none of the results in this Appendix are directly quoted in the main text. Thus, as an initial condition for the moisture mixing ratio, q, we assume Note that, in contrast to the case with potential tem- perature, F+ < 0 applies in a descending environment, and F+ > 0 in an ascending environment. Expected temporal growth of a well-mixed moisture layer is schematically shown in Figure B1: the well-mixed moisture layer deepens in response to the surface flux, H. The supplied heat flux, Ht, over time t, must be equal to an areas swept on a (q, z)-plane from the initial condition to a state at time t as indicated by the shading, thus Expected temporal growth of a well-mixed moisture layer is schematically shown in Figure B1: the well-mixed moisture layer deepens in response to the surface flux, H. q(z, t = 0) = q0 + (dq dz ) z (B1) The supplied heat flux, Ht, over time t, must be equal to an areas swept on a (q, z)-plane from the initial condition to a state at time t as indicated by the shading, thus (B1) in place of Equation (1). Here, dq∕dz (< 0) is assumed con- stant with height. Though this rather strong assumption can be relaxed, as moisture typically decreases exponen- tially with height, but more involved algebra follows. Equation (A11) reduces to Equation (11) Equation (A11) reduces to Equation (11) Using these two approximations, Δi𝜃dzm dt = −H(−). d dtΔm𝜃≃−w ( d𝜃 dz ) ≃−wΔ0𝜃 zm . (A10) (A10) In the presence of large-scale ascent, w, the height, zm, of the well-mixed layer is uplifted accordingly, thus By applying Equations (A9) and (A10) into Equation (A8), we find: By applying Equations (A9) and (A10) into Equation (A8), we find: Δi𝜃 (dzm dt −w ) = −H(−), (A5) (A5) Δ0𝜃 (dzm dt −w ) = H −H(−). (A11) (A11) which is equivalent to Equation (11). 11 F I G U R E B1 Schematic describing the well-mixed moisture layer YANO Finally, by invoking a geometrical consideration, (H −H(−))∕Δ0𝜃= −H(−)∕Δi𝜃, Equation (A11) reduces to Equation (11) APPENDIX B. DEVELOPMENT OF A WELL-MIXED MOISTURE LAYER As before, we assume a constant moisture flux, H, from the surface at z = 0. As a result, a well-mixed layer of moisture is formed and grows from the surface. Ht = Δq −F+t 2 zm. (B5) (B5) Also from a geometrical consideration suggested by Figure B1, Δq = − (dq dz ) zm + (−F+t). (B6) (B6) However, as a major difference from the potential tem- perature treatment in Section 2, the growth is inevitably associated with a development of an inversion (disconti- nuity), Δq, of the moisture at the top of the mixed layer. To close the problem with ease we assume, in turn, that the surface value, q0, is maintained constant with time, thus all the supplied moisture flux is used for deepening of the mixed layer. Thus, the inversion is defined by By substituting Equation (B6) into Equation (B5), we find: ( ) By substituting Equation (B6) into Equation (B5), we find: Ht = −1 2 (dq dz ) z2 m + (−F+ t)zm. (B7) (B7) This equation becomes equivalent to Equation (8) by replacing −dq∕dz and −F+ by d𝜃∕dz and F+, respectively. Δq = q0 −q+(zm, t). (B2) Δq = q0 −q+(zm, t). (B2) (B2)
https://openalex.org/W4287109803	https://zenodo.org/records/6371433/files/SGC-35-21-50-55-Costeleanu.pdf	English	null	CHALLENGES OF TECHNICAL TRANSLATIONS	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	3,184	ENJEUX DES TRADUCTIONS TECHNIQUES Résumé : La traduction technique signifie beaucoup plus que l’usage correct de la terminologie. C’est aussi la capacité d’écrire des textes crédibles et de transmettre l’information de façon claire et concise. Dans le présent article, la traduction technique est aperçue sur deux perspectives : d’une part, d’une perspective didactique, car la traduction peut être utilisée avec succès comme méthode d’enseignement / apprentissage en classe d’anglais langue étrangère / langage de spécialité. D’autre part, on a l’approche linguistique, étant donné qu’elle implique des concepts tels signification, équivalence, but et analyse du texte, des concepts examinés de perspective sémantique, pragmatique et stylistique. L’approche communicative a été reconnue comme la meilleure méthode pour enseigner l’anglais, car elle met l’accent sur le développement des quatre compétences : lire, écrire, parler et écouter. Selon Harmer (2001 : 84-86), trouver le terme adéquat est moins important que parler de façon fluente. Cependant, il y a des paliers où le terme adéquat est très important, l’un de ces paliers étant le langage scientifique. Mots-clés : technique, terminologie, enjeux. 1Mirela Costeleanu, University of Pitesti, mirelacosteleanu@yahoo.com CHALLENGES OF TECHNICAL TRANSLATIONS1 Abstract: Technical translation means much more than the correct use of terminology. It is also about the ability to write credible texts and to express information clearly and concidely. In the present paper, technical translation is dealt with from two perspectives: on the one hand, from a didactic perspective, as translation can be successfully used as a teaching/learning method in ESL/ESP classes. On the other hand, it is approached from a linguistic viewpoint since it incorporates such concepts as meaning, equivalence, text purpose and analysis, concepts examined in the context of semantics, pragmatics and stylistics. The Communicative Approach has been hailed as the best method to teach English as it lays great emphasis on the development of the four skills: reading, writing, speaking and listening. According to Harmer (2001: 84-86), accuracy is less important than fluency. Yet, there are fields in which accuracy is very important, one of them being science. Key words: technical, terminology, challenges Studii de gramatică contrastivă 35/2021 Studii de gramatică contrastivă 35/2021 Studii de gramatică contrastivă 35/2021 DOI: 10.5281/zenodo.6371433 1. What is technical translation? Technical translation encompasses the translation of special language texts, including not only the translation of engineering texts, but also texts in other disciplines such as economics and law. The translation of technical texts requires a good mastery of both the source and the target language. An understanding of the subject matter of the technical text is also necessary. As Jody Byrne points out, the reality of technical translations is influenced by a range of factors such as “technical communication, style, terminology, professional workflows, multimodal communication, legal requirements, technology and even psychology and pedagogy” (2009: 2-5). Klaus Schubert argues that technical translation cannot be fully described and modelled within the context of traditional Translation Studies. Technical translation takes place in a complex, highly interdisciplinary 51 Studii de gramatică contrastivă 35/2021 environment, the work of translators, the materials, the resources and the strategies used being influenced by several factors (ibidem). Learning a technical language is a very complex process which goes far beyond the correct use of terminology. This process has a lot in common with “acquiring the skills of communication for a foreign natural language” (Hann, 1992: 15). Technical translations involve a lot of challenges. Hann shows that “grammar rules change, verbs and prepositions acquire new significances, and similar terms occur with entirely different implications within brief sections of the same engineering text” (ibidem). Translation as a teaching method is seen as having a lot of disadvantages such as the students’ dependence on their first language, the emphasis on reading and writing, the neglect of speaking and listening as well as the failure to meet the learners’ practical needs. Moreover, the mere memorization of bilingual word lists does not encourage students to communicate freely in the target language. The method of translation fell into disgrace mainly because it was seen as hindering language acquisition. It was considered to discourage creativity and spontaneity because it lacks interaction. Advocates of the Communicative Approach point out that the use of the first language is counterproductive in the process of acquiring a second language and that therefore the use of translation is likely to do more damage than good, holding back students from taking the leap into expressing themselves freely in the new language (Carreres, 2006: 955). Yet, translation from English into Romanian or vice-versa plays an undeniable role in the teaching/learning of ESP. 1. What is technical translation? For Romanian engineering students studying English as a second language, the first language is the reference system in the learning process. Although it is still largely associated with the Grammar Translation Method, translation is no longer seen as an obsolete teaching method. There are many advantages to translation if it is used properly in ESP classes. The ESP teacher’s resort to translation cannot be seen as his/her failure to use only the target language in class. An argument in this regard is the fact that, as Harmer shows, “ when we learn a foreign language, we use translation almost without thinking about it, particularly at elementary and intermediate levels” (Harmer, 2001: 131). As David Atkinson points out, translation encourages learners to think about meaning, not just manipulate forms mechanically, offering them real-life activities (Atkinson, 1993: 53-54). Translation is a useful aid in ESP classes since it stimulates interactivity and encourages learners to transfer the thought process from L1 into L2. 2. Challenges of translation for engineering students crankshaft - a long metal rod, especially one in a car engine, that helps the engine turn the wheel (arbore cotit) crankshaft - a long metal rod, especially one in a car engine, that helps the engine turn the wheel (arbore cotit) camshaft - a device that causes the valves of an engine to open or close at the correct time (arbore cu came). camshaft - a device that causes the valves of an engine to open or close at the correct time (arbore cu came). If students are unaware of the function of a clutch in a car, such a definition as “a mechanism for connecting and disconnecting an engine and the transmission system in a vehicle, or the working parts of any machine” will hardly help them find the correct equivalent in Romanian (ambreiaj). On the other hand, students whose level of English is not very good may be faster in finding the Romanian corresponedent of an English technical word if they have wide scientific knowledge. The more familiar ESP students are with their field of study, the easier it will be for them to understand and acquire the technical vocabulary. Rarely do they feel the need to translate a technical text if the topic dealt with has been studied with the subject specialist in advance. ESP students also have a lot of difficulty with those words designating car parts which have different names in British English and American English: aerial/antenna, bonnet/hood, boot/trunk, indicator/ turn signal, number plate/license plate, wing/fender, accelerator/gas pedal. A large number of English technical terms are polysemantic, sometimes being part of word combinations which have no exact equivalents in Romanian. Cylinder, for instance, refers to many cylindral parts of machines and the cylindral chamber in which the steam acts upon the piston. Arm is used with the technical meaning of lever. Body, when used in technical texts, may have the following meanings: mass; box; drawer; casing. Finger means hand, pointer, ratchet. Hand is part of the compounds handbrake and handset. Head is part of the compounds headlamp and headlight. It is also used to refer to the head of a nail, or the head of a cylinder. Dog means safety piece, champ, hook. Horse is part of the compound horsepower. Work has in technical texts a different meaning from the one students are familiar with. 2. Challenges of translation for engineering students Translation activities and the use of L1 can help students understand specialised materials in the ESP classroom and improve their skills (Avand, 2009: 45). The method of translation is beneficial to elementary students, but it may be useful even at intermediate and advanced levels. Romanian engineering students prefer translation as a learning aid for several reasons. There are students who are not very comfortable with speaking freely in the classroom. These students do not regard translation as a time-consuming, boring, or inefficient activity. On the contrary, they find it useful especially in the introduction of new specialised vocabulary. Since they dislike monolingual instruction, they feel more relaxed and self-confident if they are allowed to use Romanian in bilingual activities. Since engineering is an exact science, most students need to make sure that they have understood a concept perfectly. In other words, accuracy is very important to them. When it comes to 52 Studii de gramatică contrastivă 35/2021 the introduction of new words, they would rather be given an accurate equivalent of the technical word than an explanation of it, no matter how clear and concise it is. ESP students encounter lexical, grammatical, and sociocultural difficulties when dealing with translation tasks. Lexical problems stem mainly from the lack of direct correspondence between Romanian and English technical vocabularies. Technical words are specific to a particular topic, discipline or field of study. They can be understood and acquired by studying the respective field. These words are the responsibility of the subject specialist. Such words as pedal, accelerator, piston, cylinder, distribution, combustion do not pose any problems to ESP students studying to become automotive engineers even if their scientific knowledge is poor. Their similarity with their Romanian equivalents helps Romanian students guess their meanings. In other situations, this similarity may be misleading. Thus, they may translate kinetic energy as energie chinetică, thermal energy as energie termală instead of energie cinetică and energie termică. Even students whose level of English is high may encounter problems in finding accurate equivalences between English and Romanian if their level of scientific knowledge is low. The mere definitions of such words as crankshaft and camshaft will not help an ESP student who lacks basic technical knowledge peculiar to his/her field of study to distinguish between the two terms. Studii de gramatică contrastivă 35/2021 A short text will make the difference between them obvious: “As an engine produces a couple - rotary force - the moving parts of the machine it is driving will produce resistance, due to friction and other forces. As a result, torque (twisting force) is exerted on the output shaft of the engine” (Ibbotson, 2009: 86). Besides lexical problems, ESP students often encounter grammatical problems when translating technical texts. Thus, they are likely to produce sentences like “The pliers is on the workbench” and “The pincers is here” instead of “The pliers are on the workbench” and “The pincers are here”. Besides lexical problems, ESP students often encounter grammatical problems when translating technical texts. Thus, they are likely to produce sentences like “The pliers is on the workbench” and “The pincers is here” instead of “The pliers are on the workbench” and “The pincers are here”. p Some words usually regarded as uncountable nouns (energy, mobility) behave as countable nouns in engineering contexts: Energy cannot be created or destroyed, only converted from one form to another. It needs an energy of 400 joules. Studii de gramatică contrastivă 35/2021 Studii de gramatică contrastivă 35/2021 for example, lifting a load to a certain height by crane - is called work, students have a tendency to translate work as muncă, not as lucru mecanic. When dealing with a polysemous word, students should be encouraged to try to guess its meaning from the context. The different meanings of polysemous words can be taught by starting from the primary meaning (which more often than not belongs to general English) and extending the meaning based on the general English word. for example, lifting a load to a certain height by crane - is called work, students have a tendency to translate work as muncă, not as lucru mecanic. When dealing with a polysemous word, students should be encouraged to try to guess its meaning from the context. The different meanings of polysemous words can be taught by starting from the primary meaning (which more often than not belongs to general English) and extending the meaning based on the general English word. Romanian engineering students may encounter problems with different systems of units which are used in different parts of the world. Some of them may be familiar with the conversion of kilograms into pounds, for instance, but most ESP teachers will not find it so easy. To avoid confusion, it is preferable not to change units in the translated text. Technical dictionaries are a valuable aid when doing technical translations. Unfortunately, there are situations when they are not enough. If students look up such words as quench, anneal and temper in an English-Romanian technical dictionary, they will be quite confused. Thus, both quench and temper mean a căli. Both anneal and temper mean a tempera. In reality, quenching, annealing and tempering refer to distinct types of heat treatment (quenching - metal is heated, then dipped in water or oil to cool it rapidly; annealing - metal is heated, then allowed to cool slowly; tempering - metal is heated and kept at a high temperature for a period of time). Both the ESP teacher and students may be confused by such words as torque and couple. According to technical dictionaries, they both mean cuplu. Yet, they are not synonyms. 2. Challenges of translation for engineering students In the sentence The amount of energy needed to do a task - 53 Studii de gramatică contrastivă 35/2021 Other ESP teachers see technical translation as a form of communication which involves interaction and cooperation between students. If well designed, translation activities practise not only reading and writing, but also speaking and listening. The aim of translation is the improvement of language proficiency, not the development of translation skills. Translation is a useful skill especially for people who travel a lot. Being a mediator between two people or two groups who cannot speak the same language requires translation. Technical translations are among the most demanded types of translation services. Low-quality translations may lead to malfunctioning systems or damaged devices. Those teachers who are aware that these situations are imminent in the students’ personal and professional lives will see the validity of translation as an activity in communicative classrooms. The choice of materials is very important when designing translation activities. A close cooperation with the subject specialists will help ESP teachers select texts about topics that have already been studied by students in their specialist courses. ESP teachers should know exactly when to use translation, which groups of students they should use it with and how much time to allot to it so that it shouldn’t dominate classroom practice. Designed well, translation activities in ESP classes can practise the four skills. Translation in groups can stimulate ESP students to discuss the meaning and use of language at the deepest levels while trying to find equivalents in another language. The class may be divided into two groups. Each group is given a short text to translate into the target language. Then the groups exchange their papers and they are asked to translate the other group’s version into their mother tongue. The comparison of the original text with the new version may have interesting, often amusing results. Students may be asked to look at translations which contain mistakes. They are asked to discuss the causes of errors. They may also be asked to bring in short texts which they find interesting. They can discuss and translate them. Choosing the right materials and activities will undoubtedly have a positive impact on the students’ motivation and dynamics. 3. ESP teachers’ attitude to translation Some ESP teachers have negative attitudes to translation because they associate it with the dull, lengthy activities they had to carry out when they themselves were students. They also see it as independent from the four skills which define language competence: reading, speaking, listening and writing. It prevents learners from thinking in the L2 and ESP teachers from maintaining an English-speaking environment in the class. Translation activities may be tricky for ESP teachers. They take a lot of preparation. Although nobody expects an ESP teacher to be an expert in the technical field, a good comprehension of the topic being translated is extremely valuable since technical terms do not always have direct translations in the target language. 54 References Atkinson, D., 1993, Teaching Monolingual Classes, London, Longman. Avand, A.Q., 2009, «Using translation and reading comprehension of ESP learners», Asian ESP Journal, 5(1), p. 44-60. Byrne, J., 2009, «The Coming of Age of Technical Translation: an Introduction», The Journal of Specialised Translation, p. 2-5. Carreres, A., 2006, « Strange Bedfellows: Translation and Language Teaching. The teaching of Translation into L2 in Modern Languages Degrees: Uses and Limitations», Sixth symposium on translation, terminology and interpretation, p. 955-959, http://www.cttic.org/publications_06Symposium.asp. (consulted on 7th May 2021). Dictionar tehnic englez-roman, Bucuresti, Editura Tehnica, 2002. Conclusions Although in the last 40 years or so, the use of translation has been described as a counterproductive practice, a lot of theorists and researchers have re-assessed the role of translation as an instrument to support and enhance L2 acquisition. In ESP classes, translation activities have an undisputed value if they are designed with a specific aim in mind and if they are used efficiently. References 55 Studii de gramatică contrastivă 35/2021 Studii de gramatică contrastivă 35/2021 Hann, M., 1992, The key to technical translation, Amsterdam, John Benjamins Publishing House. H J 2013 Th f E l h l h L d P Hann, M., 1992, The key to technical translation, Amsterdam, John Benjamins Publishing Hous Harmer, J., 2013, The practice of English language teaching, London, Pearson. Hann, M., 1992, The key to technical translation, Amsterdam, John Benjamins Publishing House. Harmer, J., 2013, The practice of English language teaching, London, Pearson. Ibbotson, M., 2009, Professional English in Use, Technical English for Professionals, Cambridge, Cambridge University Press. Hann, M., 1992, The key to technical translation, Amsterdam, John Benjamins Pub Harmer, J., 2013, The practice of English language teaching, London, Pearson. Harmer, J., 2013, The practice of English language teaching, London, Pearson. Ibbotson, M., 2009, Professional English in Use, Technical English for Professionals, Cambridge, Cambridge University Press. Mirela COSTELEANU is a lecturer at the University of Piteşti, Faculty of Theology, Letters, History and Arts, Department of Applied Foreign Languages and Doctor in Humanities (Philology). She has been teaching English as a foreign language for twenty years. Her area of interest includes English for Specific Purposes teaching and learning research in Romanian Higher Education institutions, didactics, pedagogy and second language acquisition, teaching technical English, translation theory and practice. She is the author of several English practical courses such as: A practical course in technical English, English for economics, English in the press, Test your English grammar and vocabulary. She has also published a number of papers and articles focused on linguistics, didactics and ESP. 56
https://openalex.org/W4286634195	https://www.frontiersin.org/articles/10.3389/fmed.2022.878966/pdf	English	null	Intrinsic Respiratory Gating for Simultaneous Multi-Mouse μCT Imaging to Assess Liver Tumors	Frontiers in medicine	2,022	cc-by	9,115	Intrinsic Respiratory Gating for Simultaneous Multi-Mouse µCT Imaging to Assess Liver Tumors Mirko Thamm 1,2, Stefanie Rosenhain 2, Kevin Leonardic 2, Andreas Höfter 2, Fabian Kiessling 1,3, Franz Osl 4, Thomas Pöschinger 4 and Felix Gremse 1,2* 1 Experimental Molecular Imaging, RWTH Aachen University, Aachen, Germany, 2 Gremse-IT GmbH, Aachen, Germany, 3 Fraunhofer Institute for Digital Medicine MEVIS, Bremen, Germany, 4 Discovery Pharmacology, Pharma Research and Early Development (pRED), Roche Innovation Center Munich, Penzberg, Germany Small animal micro computed tomography (µCT) is an important tool in cancer research and is used to quantify liver and lung tumors. A type of cancer that is intensively investigated with µCT is hepatocellular carcinoma (HCC). µCT scans acquire projections from different angles of the gantry which rotates X-ray source and detector around the animal. Motion of the animal causes inconsistencies between the projections which lead to artifacts in the resulting image. This is problematic in HCC research, where respiratory motion affects the image quality by causing hypodense intensity at the liver edge and smearing out small structures such as tumors. Dealing with respiratory motion is particularly difﬁcult in a high throughput setting when multiple mice are scanned together and projection removal by retrospective respiratory gating may compromise image quality and dose efﬁciency. In mice, inhalation anesthesia leads to a regular respiration with short gasps and long phases of negligible motion. Using this effect and an iterative reconstruction which can cope with missing angles, we discard the relatively few projections in which the gasping motion occurs. Moreover, since gated acquisition, i.e., acquiring multiple projections from a single gantry angle is not a requirement, this method can be applied to existing scans. We applied our method in a high throughput setting in which four mice with HCC tumors were scanned simultaneously in a multi-mouse bed. To establish a ground truth, we manually selected projections with visible respiratory motion. Our automated intrinsic breathing projection selection achieved an accordance of 97% with manual selection. We reconstructed volumetric images and demonstrated that our intrinsic gating method signiﬁcantly reduces the hypodense depiction at the cranial liver edge and improves the detectability of small tumors. Furthermore, we show that projection removal in a four mice scan discards only 7.5% more projections than in a single-mouse setting, i.e., four mouse scanning does not substantially compromise dose efﬁciency or image quality. Keywords: respiratory gating, µCT, mice, liver tumor, hepatocellular carcinoma, iterative reconstruction, small animal, high through put Intrinsic Respiratory Gating for Simultaneous Multi-Mouse µCT Imaging to Assess Liver Tumors To the best of our knowledge, no comparable method that combines multi-mouse scans for high throughput, intrinsic respiratory gating, and an available iterative reconstruction has been described for liver tumor imaging before. Edited by: Giorgio Treglia, Ente Ospedaliero Cantonale (EOC), Switzerland Reviewed by: Nguyen Minh Duc, Pham Ngoc Thach University of Medicine, Vietnam Stefan Sawall, German Cancer Research Center (DKFZ), Germany Correspondence: Felix Gremse fgremse@ukaachen.de Specialty section: This article was submitted to Nuclear Medicine, a section of the journal Frontiers in Medicine Received: 18 February 2022 Accepted: 08 June 2022 Published: 06 July 2022 ORIGINAL RESEARCH published: 06 July 2022 doi: 10.3389/fmed.2022.878966 ORIGINAL RESEARCH published: 06 July 2022 doi: 10.3389/fmed.2022.878966 INTRODUCTION For the assessment of liver tumors, two main issues need to be addressed particularly: soft tissue contrast and high throughput scanning to reduce the necessary time and costs. Our approach uses a model-based iterative reconstruction. Whereas this type of reconstruction is computationally intensive, it allows projections to be discarded without substitution. This is an important distinction to some previously described methods where retrospective phase binning or interpolation (13, 23) is used to re-create equidistant projections. µCT image contrast is based on the absorption of X-rays and is therefore highly suitable to examine radiodense structures like bones, with high resolution down to the sub-micrometer range (5). However, it often provides insuﬃcient soft tissue contrast, which is needed to investigate liver tissue and liver tumors (6). To overcome this problem, a radiodense contrast agent is used to increase contrast between soft tissues of interest. Because of the longer acquisition times of preclinical µCTs and the fast pharmacokinetics of clinical contrast agents, these clinical approaches cannot be simply transferred to preclinical research. Additionally, toxicological side-eﬀects of preclinical contrast agents have to be considered (3). However, liver tumors and metastases can be assessed and monitored over time using contrast agents such as ExiTron nano, eXIA, or Fenestra, which accumulate in healthy liver tissue and increase the radiodensity compared to tumor tissue (1, 7, 8). For in vivo studies that target liver tissue and liver tumors, it is important to consider the respiratory motion, because the region of interest moves with every breath due to anatomical proximity to the lung. Therefore, liver imaging suﬀers more from breathing artifacts than imaging of subcutaneous tumors, for example. In vivo imaging of mice requires special strategies because of their fast physiological motion (16). One major eﬀect of the respiratory motion is a blurring at the cranial edge of the liver. It often looks like a gradient with hypodense intensity toward the diaphragm which makes it harder to evaluate HCC tumors in that region, see Figures 1A,B. To counteract these motion artifacts, respiratory gating can be applied for improved liver imaging. A µCT scan acquires projections from equidistant angles by rotating the X-ray source and detector around the sample. Afterwards, a reconstruction algorithm computes a 3D volume from these projections. Motion inside the sample causes inconsistencies between projections which leads to artifacts in the resulting image. Thereby, motion by the heartbeat and breathing cause artifacts while scanning anesthetized animals. INTRODUCTION forced ventilation (10, 11) or a respiratory pad (12) for non- ventilated mice. For intrinsic gating [synonymous with self- gating, image-based, or raw-data-based gating (13)], the motion is calculated from the projection data itself (9, 14). An extrinsic motion signal can be applied prospectively or retrospectively (9, 15). In prospective gating, the X-ray exposure is actuated by the sought-after phase of the physiological motion (16, 17). This strategy avoids reconstruction artifacts, because it provides uniform angular projections but needs exceedingly long scan times (∼10 min) (15) and requires complicated hardware like a pulse-able radiation source or a quick shutter mechanism, which is usually not available for µCT. In retrospective gating, the motion aﬀected projections are detected after the scan. That can also be done extrinsically by applying a recorded external signal (18) or intrinsically (14). To avoid missing angles, the acquisition protocol usually requires multiple projections for every gantry angle (19) because the reconstruction would otherwise suﬀer from artifacts (15). However, this again leads to longer scan times and potentially higher X-ray exposure. The dose should be as low as possible, because radiation can aﬀect the immune system and other biological pathways of the animals, particularly in longitudinal imaging studies (20). To avoid both a high radiation dosage and long scan times, iterative reconstructions were suggested to minimize artifacts (2, 21, 22). Small animal micro computed tomography (µCT) is an important tool in cancer research. Since the animals can be examined non-invasively and repeatedly, the number of animals to be sacriﬁced in a longitudinal study can be drastically decreased. µCT is also used in combination with single photon emission computed tomography (SPECT) and positron emission tomography (PET) to provide anatomical information. However, standalone µCT is also widely used due to its fast acquisition time, high spatial resolution resulting in accurate anatomical information, relatively simple image analysis, and diverse application possibilities (1–3). A type of cancer that is intensively investigated with µCT is hepatocellular carcinoma (HCC). Now-a-days, it is the prevailing liver cancer with increasing incidence (4). Multiple HCC mouse models are used preclinically to assess the treatment response of new drugs (1). For example, Hage et al. (1) characterized two HCC models, investigated their response to immune checkpoint blockers (anti-PD-1and anti-CTLA- 4), and determined the orthotopic tumor growth by in vivo µCT imaging. Citation: Thamm M, Rosenhain S, Leonardic K, Höfter A, Kiessling F, Osl F, Pöschinger T and Gremse F (2022) Intrinsic Respiratory Gating for Simultaneous Multi-Mouse µCT Imaging to Assess Liver Tumors. Front. Med. 9:878966. doi: 10.3389/fmed.2022.878966 July 2022 \| Volume 9 \| Article 878966 Frontiers in Medicine \| www.frontiersin.org 1 Gating for Multi-Mouse µCT Thamm et al. Frontiers in Medicine \| www.frontiersin.org INTRODUCTION Scan augmentations known as cardiac and respiratory gating can reduce the problematic motion eﬀects and improve the image quality. This can help to visualize certain pathologies that would otherwise be invisible (9). Moreover, measurements of tumors are more exact with gating, because it reduces the blurring at the edge of pathological structures (2, 9). For extrinsic gating, a motion signal is obtained by an external hardware like a ventilator for Inhalation anesthesia (e.g., isoﬂurane) leads to a regular respiration with short phases of gasping breathing and long phases with barely any motion (9, 24). This makes inhalation anesthesia ideally suited for intrinsic retrospective gating (9). Usage of an iterative reconstruction allows for basically any µCT scan to discard the relatively few projections in which the gasping motion occurs and therefore still have suﬃcient data for the reconstruction. That is the basis for the approach of this study. An important parameter for our gating approach is the rejection fraction (RF), i.e., the fraction of projections which should not be included in the reconstruction. This can roughly be regarded as the sensitivity of the motion detection algorithm. The following eﬀects are expected to occur with an increasing RF, and not all are improving the image quality: July 2022 \| Volume 9 \| Article 878966 2 Thamm et al. Gating for Multi-Mouse µCT Thamm et al. Gating for Multi Mous FIGURE 1 \| Liver tumor imaging. (A) Coronal slice through a contrast-enhanced mouse liver, reconstructed without gating. W: 1,007 HU L: 443 HU. Tumors and GURE 1 \| Liver tumor imaging. (A) Coronal slice through a contrast-enhanced mouse liver, reconstructed without gating. W: 1,007 HU L: 443 HU. Tumors and ood vessels appear as dark regions. A hypodense depiction looking like a gradient at the cranial edge of the liver occurs due to contributions from the lung. The mor marked by the arrow is hardly visible. (B) Respiratory gating improves contrast at the liver edge and increases the detectability of the marked tumor. W: 1,007 U L: 443 HU. (C) The liver shows a rough/uneven surface in the 3D rendering. (D) The 3D rendering of a gated reconstruction shows a smoother surface with tectable tumors. (C+D) Are simple surface rendering done by the commercially available software mentioned under materials and methods. (E) Line proﬁle from int A to point B, covering 6.96 mm. INTRODUCTION Linear regressions over the segment that covers the liver show a steeper slope without gating (37.5 HU/mm) than with gating 4.6 HU/mm). Th ib i f h l h li d i i Th f i l j i f i d b FIGURE 1 \| Liver tumor imaging. (A) Coronal slice through a contrast-enhanced mouse liver, reconstructed without gating. W: 1,007 HU L: 443 HU. Tumors and blood vessels appear as dark regions. A hypodense depiction looking like a gradient at the cranial edge of the liver occurs due to contributions from the lung. The tumor marked by the arrow is hardly visible. (B) Respiratory gating improves contrast at the liver edge and increases the detectability of the marked tumor. W: 1,007 HU L: 443 HU. (C) The liver shows a rough/uneven surface in the 3D rendering. (D) The 3D rendering of a gated reconstruction shows a smoother surface with 1) The contribution of the lung to the liver edge region is reduced, increasing signal in the liver and contrast to the lung, decreasing the gradient toward hypodense intensities at the cranial edge of the liver. 1) The contribution of the lung to the liver edge region is reduced, increasing signal in the liver and contrast to the lung, decreasing the gradient toward hypodense intensities at the cranial edge of the liver. Therefore, an optimal rejection fraction needs to be experimentally determined. To illustrate this problem, Figure 2 shows a µCT scan of a single mouse without contrast agent. A rejection fraction of 0.2 improves the image quality and signal- to-noise ratio at the liver edge, but higher rejection fractions are suboptimal again. 2) The remaining projections are more consistent leading to less streaking artifacts. 2) The remaining projections are more consistent leading to less streaking artifacts. g A higher throughput can be accomplished by scanning multiple mice simultaneously using special mouse holders. As 3) Less projections mean less raw data, which leads to more noise. 3) Less projections mean less raw data, which leads to more noise. July 2022 \| Volume 9 \| Article 878966 Frontiers in Medicine \| www.frontiersin.org 3 Gating for Multi-Mouse µCT Thamm et al. FIGURE 2 \| Optimal rejection fraction in a µCT scan of the liver of a single mouse without contrast agent. (A vs. Reconstruction Volumetric images were reconstructed using our µCT reconstruction software on a PC with Microsoft Windows 10 equipped with an Intel Core i7-5930K processor, 32GB RAM, and an NVIDIA RTX 2080 Super GPU. The reconstruction software is commercially available (CTRecon, Gremse-IT GmbH, Aachen, Germany) and oﬀers a model-based iterative reconstruction. Since iterative reconstruction is much more computationally intensive than direct reconstruction, our iterative reconstruction software makes use of multi-GPU- acceleration, if available, and requires only a few minutes of reconstruction time per mouse when reconstructing at typical resolutions around 80 µm. For a singlemouse setting, retrospective intrinsic gating in combination with an iterative reconstruction has been used before and proven to improve tumor volume accuracy compared to non-gated images (2). However, to the best of our knowledge, no comparable method that combines multi-mouse scans for high throughput, intrinsic respiratory gating and a commercially available iterative model-based reconstruction has been described before and evaluated for liver tumor imaging. We present and test a method that can be used for multi-mouse scans to assess the liver tumor state in high throughput settings and does not require any additional measuring hardware such as breathing pads. We show that a retrospective intrinsic gating of a four-mice scan leads to the rejection of only 7.5% more projections compared to a single-mouse setting. Our model- based iterative reconstruction eliminates the need to acquire multiple projections at each angle and can create an image of competitive quality. Moreover, this method can be applied to basically all existing µCT scans. The processing steps of the gating are described in Figure 3. The software ﬁrst computes a coarse preview reconstruction using the raw data, requiring only a few seconds. Then, the user can place a sphere forming a region of interest (ROI) at the diaphragm of the mouse of interest (MOI). User interactions to place a ROI have been described before (13, 14, 28). The reconstruction software projects the sphere onto each acquired projection and computes the mean signal intensity inside the projected sphere to calculate a curve. The projected sphere intensity of the coarse reconstruction is subtracted to remove the changes that result from the rotation. Then, for low pass ﬁltering, a median ﬁlter with radius 4 is used, resulting in a smoothed curve. This curve is then subtracted to achieve a high-pass ﬁlter. Reconstruction Finally, a threshold is determined and applied to reject a number of breathing projections according to the rejection fraction. This is similar to previous approaches (28). However, in our case with the 4 mice, the reconstruction of the image of the MOI can also be aﬀected by motion that is caused by other mice behind or in front of the MOI. That does not apply to every breathing INTRODUCTION B) The breathing of the mouse causes a hypodense depiction of the cranial liver parts which is reduced using respiratory gating. W: 2,487 HU L: 50 HU. (C) The signal was measured at the marked cranial liver region for different rejection fractions (RF) showing an optimum at RF=0.2. (µCT device U-CT, MILabs B.V., Utrecht, the Netherlands, continuous rotation mode, tube voltage of 65 kV exposure time 75 ms, tube current 0.13 mA). The optimum is a compromise for which inconsistent projections are removed with the trade-off that less data is available for reconstruction. FIGURE 2 \| Optimal rejection fraction in a µCT scan of the liver of a single mouse without contrast agent. (A vs. B) The breathing of the mouse causes a hypodense depiction of the cranial liver parts which is reduced using respiratory gating. W: 2,487 HU L: 50 HU. (C) The signal was measured at the marked cranial liver region for different rejection fractions (RF) showing an optimum at RF=0.2. (µCT device U-CT, MILabs B.V., Utrecht, the Netherlands, continuous rotation mode, tube voltage of 65 kV exposure time 75 ms, tube current 0.13 mA). The optimum is a compromise for which inconsistent projections are removed with the trade-off that less data is available for reconstruction. automated breathing projection algorithm. Reconstructions were then performed for diﬀerent rejection fractions to assess the eﬀect on image quality. already described before, some devices can hold two (25) or four (26, 27) mice. High throughput is a conﬂicting goal with high image quality though, because multiple mice also multiply the overall physiological motion brought into the scan by each animal. Multiple respiratory pads can be used with a multi-mouse bed, but they take up valuable space inside the µCT and do not provide the information about motion contributions across mice, as described below. Additionally, the required external hardware is expensive, error prone, and requires time for setup before scanning. Frontiers in Medicine \| www.frontiersin.org Image Analysis Images were visualized and analyzed using the software Imalytics Preclinical 3.0 (Gremse-IT GmbH, Aachen, Germany) (30). To form a ground truth for further analysis, we manually examined each projection of every scan for each mouse and compared it to the preceding and following projections. In this way, we could visually detect respiratory motion and determine whether it aﬀects the MOI. During the manual review, we decided for each projection how much the possible respiratory motion of each mouse aﬀects the MOI. We noted this in integral numbers from 0–10. A clear full motion of the diaphragm of the MOI itself corresponds to 10, as does a corresponding motion of another mouse that overlaps with the MOI in the projection under consideration. We have devalued a weaker motion or a very slight overlap accordingly. If there is no overlap with the MOI, a breathing motion is irrelevant and was noted with 0. To simulate gating, which can only accept or discard projections, we deﬁned the following threshold: Projections with a motion value ≥3 are discarded. The iterative reconstruction itself involves a forward model which computes the projections using line integrals which are implemented using GPU-accelerated texture-based trilinear ﬁltering. A beam hardening model is applied to account for x-ray source distribution, tissue absorption and detector response curve. For this study we apply a Gaussian smoothing (1.34 stddev) of the measured projections followed by 3x3 software binning to achieve an appropriate balance between noise and resolution (∼150 µm) for this application. The weighted squared diﬀerence between simulated and measured pixels is used as cost term where the pixel weight is based on the pixel intensity assuming a poisson-like noise model. To minimize the cost term, the linear conjugate gradient method is used with 30 iterations where the gradient is computed using GPU-accelerated adjoint algorithmic diﬀerentiation (29). For quantitative inspection of the image quality, we set the following measurement points or areas in each volume: • Start- and endpoint for a line proﬁle over the liver starting at the cranial edge (see Figure 7). We reconstructed volumes of each mouse with rejection fractions from 0 to 0.6 in steps of 0.05. Therefore, our data consists of 156 (= 3 scans times 4 mice per scan times 13 rejection fractions) volumetric images in total. Each volume was reconstructed with a voxel size of 80 µm. MATERIALS AND METHODS The raw data basis for this study consists of three scans. For each scan four mice with liver tumors were placed in a multi- mouse bed and thus, scanned simultaneously. We examined each projection of each scan to manually select breathing projections and establish a ground truth for evaluation of our July 2022 \| Volume 9 \| Article 878966 4 Gating for Multi-Mouse µCT Thamm et al. FIGURE 3 \| Gated reconstruction approach. A coarse preview is reconstructed from the raw data and the user can mark the diaphragm with a sphere (ROI). From the signal intensity inside the projected sphere, the algorithm computes a motion score for each projection and rejects a fraction of the projections. From the remaining projections, a reconstruction is computed. This process is repeated for each mouse of a multi-mouse scan. FIGURE 3 \| Gated reconstruction approach. A coarse preview is reconstructed from the raw data and the user can mark the diaphragm with a sphere (ROI). From the signal intensity inside the projected sphere, the algorithm computes a motion score for each projection and rejects a fraction of the projections. From the remaining projections, a reconstruction is computed. This process is repeated for each mouse of a multi-mouse scan. dimensions are 777x442x752 (scan 1), 800x427x800 (scan 2) and 800x465x800 (scan 3). cycle though. Only those projections are aﬀected in which the breathing mouse and the MOI overlap, as it is visualized in Figure 4. To clarify, even if four mice are scanned at once, a speciﬁc reconstruction is performed for each individual mouse. Each reconstruction is performed using a selection of projections speciﬁc to that mouse. The above-mentioned threshold is deﬁned by the rejection fraction (RF) which indicates the fraction of the projections omitted from the reconstruction. At a rejection of 0.2, for example, 20% of projections in which the motion parameter has the lowest values are rejected. Frontiers in Medicine \| www.frontiersin.org Image Analysis The reconstruction • 3 spherical regions of 1 mm diameter at the cranial edge of the liver (see Figure 8). • 3 spherical regions of 1 mm diameter in a central area of the liver but outside tumors (see Figure 8). July 2022 \| Volume 9 \| Article 878966 5 Thamm et al. Gating for Multi-Mouse µCT GURE 4 \| Overlap of respiratory motion. We determined the motion score for mouse 1 (MOI). All curves are derived from the manual breathing examination of all jections. (A) The x-ray source and detector rotate around the sampled multi-mouse bed. (B,C) Show the different resulting projections. (D1) Shows the motion ore for a simulated 4-mice-scan with overlap-based projection rejection that results when only those respiratory motions are considered which overlap the MOI. 2) Simulates a 4-mice scan with breathing pads. Lacking the information, in which mice overlap in which projection, every respiratory motion needs to be nsidered. The resulting cumulative curve of the motion score shows irrelevant contribution of other mice leading to unnecessary projection rejection. segmentations of liver tumors (see Figure 9) Micro Computed Tomography (µCT) FIGURE 4 \| Overlap of respiratory motion. We determined the motion score for mouse 1 (MOI). All curves are derived from the manual breathing examination of all projections. (A) The x-ray source and detector rotate around the sampled multi-mouse bed. (B,C) Show the different resulting projections. (D1) Shows the motion score for a simulated 4-mice-scan with overlap-based projection rejection that results when only those respiratory motions are considered which overlap the MOI. (D2) Simulates a 4-mice scan with breathing pads. Lacking the information, in which mice overlap in which projection, every respiratory motion needs to be considered. The resulting cumulative curve of the motion score shows irrelevant contribution of other mice leading to unnecessary projection rejection. • 3 segmentations of liver tumors (see Figure 9). • 1 segmentation of a homogenous area of the mouse bed. Image acquisition was performed using a micro-computed tomography device (U-CT, MILabs B.V., Utrecht, the Netherlands). Anesthetized mice were placed in a commercially available multi-mouse bed (MILabs B.V., Utrecht, the Netherlands) and a scan of liver and lungs was performed. In a full-rotation in step-and-shoot mode, 1440 projections (1944 x1536 pixels) were acquired with an X-ray tube voltage of 55 kV, tube current 0.17 mA, exposure time of 75 ms, source isocenter distance of 117.578 mm, and source detector distance of 297.459 mm. The beam was hardened by the ﬁlters: Aluminum 100 µm (ﬁxed ﬁlter) + aluminum 400 µm (from a ﬁlter wheel). For each scan a dose of 344 mGy was applied according to the Effect of Overlapping Mice on Projection Rejection A µCT scan acquires projections from many, often equidistant, angles. Figure 4A illustrates how the rotating X-ray source and detector result in diﬀerent projections (B–C). We manually examined each projection of every scan for each mouse respectively to detect respiratory motion. We used the manual labeling to simulate the projection selection for the following cases: FIGURE 5 \| Used projections according to manual motion detection. We averaged the manual examination of all twelve mice. 1 mouse: 85.22 %; Only the MOI is considered, which emulates a single-mouse scan. 4 mice opt: 77.78 %; all mice but only their relevant motion is considered, which simulates a 4-mice-scan with overlap-based projection rejection. 4 mice add.: 53.38 %; each motion is added up without considering the actual overlap, which simulates a 4-mice scan with breathing pads and conservative projection selection. The values differ signiﬁcantly (p < 0.0001 as indicated by *, one-way ANOVA). 1. Single-mouse scan. Only the respiratory motion of the MOI itself is considered. Therefore, the same projections are rejected as for a single-mouse scan. 2. 4-mice scan with overlap-based projection rejection. When the projection of the MOI overlaps the projection of another mouse, the respiratory motion of the other mouse should be considered for gating, leading to projection rejections for the MOI. On the other hand, respiratory motion of other mice which do not overlap can be ignored as far as the MOI is concerned. Our algorithmic approach aims to allow for an optimized gating leading to a 4-mice scan with overlap- based projection rejection. This is shown in Figure 4D1. The motion parameter for the MOI (black) has only slightly more deﬂections than the motion curve of the MOI itself (red). The remaining curves show the breathing motion of the other mice, as far as they are relevant for the MOI, i.e., they happen in an overlapping projection. manufacturers information which was assessed for our scan protocol using a phantom. To induce anesthesia, 4% isoﬂurane in oxygen-enriched air using a dedicated vaporizer was applied to the animals. During imaging, the isoﬂurane concentration was reduced to 2.0% and maintained on this level. This inhalation anesthesia caused the above-mentioned regular respiration with short gasps and long phases of nearly no motion. Eyes were protected from dehydration with Bepanthen eye ointment. Effect of Overlapping Mice on Projection Rejection Liver contrast was enhanced by intravenous injecting of 100 µl of ExiTron nano 6000 (Viscover, Miltenyi Biotec, Bergisch Gladbach, Germany) at least 4 h before ﬁrst image acquisition. This contrast agent causes a long-lasting enhancement of healthy liver tissue, allowing to track tumor which appear as hypodense structures over months. 3. 3. 4-mice scan with breathing pads. A standard setup with respiratory pads misses the information which mice overlap in which projections. Therefore, a conservative gating would discard every projection in which at least one mouse shows respiratory motion. Figure 4D2 simulates a 4-mice scan with breathing pads. Every respiratory motion of each mouse is considered for the motion parameter which shows many deﬂections. This approach has the disadvantage that more data is discarded than necessary. Another possibility would be to only discard the projections in which the MOI breathes. However, this would still lead to artifacts when motion occurs in a mouse that at the time of this projection overlaps the MOI. Statistical Analysis Comparisons between two groups were performed using the Student’s t-test. For a comparison of more than two groups, statistical analysis was performed by one-way analysis of variance (ANOVA). Statistical signiﬁcance of p < 0.001 is indicated ∗∗∗and p < 0.0001 is indicated ∗∗∗∗. For statistics, linear regression and graphs the software GraphPad Prism (version 9.2.0, GraphPad Software Inc., San Diego, CA, USA) was used. The line charts were rendered with Microsoft Excel 365 (Redmond, WA, USA). July 2022 \| Volume 9 \| Article 878966 Frontiers in Medicine \| www.frontiersin.org 6 Thamm et al. Gating for Multi-Mouse µCT FIGURE 5 \| Used projections according to manual motion detection. We averaged the manual examination of all twelve mice. 1 mouse: 85.22 %; Only the MOI is considered, which emulates a single-mouse scan. 4 mice opt: 77.78 %; all mice but only their relevant motion is considered, which simulates a 4-mice-scan with overlap-based projection rejection. 4 mice add.: 53.38 %; each motion is added up without considering the actual overlap, which simulates a 4-mice scan with breathing pads and conservative projection selection. The values differ signiﬁcantly (p < 0.0001 as indicated by , one-way ANOVA). For the generation of the orthotopic iAST mouse model, 5x108 infectious units (IU) of adenovirus (Ad.Cre) expressing Cre recombinase (Vector BioLabs, Malvern, PA, USA) were injected intravenously into 6–8 week old inducible AST (iAST) mice as described in Stahl et al. (31). The iAST mice express the SV40 large T antigen with a hepatocyte-speciﬁc albumin promoter. Conditional expression is regulated by a loxP ﬂanked stop cassette. After the experiments, mice were euthanized by cervical dislocation. A body weight loss of 20% and more was deﬁned as an endpoint criterion. Frontiers in Medicine \| www.frontiersin.org Limitations and Future Work While we evaluated a 4-mice-bed and compared the results to single-mouse scans, it would be interesting to also test 2- or 3-, or 5-mice-beds to ﬁnd the best compromise between throughput and image quality. We did not make a direct comparison between iterative reconstruction and ﬁltered back projection (FBP). This would be interesting to better investigate the tradeoﬀbetween the expected better quality of iterative reconstruction and the signiﬁcantly lower computational requirements of FBP. With FBP, one could also evaluate diﬀerent approaches to compensate for the missing projections. A full reconstruction with FBP takes about as long as a single pass of iterative reconstruction. We used 30 iterations each for this study. Future work could vary this number and investigate the impact on quality and computation time. Prerequisites The algorithm considers the ROI at the diaphragm of the MOI. In case of an overlapping motion, the signal intensityof the projection changes there as well which is recognized. We can state that the algorithm, which was originally designed for a single-mouse scan also detects respiratory motion in overlapping mice well. An essential prerequisite of our method is the described anesthesia. We demonstrated in section 3.1 that in a four mice scan only about 21% of the projections are aﬀected by respiratory motion due to the gasping breathing. This may change with the dosage of the anesthetics. While this was not analyzed systematically, induction of the required gasping breathing behavior may require a comparatively strong anesthesia. The advantages of the presented gating method (less wasted X-ray dosage and shorter anesthesia) must therefore be weighed against the disadvantages of the potentially more stressful anesthesia. However, in our experience, a somewhat stronger anesthesia is acceptable for the short µCT scanning duration of a few minutes and is also beneﬁcial to reduce the risk that a mouse wakes up during the scan which is stressful for the animal and operator and typically results in repetition of the failed scan. Mice and Cell Lines All animal experiments were approved and conducted according to the regulations from the Government of Upper Bavaria (Regierung von Oberbayern; Approval Number: ROB55.2- 2532.Vet_03-15-89) and performed in accordance with the Federation for Laboratory Animal Science Associations (FELASA). The animal facility has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). We compared how many projections can be included in the reconstruction averaged across all data sets. The results are shown in Figure 5. Particularly noteworthy is the small diﬀerence between the simulated single-mouse scan (85.22%) and Female mice (iAST transgenic mice) were obtained from Charles River Laboratories (Sulzfeld, Germany). Frontiers in Medicine \| www.frontiersin.org July 2022 \| Volume 9 \| Article 878966 7 Gating for Multi-Mouse µCT Thamm et al. the 4-mice scan with overlap-based projection rejection (77.78%). This diﬀerence is <7.5% (absolute, or 9.6% relative). If, on the other hand, a 4-mice scan with breathing pads is simulated by considering the respiratory motion of all mice, only 53.38% of the projections could be used for the reconstruction. A noticeable diﬀerence that would greatly aﬀect the image quality. was found at 15% RF. This can be explained by increased noise at higher RFs due to the exclusion of too many projections. At a rejection fraction of 20%, our gating resulted in a signiﬁcant increase of the CNR (p < 0.001, paired t-test, Figure 9). Image Intensity at Liver Edge and Center The gradient toward hypodense intensities at the cranial edge of the liver is a problem for liver imaging that can be addressed with respiratory gating. In slices, it appears as a gradient that becomes darker toward the diaphragm, in 3D images as a frayed surface (Figure 1). We also exempliﬁed that linear regression over the line proﬁle of the liver slopes in the cranial direction, which can be improved by gating. We investigated this for all samples (Figure 7) and could determine that the slope is signiﬁcantly reduced with gating (RF = 0.2). The demands on the computing power of the hardware are high for the presented method. An iterative reconstruction needs many computing steps. Our reconstruction requires a reasonable amount of time (∼12 minutes per mouse on our sytem) because the calculation steps are parallelized using GPU-Accelerated Adjoint Algorithmic Diﬀerentiation (29). Further speed-ups are possible by using more and stronger GPUs. Furthermore, four reconstructions with full ﬁeld of view are required per scan, i.e., one per mouse. Since the line proﬁles appeared to be quite noisy and were aﬀected by blood vessels and tumors they passed through, we also assessed the eﬀect with regions that we placed in apparently homogenous liver regions. We determined the diﬀerence between the mean CT-value of three spherical regions in the center of the liver and three spherical regions at the edge of the liver. These regions were placed for each mouse and the intensities were computed for a range of rejection fractions. This diﬀerence was found to be smaller for each mouse with gating (RF = 0.2) than without gating. This is illustrated in Figure 8 and we found that the hypodensity at the liver edge was signiﬁcantly (p < 0.0001, paired t-test) reduced by gating. The eﬀect increases with the rejection fraction, but no noticeable improvement occurs for values above 0.2 (Figure 8B). Even for high rejection fractions, the liver center remains somewhat more radiodense, however. We do not know whether this is a result of the contrast agent distribution, vascularization, or a remaining breathing motion artifact. Evaluation of Automated Projection Selection We provided a method to improve µCT reconstructions, while maintaining a high throughput setting. Most notably is the ease of use and the possibility to apply the method to already existing scans since gated acquisition is not necessary and acquiring multiple projections from a single gantry angle is not a requirement. The manual labeling of the motion in all projections allows an evaluation of the automated projection selection algorithm. The results are shown in Figure 6. The best match with the manually determined ground truth is in our use case a rejection fraction of 0.2. This coincides expectedly with the result in Figure 5. Detectability of Tumors The overall goal of this study is to improve liver tumor imaging. Therefore, we examined liver vs. tumor contrast-to-noise-ratio (CNR). The voxel noise was derived from a homogenous area in the mouse bed. Contrast and CNR were plotted over the rejection fraction in Figure 9. The contrast increases with RF and saturates at 25%. The optimum RF for tumor detectability in our scenario A previously described method for single-mouse scans also places an ROI on the diaphragm for intrinsic retrospective gating Frontiers in Medicine \| www.frontiersin.org July 2022 \| Volume 9 \| Article 878966 8 Gating for Multi-Mouse µCT Thamm et al. GURE 6 \| Optimal Rejection Fraction (RF). (A) The best correspondence between the manual evaluation and the algorithmic gating is at RF = 0.2. The success rate 96.92%. (B) The reconstruction software provides a projection rejection map which is depicted as red stripes over the projection sinogram. The peaks of manually etected motion (see Figure 4B) match the algorithmically created stripes in the map. GURE 7 \| Evaluation of line proﬁles. (A) Start- and endpoint for the evaluated line proﬁle. The breathing results in a motion of the lungs and liver as shown by the hite arrow. Image reconstructed with RF = 0.2. W: 4,014 HU L: 668 HU (B) Lineproﬁle from point A to point B. Linear show a steeper slope without gating (25.5 U/mm) than with gating (6.5 HU/mm). The lineproﬁle covers 7.2 mm. (C) The slope is signiﬁcantly reduced by gating (for RF = 0.2, n = 12, p < 0.0001 as indicated y , paired t-test). d discards motion aﬀected projections (13). Because that ethod uses a Feldkamp type volumetric reconstruction, missing gular projections were substituted with projections from other ations or interpolated from neighboring projections However by means of a model-based reconstruction in combination with motion compensating vector ﬁelds (32) or motion estimation using a non-rigid registration method (21). In both cases, the motion aﬀected projections are reshaped so that they can be FIGURE 6 \| Optimal Rejection Fraction (RF). (A) The best correspondence between the manual evaluation and the algorithmic gating is at RF = 0.2. The success rate is 96.92%. (B) The reconstruction software provides a projection rejection map which is depicted as red stripes over the projection sinogram. The peaks of manually detected motion (see Figure 4B) match the algorithmically created stripes in the map. Detectability of Tumors FIGURE 8 \| Gating reduces the hypodensity at the liver edge. (A) Evaluated regions at the center of the liver C1−3 and near the edge toward the diaphragm E1−3. The gasping breathing results in a motion of the lungs and liver as shown by the white arrow. At the cranial liver edge (E1−3), the contribution of motion projections causes a hypodense depiction. Image reconstructed with RF = 0.2. W: 4,014 HU L: 668 HU (B) The hypodense depiction at the liver edge is reduced with increasing rejection fractions. For RF > 0.2 no noticeable improvement is found. (C) The difference between liver center and edge CT-values is signiﬁcantly reduced by gating (for RF = 0.2, n = 12, p < 0.0001 as indicated by , paired t-test). FIGURE 8 \| Gating reduces the hypodensity at the liver edge. (A) Evaluated regions at the center of the liver C1−3 and near the edge toward the diaphragm E1−3. The gasping breathing results in a motion of the lungs and liver as shown by the white arrow. At the cranial liver edge (E1−3), the contribution of motion projections causes a hypodense depiction. Image reconstructed with RF = 0.2. W: 4,014 HU L: 668 HU (B) The hypodense depiction at the liver edge is reduced with increasing rejection fractions. For RF > 0.2 no noticeable improvement is found. (C) The difference between liver center and edge CT-values is signiﬁcantly reduced by gating (for RF = 0.2, n = 12, p < 0.0001 as indicated by , paired t-test). FIGURE 9 \| Contrast of liver vs. tumor tissue. (A) An exemplary coronal µCT slice through the liver. One of the spheres at the liver edge E1 and one of the spheres at the center C1 can be seen. The other measurement spheres are in other slices. Also visible is the segmentation of two tumors T1 and T2. The third segmented tumor is not visible in this slice. The artifact at the top of the image is due to the fact that this is also the edge of the volumetric image, where less data is available for reconstruction. W: 4,429 HU L: 670 HU (B) Averaged over all mouse samples, the contrast between liver and tumor increases with RF and reaches saturation at RF = 0.25. Detectability of Tumors FIGURE 6 \| Optimal Rejection Fraction (RF). (A) The best correspondence between the manual evaluation and the algorithmic gating is at RF = 0.2. The success rate is 96.92%. (B) The reconstruction software provides a projection rejection map which is depicted as red stripes over the projection sinogram. The peaks of manually detected motion (see Figure 4B) match the algorithmically created stripes in the map. FIGURE 7 \| Evaluation of line proﬁles. (A) Start- and endpoint for the evaluated line proﬁle. The breathing results in a motion of the lungs and liver as shown by the white arrow. Image reconstructed with RF = 0.2. W: 4,014 HU L: 668 HU (B) Lineproﬁle from point A to point B. Linear show a steeper slope without gating (25.5 HU/mm) than with gating (6.5 HU/mm). The lineproﬁle covers 7.2 mm. (C) The slope is signiﬁcantly reduced by gating (for RF = 0.2, n = 12, p < 0.0001 as indicated by , paired t-test). FIGURE 7 \| Evaluation of line proﬁles. (A) Start- and endpoint for the evaluated line proﬁle. The breathing results in a motion of the lungs and liver as shown by the white arrow. Image reconstructed with RF = 0.2. W: 4,014 HU L: 668 HU (B) Lineproﬁle from point A to point B. Linear show a steeper slope without gating (25.5 HU/mm) than with gating (6.5 HU/mm). The lineproﬁle covers 7.2 mm. (C) The slope is signiﬁcantly reduced by gating (for RF = 0.2, n = 12, p < 0.0001 as indicated by , paired t-test). and discards motion aﬀected projections (13). Because that method uses a Feldkamp type volumetric reconstruction, missing angular projections were substituted with projections from other rotations or interpolated from neighboring projections. However, the authors concluded that in general, dose usage is better in prospective methods, because all acquired projections contribute to image reconstruction (13). This was more recently addressed by means of a model-based reconstruction in combination with motion compensating vector ﬁelds (32) or motion estimation using a non-rigid registration method (21). In both cases, the motion aﬀected projections are reshaped so that they can be included in the reconstruction to preferably use all acquired data. The method we presented might also further increase its dose eﬃciency with similar additions. July 2022 \| Volume 9 \| Article 878966 Frontiers in Medicine \| www.frontiersin.org 9 Gating for Multi-Mouse µCT Thamm et al. REFERENCES 9. Bartling SH, Kuntz J, Semmler W. Gating in small-animal cardio-thoracic CT. Methods San Diego Calif. (2010) 50:42–9. doi: 10.1016/j.ymeth.2009.07.006 1. Hage C, Hoves S, AshoﬀM, Schandl V, Hört S, Rieder N, et al. Characterizing responsive and refractory orthotopic mouse models of hepatocellular carcinoma in cancer immunotherapy. PLoS ONE. (2019) 14:e0219517. doi: 10.1371/journal.pone.0219517 1. Hage C, Hoves S, AshoﬀM, Schandl V, Hört S, Rieder N, et al. Characterizing responsive and refractory orthotopic mouse models of hepatocellular carcinoma in cancer immunotherapy. PLoS ONE. (2019) 14:e0219517. doi: 10.1371/journal.pone.0219517 10. Johnson EM, Price RE, Kurie JM, Rivera BS, Cody DD. A new method for respiratory gating during microcomputed tomography of lung in mice. J Am Assoc Lab Anim Sci. (2008) 47:46–56. Available online at: https://www. ingentaconnect.com/content/aalas/jaalas/2008/00000047/00000004/art00006 2. Blocker SJ, Holbrook MD, Mowery YM, Sullivan DC, Badea CT. The impact of respiratory gating on improving volume measurement of murine lung tumors in micro-CT imaging. PLoS ONE. (2020) 15:e0225019. doi: 10.1371/journal.pone.0225019 11. Walters EB, Panda K, Bankson JA, Brown E, Cody DD. Improved method of in vivo respiratory-gated micro-CT imaging. Phys Med Biol. (2004) 49:4163–72. doi: 10.1088/0031-9155/49/17/023 2. Blocker SJ, Holbrook MD, Mowery YM, Sullivan DC, Badea CT. The impact of respiratory gating on improving volume measurement of murine lung tumors in micro-CT imaging. PLoS ONE. (2020) 15:e0225019. doi: 10.1371/journal.pone.0225019 12. Grasruck M, Bartling S, Dinkel J, Kiessling F, Semmler W, Stierstorfer K, et al. Motion gated small animal imaging with a ﬂat-panel CT. Phys Med Imag. (2008) 6913:69134D. doi: 10.1117/12.769386 3. Mannheim JG, Schlichthaerle T, Kuebler L, Quintanilla-Martinez L, Kohlhofer U, Kneilling M, et al. Comparison of small animal CT contrast agents. Contrast Media Mol Imaging. (2016) 11:272–84. doi: 10.1002/cmmi.1689 3. Mannheim JG, Schlichthaerle T, Kuebler L, Quintanilla-Martinez L, Kohlhofer U, Kneilling M, et al. Comparison of small animal CT contrast agents. Contrast Media Mol Imaging. (2016) 11:272–84. doi: 10.1002/cmmi.1689 13. Dinkel J, Bartling SH, Kuntz J, Grasruck M, Kopp-Schneider A, Iwasaki M, et al. Intrinsic gating for small-animal computed tomography: a robust ECG-less paradigm for deriving cardiac phase information and functional imaging. Circ Cardiovasc Imaging. (2008) 1:235–43. doi: 10.1161/CIRCIMAGING.108.784702 4. Uehara T, Pogribny IP, Rusyn I. The DEN and CCl4 -induced mouse model of ﬁbrosis and inﬂammation-associated hepatocellular carcinoma. Curr Protoc Pharmacol. (2014) 66:14.30.1–10. doi: 10.1002/0471141755.ph1430s66 5. Kampschulte M, Langheinirch AC, Sender J, Litzlbauer HD, Althöhn U, Schwab JD, et al. Nano-computed tomography: technique and applications. RöFo. (2016) 188:146–54. doi: 10.1055/s-0041-106541 14. ETHICS STATEMENT The animal study was reviewed and approved by the Government of Upper Bavaria (Regierung von Oberbayern). The animal study was reviewed and approved by the Government of Upper Bavaria (Regierung von Oberbayern). The productivity of our method could be improved by reducing the required user interventions. It would be desirable if the software itself were able to determine the ROI, which has already been described (28). Work in progress is aiming to automatically detect and segment the lung toward this end. Instead of presetting a ﬁxed RF before reconstruction, the algorithm itself could ﬁnd an optimal RF for each scan. DATA AVAILABILITY STATEMENT The datasets presented in this article are not readily available because they are property of Roche Diagnostics. FUNDING The authors would like to thank the Federal Government of North-Rhine Westphalia, and the European Union (EFRE), the German Research Foundation (GR 5027/2-1 and CRC1382 project ID 403224013 - SFB 1382, project Q1) for funding. The authors would like to thank the Federal Government of North-Rhine Westphalia, and the European Union (EFRE), the German Research Foundation (GR 5027/2-1 and CRC1382 project ID 403224013 - SFB 1382, project Q1) for funding. Detectability of Tumors (C) Averaged over all mouse samples an optimum contrast to noise ratio of 3.659 was determined at RF = 0.15, similar to 3.657 at RF = 0.2. The noise was derived from a homogenous area of the mouse bed. (D) For the twelve reconstructions, the CNR is signiﬁcantly (p < 0.001 as indicated by , paired t-test) improved by the gating (RF = 0.2). FIGURE 9 \| Contrast of liver vs. tumor tissue. (A) An exemplary coronal µCT slice through the liver. One of the spheres at the liver edge E1 and one of the spheres at the center C1 can be seen. The other measurement spheres are in other slices. Also visible is the segmentation of two tumors T1 and T2. The third segmented tumor is not visible in this slice. The artifact at the top of the image is due to the fact that this is also the edge of the volumetric image, where less data is available for reconstruction. W: 4,429 HU L: 670 HU (B) Averaged over all mouse samples, the contrast between liver and tumor increases with RF and reaches saturation at RF = 0.25. (C) Averaged over all mouse samples an optimum contrast to noise ratio of 3.659 was determined at RF = 0.15, similar to 3.657 at RF = 0.2. The noise was derived from a homogenous area of the mouse bed. (D) For the twelve reconstructions, the CNR is signiﬁcantly (p < 0.001 as indicated by **, paired t-test) improved by the gating (RF = 0.2). July 2022 \| Volume 9 \| Article 878966 Frontiers in Medicine \| www.frontiersin.org 10 Thamm et al. Thamm et al. Gating for Multi-Mouse µCT Requests to access the datasets should be directed to FG, fgremse@ukaachen.de. For further improvements of the image quality, diﬀerent scan protocols could be investigated, e.g., with diﬀerent voltages, exposure times, or angle steps. Moreover, a continuous rotation scan protocol could be used. Because the X-ray source radiates continuously, less dose would be wasted for stop and go, at the cost of some motion blur caused by the rotation. CONCLUSION We can conclude that 4-mouse gating is possible with only a small loss of data (rejected projections) compared to single- mouse gating. This means that gating and high throughput are compatible. Our gating approach improves image quality i.e., the detectability of tumors in a tumor bearing liver for a 4- mouse scan without substantial compromise to dose eﬃciency compared to a single-mouse scan. AUTHOR CONTRIBUTIONS MT performed the gated reconstructions, analyzed the data, and wrote the original manuscript. SR and FG helped with the statistical analysis. AH, KL, and FG implemented the iterative reconstruction software. FO and TP planned and conducted the animal experiments. SR, KL, AH, FK, FO, TP, and FG reviewed the article. FG supervised the study and revised the article. All authors contributed to the article and approved the submitted version. REFERENCES A novel prior- and motion-based compressed sensing method for small-animal respiratory gated CT. PLoS ONE. (2016) 11:e0149841. doi: 10.1371/journal.pone.0149841 32. Brehm M, Sawall S, Maier J, Sauppe S, Kachelrieß M. Cardiorespiratory motion-compensated micro-CT image reconstruction using an artifact model-based motion estimation. Med Phys. (2015) 42:1948–58. doi: 10.1118/1.4916083 22. Sawall S, Bergner F, Lapp R, Mronz M, Karolczak M, Hess A, et al. Low-dose cardio-respiratory phase-correlated cone-beam micro-CT of small animals. Med Phys. (2011) 38:1416–24. doi: 10.1118/1.3551993 23. Ertel D, Kyriakou Y, Lapp RM, Kalender WA. Respiratory phase-correlated micro-CT imaging of free-breathing rodents. Phys Med Biol. (2009) 54:3837– 46. doi: 10.1088/0031-9155/54/12/015 Conﬂict of Interest: FG is the owner of Gremse-IT GmbH, a spin-out of the RWTH Aachen University, which commercializes software for biomedical image analysis. TP and FO are employees of Roche Diagnostics GmbH. 24. Li F, Guo CJ, Huang C-C, Yu G, Brown SM, Xu S, et al. Transient receptor potential A1 activation prolongs isoﬂurane induction latency and impairs respiratory function in mice. Anesthesiology. (2015) 122:768–75. doi: 10.1097/ALN.0000000000000607 The remaining 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. 25. Seidel J, Bernardo ML, Wong KJ, Xu B, Williams MR, Kuo F, et al. Simultaneous ECG-gated PET Imaging of multiple mice. Nucl Med Biol. (2014) 41:582–6. doi: 10.1016/j.nucmedbio.2014.03.015 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. 26. Greenwood HE, Nyitrai Z, Mocsai G, Hobor S, Witney TH. High throughput PET/CT imaging using a multiple mouse imaging system. J Nucl Med OﬀPubl Soc Nucl Med. (2019) 61:292–7. doi: 10.2967/jnumed.119.228692 27. Yagi M, Arentsen L, Shanley RM, Hui SK. High-throughput multiple-mouse imaging with micro-PET/CT for whole-skeleton assessment. Phys Medica PM Int J Devoted Appl Phys Med Biol OﬀJ Ital Assoc Biomed Phys AIFB. (2014) 30:849–53. doi: 10.1016/j.ejmp.2014.06.003 Copyright © 2022 Thamm, Rosenhain, Leonardic, Höfter, Kiessling, Osl, Pöschinger and Gremse. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). REFERENCES Bartling SH, Dinkel J, Stiller W, Grasruck M, Madisch I, Kauczor H-U, et al. Intrinsic respiratory gating in small-animal CT. Eur Radiol. (2008) 18:1375–84. doi: 10.1007/s00330-008-0903-3 6. Töpperwien M, Krenkel M, Vincenz D, Stöber F, Oelschlegel AM, Goldschmidt J, et al. Three-dimensional mouse brain cytoarchitecture revealed by laboratory-based x-ray phase-contrast tomography. Sci Rep. (2017) 7:42847. doi: 10.1038/srep42847 15. Johnston SM, Perez BA, Kirsch DG, Badea CT. Phase-selective image reconstruction of the lungs in small animals using micro-CT. Proc SPIE– Int Soc Opt Eng. (2010) 7622:76223G.1–9. doi: 10.1117/12.844359 7. Boll H, Figueiredo G, Fiebig T, Nittka S, Doyon F, Kerl HU, et al. Comparison of Fenestra LC, ExiTron nano 6000, and ExiTron nano 12000 for micro- CT imaging of liver and spleen in mice. Acad Radiol. 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Chavarrías C, Vaquero JJ, Sisniega A, Rodríguez-Ruano A, Soto-Montenegro ML, García-Barreno P, et al. Extraction of the respiratory signal from small- animal CT projections for a retrospective gating method. Phys Med Biol. (2008) 53:4683–95. doi: 10.1088/0031-9155/53/17/015 30. Gremse F, Stärk M, Ehling J, Menzel JR, Lammers T, Kiessling F. Imalytics preclinical: interactive analysis of biomedical volume data. Theranostics. (2016) 6:328–41. doi: 10.7150/thno.13624 31. Stahl S, Sacher T, Bechtold A, Protzer U, Ganss R, Hämmerling GJ, et al. Tumor agonist peptides break tolerance and elicit eﬀective CTL responses in an inducible mouse model of hepatocellular carcinoma. Immunol Lett. (2009) 123:31–7. doi: 10.1016/j.imlet.2009. 01.011 20. Boone JM, Velazquez O, Cherry SR. Small-animal X-ray dose from micro-CT. Mol Imaging. (2004) 3:149–58. doi: 10.1162/1535350042380326 21. Abascal JFPJ, Abella M, Marinetto E, Pascau J, Desco M. Frontiers in Medicine \| www.frontiersin.org July 2022 \| Volume 9 \| Article 878966 REFERENCES 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. 28. Kuntz J, Dinkel J, Zwick S, Bäuerle T, Grasruck M, Kiessling F, et al. Fully automated intrinsic respiratory and cardiac gating for small animal CT. Phys Med Biol. (2010) 55:2069–85. doi: 10.1088/0031-9155/55/7/018 29. Gremse F, Höfter A, Razik L, Kiessling F, Naumann U. GPU-accelerated adjoint algorithmic diﬀerentiation. Comput Phys Commun. (2016) 200:300– 11. doi: 10.1016/j.cpc.2015.10.027 July 2022 \| Volume 9 \| Article 878966 Frontiers in Medicine \| www.frontiersin.org 12
https://openalex.org/W2954877969	https://europepmc.org/articles/pmc6593486?pdf=render	English	null	Serious kidney disease in pregnancy: an Australian national cohort study protocol	BMC nephrology	2,019	cc-by	6,340	Abstract Background: Maternal kidney disease (acute kidney injury (AKI), advanced chronic kidney disease (CKD), dependence on dialysis or a kidney transplant) has a substantial impact on pregnancy, with risks of significant perinatal morbidity. These pregnancies require integrated multidisciplinary care to manage a complex and often challenging clinical situation. The ability to deliver optimal care is currently hindered by a lack of understanding around prevalence, management and outcomes in Australia. This study aims to expand an evidence base to improve clinical care of women with serious kidney impairment in pregnancy. Methods/design: The “Kidney Disease in Pregnancy Study” is a national prospective cohort study of women with stage 3b-5 CKD (including dialysis and transplant) and severe AKI in pregnancy, using the Australasian Maternity Outcomes Surveillance System (AMOSS). AMOSS incorporates Australian maternity units with > 50 births/year (n = 260), capturing approximately 96% of Australian births. We will identify women meeting the inclusion criteria who give birth in Australia between 1st August 2017 and 31st July 2018. Case identification will occur via monthly review of all births in Australian AMOSS sites and prospective notification to AMOSS via renal or obstetric clinics. AMOSS data collectors will capture key clinical data via a web-based data collection tool. The data collected will focus on the prevalence, medical and obstetric clinical care, and maternal and fetal outcomes of these high-risk pregnancies. Discussion: This study will increase awareness of the issue of serious renal impairment in pregnancy through engagement of 260 maternity units and obstetric and renal healthcare providers across the country. The study results will provide an evidence base for pre-pregnancy counselling and development of models of optimal clinical care, clinical guideline and policy development in Australia. Understanding current practices, gaps in care and areas for intervention will improve the care of women with serious renal impairment, women with high-risk pregnancies, their babies and their families. Keywords: Cohort study, Population, Pregnancy, Pregnancy outcome, Chronic kidney disease, Acute kidney injury, Kidney transplantation, Renal dialysis Serious kidney disease in pregnancy: an Australian national cohort study protocol Nadom Safi1, Elizabeth Sullivan1,2* , Zhuoyang Li1, Mark Brown3, William Hague4, Stephen McDonald5,6,7, Michael J. Peek8, Angela Makris9, Angela M. O’Brien1 and Shilpanjali Jesudason6,7 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Safi et al. BMC Nephrology (2019) 20:230 https://doi.org/10.1186/s12882-019-1393-z Safi et al. BMC Nephrology (2019) 20:230 https://doi.org/10.1186/s12882-019-1393-z Background population rise in diabetes, obesity and older age in pregnancy [4]. Over 5% of Australian women of child- bearing age have albuminuria or abnormal estimated GFR (eGFR) indicating CKD [2]. In Australia in 2016, 25% of all women with a functioning renal transplant and 51% of all female incident end-stage kidney disease (ESKD) cases were in women of reproductive age (15– 44 years) [5]. Parenthood is a major concern for women with CKD, and women experience important fears and uncertainties about risks to their own and their baby’s health [6, 7]. Women of childbearing age with CKD may face difficult decisions about parenthood while Chronic Kidney Disease (CKD) is an under-recognised, serious and increasing major health problem, which dis- proportionately affects Indigenous peoples and lower so- cioeconomic groups [1–3] . The prevalence of CKD amongst Australian mothers is rising due to the 2Faculty of Health and Medicine, University of Newcastle, 130 University Drive, Callaghan 2308, NSW, Australia Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Safi et al. BMC Nephrology (2019) 20:230 Page 2 of 7 Page 2 of 7 and more than 50% of such babies have birth weights less than the 10th centile [10]. Outcomes are better for women who commence dialysis after conception com- pared with those who conceive while receiving chronic dialysis [10, 28]. Even after successful kidney transplant- ation, these pregnancies continue to have high rates of pre-eclampsia (> 25%), prematurity and poor fetal growth (> 50%) [12, 13, 29]. Worse graft function (serum creatinine > 110 μmol/L) during pregnancy can predict risk of pre-eclampsia and kidney function trajectory in- cluding significantly higher rates of graft loss within 3 years of giving birth [29]. experiencing significant comorbidity and impairment to activities of daily living. Navigating paths to parenthood via complex shared decision-making can be challenging and accurate outcome data would enable evidence-based risk stratification [7]. Background There is a knowledge gap regarding the burden of CKD in pregnancy in the Australian context. Determin- ing the prevalence of kidney disease in pregnancy has not been easy, due to varying definitions, changing sta- ging systems and poor capture of renal markers (serum creatinine and urine protein) in pregnancy cohorts [8, 9]. The Australia and New Zealand dialysis and trans- plant registry (ANZDATA) reports pregnancy rates for chronic dialysis and transplant recipients of 3.3/1000 person-years and 20/1000 person-years respectively but is likely under-reported [10–12]. Rates of advanced stage CKD (Stage 3b-4), and non-chronic Acute Kidney Injury (AKI) occurring in pregnancy in Australia are unknown. This information is important when developing mater- nity and renal services. The incidence of Acute Kidney Injury (AKI) in preg- nancy has decreased in recent years in developed coun- tries, mainly due to improvement in peripartum sepsis care [30]. However, AKI still represents a potentially catastrophic event in pregnancy, caused predominantly by pre-eclampsia [31]. There is also enhanced awareness and understanding of newer conditions such as atypical haemolytic uraemic syndrome [32]. AKI in one preg- nancy is associated with worse outcomes in the next [33]. Approximately 1/10,000 pregnancies are compli- cated by AKI that requires dialysis, which was associated with poor perinatal outcomes and increased maternal death rate in one Canadian study [34]. There are no Australian data regarding frequency, causes and out- comes of pregnancy-related AKI, and this remains an important area to investigate. There is currently no data on how significant kidney disease in pregnancy (CKD stage 3b-5, dialysis, trans- plant or AKI) impacts upon pregnancy care and how it is managed in Australia. Registry and perinatal datasets currently active in Australia collect some data items and past analyses of the ANZDATA registry have provided some information on pregnancy rates and basic preg- nancy outcomes [10, 13]. However, these provide min- imal insights into obstetric and perinatal adverse events, therapy and interventions required during pregnancy, models of care delivered, and utilisation of health care resources including hospitalisation and other events. Severe CKD and AKI in pregnancy, while uncommon, cause a high burden of maternal and perinatal morbidity [14, 15, 34]. A lack of information regarding outcomes for women with these conditions in Australia including the maternal mortality and morbidity, model of care, perinatal outcomes, and access to services, presently limits the health service providers’ ability to counsel and manage affected women. Methods/design Study objective 5. Newly identified renal function impairment with any serum creatinine reading of > 150 μmol/L on 2 readings at least 24 h apart during pregnancy Using the existing AMOSS maternity surveillance sys- tem embedded in 260 maternity hospitals in Australia we will identify cases of significant CKD (Stage 3b-5 in- cluding dialysis and transplant) and severe AKI in pregnancy. Table 1 Inclusion criteria CKD and AKI will have a substantial pregnancy related and pre-existing medical morbidity, and that babies born to these mothers will have higher prematurity and con- sequent perinatal morbidity. Adverse outcomes may be related to modifiable factors in pre-pregnancy and ante- natal care, including access to specialised services, im- proved blood pressure management and dialysis treatment, fetal surveillance and timing of delivery. The study findings will inform a guideline and policy devel- opment and improve counselling for this high risk cohort. All women in Australia who gave birth: All women in Australia who gave birth: a. Between 1 August 2017 and 31 July 2018; AND b. At least 20 weeks’ gestation and/or at least 400 g birth weight; AND c. Who present with at least one of the following criteria: 1. A working kidney transplant (all women regardless of kidney transplant function) 2. Receiving any long-term dialysis before conception and continuing any dialysis during pregnancy. 3. Starting any dialysis during pregnancy (any dialysis - either once off, temporary or permanent dialysis) 4. Known pre-conception eGFR < 45 ml/min/1.73m2 (known to have a serum creatinine > 130–150 μmol/L before conception, regardless of the serum creatinine reading during pregnancy) 5. Newly identified renal function impairment with any serum creatinine reading of > 150 μmol/L on 2 readings at least 24 h apart during g a. Between 1 August 2017 and 31 July 2018; AND b. At least 20 weeks’ gestation and/or at least 400 g birth weight; AND c. Who present with at least one of the following criteria: 1. A working kidney transplant (all women regardless of kidney transplant function) 2. Receiving any long-term dialysis before conception and continuing any dialysis during pregnancy. 3. Starting any dialysis during pregnancy (any dialysis - either once off, temporary or permanent dialysis) 2 4. Known pre-conception eGFR < 45 ml/min/1.73m2 (known to have a serum creatinine > 130–150 μmol/L before conception, regardless of the serum creatinine reading during pregnancy) 4. Known pre-conception eGFR < 45 ml/min/1.73m2 (known to have a serum creatinine > 130–150 μmol/L before conception, regardless of the serum creatinine reading during pregnancy) Assessment of renal function in pregnancy – selection of inclusion criteria Serum creatinine is a widely used marker of renal func- tion in non-pregnant patients, which varies depending on muscle mass, age, and ethnicity. Renal function in non-pregnant patients is evaluated by calculating the es- timated glomerular filtration rate (eGRF), with formulae derived from populations using gender, age, ethnicity and serum creatinine measurement. The eGFR is most commonly calculated using the CKD-EPI formula and is routinely provided when reporting serum creatinine in the adult population by laboratories in Australia [37]. However, eGFR formulae are not valid for use during pregnancy, where serum creatinine usually falls due to renal physiological changes, and where a serum creatin- ine > 80-90 mmol/L usually reflects a degree of renal im- pairment. Therefore, in this study, eGFR will only be used for pre-pregnancy serum creatinine values. As shown in Table 2, in women aged 18–45 years, a pre- pregnancy serum creatinine of 130–150 micromol/L or 1. To determine the prevalence, and geographical distribution. 2. Utilise survey data to determine pregnancy outcomes (maternal and perinatal), renal outcomes, models of care, and maternity management Background The model of care required for such high risk pregnancies may not be equitably avail- able to all women at higher risk, particularly those who are Indigenous, geographically isolated, from a lower so- cioeconomic status (SES) or from a culturally and lin- guistically diverse (CALD) group. This knowledge is important to obtain as pregnancy in women with CKD is associated with worse obstetric and perinatal outcomes including preterm birth, small for gestational age, and pre-eclampsia and gestational hyper- tension [14, 15]. The risk is increased, even in women with Stage 1 CKD [16, 17] and continues to rise as CKD stage advances [15, 18, 19]. Pre-conception eGFR< 40 ml/min, poorly controlled hypertension and proteinuria are factors associated with higher rates of obstetric and perinatal complications [20–23]. As pre-conception renal function worsens, the risk of “stage shift” or deteri- oration in renal function also increases, and may neces- sitate dialysis during pregnancy [9, 24, 25]. The aim of this study is to determine the Australian prevalence, pregnancy-related and pre-existing maternal and renal morbidity and perinatal outcomes associated with serious maternal kidney disease in pregnancy; and to describe patterns of presentation, pregnancy manage- ment and models of care and adequacy of healthcare de- livery in these pregnancies. We will achieve this by development of a new framework for capture of mater- nal and fetal outcomes for cases of significant kidney disease in pregnancy, using the existing Australasian Maternity Outcomes Surveillance System (AMOSS) re- search platform which is embedded in many maternity units across Australia. We hypothesise that mothers with Pregnancies in women receiving chronic dialysis are rare due to the reduced fertility of women with advanced kidney failure, and are particularly challenging in terms of maternal care [8]. The recognition that intensive dia- lysis improves outcomes has led to changes in clinical practice and counselling for women with advanced CKD [19, 24, 26, 27]. Australian women receiving chronic dia- lysis have a 27% fetal loss rate compared with 7% among all pregnancies [10]. Among live births, 75% are preterm Page 3 of 7 Safi et al. BMC Nephrology (2019) 20:230 Safi et al. BMC Nephrology (2019) 20:230 Safi et al. BMC Nephrology (2019) 20:230 Table 1 Inclusion criteria Study design A national, prospective cohort study using the Australa- sian Maternity Outcomes Surveillance System (AMOSS) . The AMOSS methods have been described in detail elsewhere [35, 36]. In brief, AMOSS is an established surveillance and research system across maternity units in Australia and New Zealand to study rare but clinically important and severe conditions in pregnancy, which place a disproportionate burden on women, their fam- ilies and healthcare systems. AMOSS has access to al- most all (95% [260/275]) eligible Australian maternity units with more than 50 births/year, capturing an esti- mated 96% of women giving birth in a hospital in Australia. Table 2 Pre-pregnancy serum creatinine and eGFR Age eGFRa based on serum creatinine Table 2 Pre-pregnancy serum creatinine and eGFR Age eGFRa based on serum creatinine 150 micromol/L Table 2 Pre-pregnancy serum creatinine and eGFR Table 2 Pre-pregnancy serum creatinine and eGFR Age eGFRa based on serum creatinine 150 micromol/L 18 44b 25 41b 30 40b 35 39b 40 37b 45 36b 50 35b Table 2 Pre-pregnancy serum creatinine and eGFR Age eGFRa based on serum creatinine 150 micromol/L 18 44b 25 41b 30 40b 35 39b 40 37b 45 36b 50 35b aeGFR(ml/min/1.73m2) calculated for adult females at various ages within the childbearing age range, using the CKD-EPI formula [45] without ethnicity coefficients beGFR < 45 ml/min/1.73m2 indicates Stage 3b CKD. Therefore, all women with pre-pregnancy serum creatinine ≥150micromol/L will have CKD Stage 3b or higher Study population, case definition and inclusion criteria The study population will include all women giving birth in participating Australian AMOSS sites between 1st August 2017 and 31st July 2018. Cases of serious kidney disease giving birth in participating Australian AMOSS sites during the study period will be collected. Table 1 shows the inclusion criteria. Birth is defined as the birth of one or more live or stillborn infants of at least 20 weeks gestation or at least 400 g birthweight. Women not giving birth (e.g. pregnancy ending in miscarriage or termination of pregnancy < 20 weeks gestation) or with early-stage CKD and less severe AKI will be excluded from the study. Safi et al. BMC Nephrology (2019) 20:230 Safi et al. BMC Nephrology (2019) 20:230 Page 4 of 7 4. Medication and other therapies: medication used prior to pregnancy; details of antihypertensive and immunosuppressant medications. 4. Medication and other therapies: medication used prior to pregnancy; details of antihypertensive and immunosuppressant medications. Study outcomes Primary outcomes: Prevalence rates of maternal AKI and CKD in the general AMOSS birth (live or stillbirths) cohort as per inclusion criteria. The rate will be expressed as per 1000 maternities based on birth rates for AMOSS hospitals during the study period. Secondary outcomes will include: 1) Maternal obstetric outcomes and complications – a range of outcomes dur- ing antenatal, delivery and post-partum stages; 2) Mater- nal renal outcomes including renal function, graft function and commencement of dialysis; 3) Adequacy and complications of dialysis therapy; 4) Transplant- related complications including graft dysfunction and in- fection; 5) Perinatal outcomes including birth outcomes and adverse outcomes especially related to preterm birth (Additional file 3). The second survey has been developed for this study will capture specific renal information (Additional file 2): 1. General renal history: presentation; the underlying cause of kidney disease; primary diagnosis including kidney biopsy; biochemical data; proteinuria; blood pressure; details of kidney disease management; timing and model of antenatal care; and access to specialist obstetric, obstetric medicine and nephrology services. Study design more will reflect eGFR < 45 ml/min/1.73m2 (CKD Stage 3b or higher) [38]. The data collector will obtain pre- pregnancy serum creatinine measurements wherever available, for all cases. 5. Antenatal, birth and postpartum care: blood pressure measurements; antenatal screening and fetal growth scans; antenatal events, including diagnoses of pre-eclampsia, placental abruption; blood transfusion; and birth and post-partum management. 5. Antenatal, birth and postpartum care: blood pressure measurements; antenatal screening and fetal growth scans; antenatal events, including diagnoses of pre-eclampsia, placental abruption; blood transfusion; and birth and post-partum management. 5. Antenatal, birth and postpartum care: blood pressure measurements; antenatal screening and fetal growth scans; antenatal events, including diagnoses of pre-eclampsia, placental abruption; blood transfusion; and birth and post-partum management. AMOSS data collectors based in each AMOSS partici- pating site will complete two web-based data collection forms for all women who fulfil the inclusion criteria for the study (Fig. 1). The first survey form is the general AMOSS data collection form utilised in all AMOSS stud- ies, obtaining demographic characteristics such as mater- nal age, hospital sector, comorbidity, smoking, and BMI, and captured data on medical and obstetric history, ob- stetric interventions and information about type of care, and location of care. In addition this general survey col- lects information regarding baby outcomes including birth status, gestation, gender, birthweight, admission to inten- sive or specialised care units, congenital abnormalities, and neonatal morbidity and mortality (Additional file 1). Data collection gy 2. Transplant history: transplant source; transplant biopsy; anti-rejection treatment and therapeutic monitoring. Electronic survey data will be collected via an encrypted web-based data management system (the ‘AMOSS sys- tem’). Record data will be kept on a secure server with daily backup to an offsite facility. Where a data collector is not able to complete a survey using the web-based survey system, they may handwrite survey responses g 3. Dialysis: commencement of dialysis during pregnancy; dialysis modality; vascular access and complications. Fig. 1 AMOSS surveillance and data collection workflow. Hospital case identification. Clinical management/Hospital based obstetric and other information systems. Morbidity and/or clinical review meetings. Direct and indirect notification Fig. 1 AMOSS surveillance and data collection workflow. Hospital case identification. Clinical management/Hospital based obstetric and other information systems. Morbidity and/or clinical review meetings. Direct and indirect notification Safi et al. BMC Nephrology (2019) 20:230 Page 5 of 7 Page 5 of 7 using the PDF versions of surveys, and scan/send to AMOSS where the survey will be entered on their behalf by the AMOSS team. pattern of missing data for key study and outcome fac- tors with high proportion of missing data. Multiple im- putation will be performed for variables qualified as ‘missing at random’ [42]. For variables with pattern of missing data unlikely to be ‘missing at random’, a ‘proxy’ category for missing data will be created for variables with > 2% missing and a sensitivity analysis will be con- ducted to explore the effect of missing data. A p-value < 0.05 or a confidence interval not including unity will be considered statistically significant. All statistical analyses will be performed using SPSS 24.0 software (Armonk, NY, USA: IBM Corp.). AMOSS provides continuous education and support for data collectors before the start of data collection. This is achieved by disseminating the renal study proto- col using the AMOSS newsletter, emails, phone calls and presentations at hospitals and conferences. The AMOSS team and investigators will provide support for all data collectors regarding enquiries during the study via emails, phone calls or site visits as requested. Sample size Based on current international literature, which esti- mates CKD Stage 3b-5 rate of 2 to 12 per 10,000; severe AKI requiring dialysis as 1 per 10,000 pregnancies; esti- mated 295,530 births ≥20 weeks gestation/year in par- ticipating Australian AMOSS sites [34, 39–41]; and allowing for potential early pregnancy losses; we esti- mate identifying between 89 and 384 cases in 1 year. The strengths of this study come from the AMOSS re- search model, which encompasses 96% of hospital births in Australia, and where cases can be identified prospect- ively and data collected after birth. In addition, multiple sources to identify eligible participants will increase the ability to capture cases, thereby minimising missed cases. The main challenge in this study will be the data collection for women who (1) receive their obstetric and renal care at different locations, or (2) receive renal care at a non-AMOSS site. To overcome these challenges, ethics approval was granted at most sites for the AMOSS Project Coordinator to receive minimal identi- fying details of prospective patients receiving renal care at one site and pass those details on to the onsite AMOSS data collector at the woman’s maternity hos- pital. For women receiving renal care at a non-AMOSS site, separate ethical and/or governance approval for data collection at the specialist clinic and matching with maternity data will be sought on a case-by-case basis. Discussion We will identify study participants through multiple sources: AMOSS surveillance; notification from nephrol- ogists, obstetric physicians and obstetricians; audit com- mittees; and review of routine data collection within hospitals. This AMOSS study will highlight the issue of serious kidney disease in pregnancy in Australia, with height- ened awareness in approximately 260 maternity units and among obstetric and renal healthcare providers across the country. The study will focus on cases of ser- ious kidney disease in pregnancy that, while uncommon, can cause significant morbidity for mothers and babies, present clinical complexity for clinicians, and place a disproportionate burden on health care resources. The study will fill an important knowledge gap on the preva- lence and models of care for this high-risk cohort of women. Statistical analysis We anticipate our analysis will largely utilise descriptive statistics (frequency (percentage) for categorical data and mean (standard deviation) or median (interquartile range) for continuous data) will be used to summarise the demographic characteristics, kidney disease data, maternal and baby outcomes of women with advanced CKD, dialysis, transplant and AKI. Cases will be reviewed in depth to map patient care models. Maternal and baby outcomes will be compared with Australian norms. If sample size allows, odds ratios will be esti- mated via logistic regression for outcomes and adjusted for relevant parameters including demographic data (maternal age, ethnicity); renal function, proteinuria and blood pressure at conception; time from commencement of dialysis or transplant; and dialysis modality and hours (Additional file 4). Variables associated with outcomes in the univariable analysis (p < 0.2) and other factors identi- fied in the literature as potentially predictive will be en- tered into multivariable logistic regression models. Final models will be determined by taking into account the final sample size for each study group, likely causal path- way, collinearity, and clinical significance. Analysis of variance (ANOVA) and generalised linear models will be used to examine the change in maternal kidney function during pregnancy. We will examine the distribution and The results of this study will be immediately relevant and enable translation into clinical practice. Specifically, we will. a) Improve the evidence base for pre-pregnancy coun- selling and pregnancy management, and will pro- mote this as an important pathway towards improved outcomes. We will identify healthcare professional groups to target for strategic education Safi et al. BMC Nephrology (2019) 20:230 Page 6 of 7 Safi et al. BMC Nephrology (2019) 20:230 Page 6 of 7 in specialist and primary care, and mechanisms to target high-risk women antenatally. Previous AMOSS studies have led to important improve- ments in safety and quality of care for pregnant women suffering from rare and under-studied con- ditions, where there is often a lack of data and clin- ical experience to guide management. manuscript. NS, ES, ZL, AO and SJ managed the operation of the study. NS, ZL and SJ drafted the manuscript. manuscript. NS, ES, ZL, AO and SJ managed the operation of the study. NS, ZL and SJ drafted the manuscript. Additional files Received: 5 July 2018 Accepted: 23 May 2019 Received: 5 July 2018 Accepted: 23 May 2019 Received: 5 July 2018 Accepted: 23 May 2019 Received: 5 July 2018 Accepted: 23 May 2019 Additional file 1: General Information: Case report form is used to collect general maternal and neonatal data. (PDF 411 kb) Additional file 2: Renal Disease in Pregnancy: Case report form is used to collect data specifically related to renal disease in pregnancy. (PDF 511 kb) Additional file 3: Main outcome measurements: A list of the main outcome measures that will form the basis of data analysis. (DOCX 12 kb) Additional file 4: Selected covariates: The list of variables that we expect to be associated with the outcomes. (DOCX 13 kb) Availability of data and materials Not applicable. b) Promote clinical guideline and policy development in Australia by providing an evidence base describing current models of care, and highlighting gaps in care or risk factors for adverse outcomes. Currently, there are no Australian guidelines or policies to facilitate delivery of specialised pregnancy care to women with renal disease. Abbreviations AKI A Kid 4. Hilder L, Zhichao Z, Parker M, Jahan S, Chambers G. Australia's mothers and babies 2012. Perinatal statistics series no. 30. Cat. no. PER 69. Canberra: AIHW; 2014. 4. Hilder L, Zhichao Z, Parker M, Jahan S, Chambers G. Australia's mothers and babies 2012. Perinatal statistics series no. 30. Cat. no. PER 69. Canberra: AIHW; 2014. AKI: Acute Kidney Injury; AMOSS: Australasian Maternity Outcomes Surveillance System; BMI: Body Mass Index; CKD: Chronic Kidney Disease; eGFR: Estimated Glomerular Filtration Rate 5. Australia and New Zealand Dialysis and Transplant Registry, 40th Annual Report [http://www.anzdata.org.au/v1/report_2017.html] accessed 22 May 2018. 5. Australia and New Zealand Dialysis and Transplant Registry, 40th Annual Report [http://www.anzdata.org.au/v1/report_2017.html] accessed 22 May 2018. Acknowledgements AMOSS participating sites and data collectors, and former AMOSS assistant, Kirisha Turnbull. 6. Tong A, Brown MA, Winkelmayer WC, Craig JC, Jesudason S. Perspectives on pregnancy in women with CKD: a Semistructured interview study. Am J Kidney Dis. 2015;66(6):951–61. 6. Tong A, Brown MA, Winkelmayer WC, Craig JC, Jesudason S. Perspectives on pregnancy in women with CKD: a Semistructured interview study. Am J Kidney Dis. 2015;66(6):951–61. Author details 1 d) Use this study to form the foundation for developing new systems for ongoing data capture regarding maternal kidney disease, including the potential to re-purpose the data collection instru- ment into an ongoing registry. 1Faculty of Health, University of Technology Sydney, 235 Jones Street Ultimo, Sydney, NSW 2007, Australia. 2Faculty of Health and Medicine, University of Newcastle, 130 University Drive, Callaghan 2308, NSW, Australia. 3Department Renal Medicine and Medicine, St. George Hospital and University of New South Wales, Kogarah, Sydney, Australia. 4Robinson Research Institute, University of Adelaide, Women’s and Children’s Hospital, Adelaide 5006, SA, Australia. 5ANZDATA Registry, South Australia Health and Medical Research Institute, Adelaide, Australia. 6Adelaide Medical School, University of Adelaide, Adelaide, SA, Australia. 7Central and Northern Adelaide Renal and Transplantation Service (CNARTS), Royal Adelaide Hospital, Adelaide, SA, Australia. 8The Canberra Hospital, The Australian National University, Bdg 11, Level 2, Yamba Dve, Garran, Canberra 2605, ACT, Australia. 9University of Western Sydney and the University of New South Wales, Sydney, Australia. Given that no other studies have explored serious mater- nal kidney disease in the Australian birth cohort, we antici- pate the results will be of great interest to the obstetric and renal community nationally and internationally. References 1. Australia and New Zealand Dialysis and Transplant Registry, 39th Annual Report [http://www.anzdata.org.au/v1/report_2016.html] accessed 18 October 2017. 1. Australia and New Zealand Dialysis and Transplant Registry, 39th Annual Report [http://www.anzdata.org.au/v1/report_2016.html] accessed 18 October 2017. 2. Australian Institute of Health and Welfare: Cardiovascular disease, diabetes and chronic kidney disease—Australian facts: Prevalence and incidence. Cardiovascular, diabetes and chronic kidney disease series no. 2. Cat. no. CDK 2. In. Canberra: AIHW; 2014. 2. Australian Institute of Health and Welfare: Cardiovascular disease, diabetes and chronic kidney disease—Australian facts: Prevalence and incidence. Cardiovascular, diabetes and chronic kidney disease series no. 2. Cat. no. CDK 2. In. Canberra: AIHW; 2014. 3. Dharmarajan SH, Bragg-Gresham JL, Morgenstern H, Gillespie BW, Li Y, Powe NR, Tuot DS, Banerjee T, Burrows NR, Rolka DB, et al. State-level awareness of chronic kidney disease in the U.S. Am J Prev Med. 2017;53(3):300–7. 3. Dharmarajan SH, Bragg-Gresham JL, Morgenstern H, Gillespie BW, Li Y, Powe NR, Tuot DS, Banerjee T, Burrows NR, Rolka DB, et al. State-level awareness of chronic kidney disease in the U.S. Am J Prev Med. 2017;53(3):300–7. Funding R l Ad Royal Adelaide Hospital Research Fund 2016 Clinical Project Grant. The funding body had no role in study design, data collection, data analysis, data interpretation, or manuscript writing. Ethics approval and consent to participate Ethics approval for this study (Ref: HREC/09/CIPHS/21) was granted on 29 July 2016 by NSW Population and Health Services Research Ethics Committee and multiple Human Research Ethics Committees across Australia [43]. Consent is not obtained from individual women participating in the study, as their identity will not be disclosed. Individual sites granted their consent to collect and provide to AMOSS summarised non-identifiable data after an event has occurred. In addition, no photos or videos of participants are collected. Consent from participants was requested to be waived from and approved by the NSW Population & Health HREC and other ethics committees. c) Disseminate our findings to key stakeholders (clinical providers - medical, midwifery, nursing, allied health, government policy planners and patient/consumer groups) involved in the care of women with CKD and AKI, women with high-risk pregnancy, their babies and their families. 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J Nephrol. 2017;30(6):767–71. 9. Piccoli GB, Cabiddu G, Attini R, Vigotti FN, Maxia S, Lepori N, Tuveri M, Massidda M, Marchi C, Mura S, et al. Risk of adverse pregnancy outcomes in women with CKD. J Am Soc Nephrol. 2015;26(8):2011–22. 33. Tangren JS, Powe CE, Ankers E, Ecker J, Bramham K, Hladunewich MA, Karumanchi SA, Thadhani R. Pregnancy outcomes after clinical recovery from AKI. J Am Soc Nephrol. 2017;28(5):1566–74. 10. Jesudason S, Grace BS, McDonald SP. Pregnancy outcomes according to dialysis commencing before or after conception in women with ESRD. Clin J Am Soc Nephrol. 2014;9(1):143–9. 34. Hildebrand AM, Liu K, Shariff SZ, Ray JG, Sontrop JM, Clark WF, Hladunewich MA, Garg AX. Characteristics and outcomes of AKI treated with Dialysis during pregnancy and the postpartum period. J Am Soc Nephrol. 2015; 26(12):3085–91. 11. Shahir AK, Briggs N, Katsoulis J, Levidiotis V. An observational outcomes study from 1966-2008, examining pregnancy and neonatal outcomes from dialysed women using data from the ANZDATA registry. Nephrology (Carlton). 2013;18(4):276–84. 35. Farquhar CM, Li Z, Lensen S, McLintock C, Pollock W, Peek MJ, Ellwood D, Knight M, Homer CS, Vaughan G, et al. Incidence, risk factors and perinatal outcomes for placenta accreta in Australia and New Zealand: a case-control study. BMJ Open. 2017;7(10):e017713. 12. Wyld ML, Clayton PA, Jesudason S, Chadban SJ, Alexander SI. Pregnancy outcomes for kidney transplant recipients. Am J Transplant. 2013;13(12): 3173–82. 36. Sullivan EA, Javid N, Duncombe G, Li Z, Safi N, Cincotta R, Homer CSE, Halliday L, Oyelese Y. Vasa Previa diagnosis, clinical practice, and outcomes in Australia. Obstet Gynecol. 2017;130(3):591–8. 13. Authors’ contributions Sterne JAC, White IR, Carlin JB, Spratt M, Royston P, Kenward MG, Wood AM, Carpenter JR. 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https://openalex.org/W1999107935	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0017188&type=printable	English	null	Negative Refraction Angular Characterization in One-Dimensional Photonic Crystals	PloS one	2,011	cc-by	7,623	Introduction Photonic crystals can be considered as multidimensional periodic gratings, in which the features of refraction at flat surfaces are dominated by Bragg diffraction effects. The refraction angle from positive to negative can be tailored based on photonic band theory [1]. Numerous studies on diffraction gratings and periodic planar waveguides, essentially the one-dimensional counterparts for the photonic structures, led to the observation of a vast variety of anomalous refraction effects, including ‘‘birefringence’’ [2–6]. These systems have undergone extensive and systematic study based on the wave vector diagram formalism. This formalism has proven to be an excellent tool in explaining the unusual refractive properties for the one-dimensional diffraction grating system. In the late 1990s, diffraction characteristics that appeared to be negative refraction were explained in terms of the dispersion surfaces of photonic bands and prism, lens, and collimation effects based on refraction were predicted [7–9]. Specifically, it has been demonstrated that light propagation in strongly modulated 2D/3D photonic crystals becomes refraction-like in the vicinity of the photonic bandgap, even in the presence of strong multiple diffraction [4]. In these conditions, it is possible to define an effective phase refractive index to explain the propagation inside the photonic crystal using the conventional Snell’s law. Since such effective index is determined by This behavior can be understood by using the effective-mass model in electron-band theory. In the photonic case a Bloch photon, near the bandgaps, can be considered as free, and be regarded as a refracted photon inside of a medium with an effective refractive index. These particular index states only appear close the photonic bandgap in a similar way as the effective mass states in a semiconductor. The same conclusion has been reached by others groups [10]. For instance, the effective dielectric constant of a 2D photonic crystal in all optical bands, for both TE and TM polarizations, was calculated. It has been found that near the gamma point (center of the Brillouin zone), the dispersion relationship for the TM mode is independent of the propagation direction, while the TE mode in general depends on the electromagnetic waves propagation direction. Therefore, for a 2D photonic crystal, there always exists an effective dielectric index for the TM mode near the gamma point. However, it cannot be defined as an effective refractive index for TE mode unless the photonic crystal is highly symmetric. Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: je.lugo.arce@umontreal.ca the photonic band structure, it can be negative and less than unity, which leads to negative refraction [9]. Jesus Eduardo Lugo*, Rafael Doti, Jocelyn Faubert Visual Psychophysics and Perception Laboratory, School of Optometry, University of Montreal, Montreal, Quebec, Canada Abstract Background: Photonic crystals are artificial structures that have periodic dielectric components with different refractive indices. Under certain conditions, they abnormally refract the light, a phenomenon called negative refraction. Here we experimentally characterize negative refraction in a one dimensional photonic crystal structure; near the low frequency edge of the fourth photonic bandgap. We compare the experimental results with current theory and a theory based on the group velocity developed here. We also analytically derived the negative refraction correctness condition that gives the angular region where negative refraction occurs. Methodology/Principal Findings: By using standard photonic techniques we experimentally determined the relationship between incidence and negative refraction angles and found the negative refraction range by applying the correctness condition. In order to compare both theories with experimental results an output refraction correction was utilized. The correction uses Snell’s law and an effective refractive index based on two effective dielectric constants. We found good agreement between experiment and both theories in the negative refraction zone. Conclusions/Significance: Since both theories and the experimental observations agreed well in the negative refraction region, we can use both negative refraction theories plus the output correction to predict negative refraction angles. This can be very useful from a practical point of view for space filtering applications such as a photonic demultiplexer or for sensing applications. Citation: Lugo JE, Doti R, Faubert J (2011) Negative Refraction Angular Characterization in One-Dimensional Photonic Crystals. PLoS ONE 6(4): e17188. doi:10.1371/journal.pone.0017188 R, Faubert J (2011) Negative Refraction Angular Characterization in One-Dimensional Photonic Crystals. PLoS ONE 6(4): e17188 0017188 Editor: Matteo Rini, Joint Research Centre - European Commission, Germany Editor: Matteo Rini, Joint Research Centre - European Commission, Germany Received August 19, 2010; Accepted January 24, 2011; Published April 6, 2011 Received August 19, 2010; Accepted January 24, 2011; Published April 6, 2011 Copyright: 2011 Lugo 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. Funding: This work was supported by NSERC-Essilor Research Chair and an NSERC operating grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Introduction 1 layers appear to be collapsed. Nevertheless, negative refraction was observed in all our experiments where several regions were scanned. Scanning electron microscopy (SEM) was used to measure the films thicknesses which were 326611 nm (a) and 435611 nm (b). the effective mass is negative. Recently, we have simulated a lossless 1D PC structure and showed that negative refraction could be present near the low frequency edge of at least the second, fourth and sixth bandgaps [12]. The same conclusion was reached by other groups [13–15]. Furthermore, we experimentally demonstrated negative refraction in strongly modulated porous silicon 1D-PC in the visible and near infrared regions. However, in [12] negative refraction was explored with only one angle of incidence. Therefore, a complete angular characterization is still missing. Moreover, in regards to the theory of negative refraction in 1D PCs, the existence of antiparallel energy and phase velocity has been thoroughly analyzed in [16]. The existence of negative refraction in 2D PCs is substantially different from the one- dimensional case because 2D PCs with a negative slope band demonstrates negative refraction beam propagation. This is not true for 1D PCs because the correctness propagation condition needs to be fulfilled. The ‘‘correctness’’ of propagation in 1D PCs implies that the correct physical conditions, required to observe negative refraction, are met. The analysis presented in [16], for 2DPCs, only tackles negative refraction for on-plane propagation where the crystal is periodic. The exact analogy for negative refraction propagation between 2D PCs and 1D PCs is the normal incidence case, where the 1D PCs are periodic in that particular direction. Nonetheless the aforementioned correctness condition should also be applied in the 2D PCs case when you have off-plane propagation, a point that we will discuss later. In this paper, we experimentally completed the angular characterization of negative refraction in a 1D PC structure, near the low frequency edge of the fourth photonic bandgap. We compared the experimental results with current negative refraction theory in 1D PCs [11] and with a theory developed here, based on the group velocity. We confronted both negative refraction theories and found good agreement between them with differences up to 4 degrees, within the explored incidence angle interval. We analytically derived the correctness condition and showed that for the experimental conditions we used, the correctness condition is fulfilled up to an incidence angle of 15 degrees. Introduction By using similar arguments presented in [9], Kavokin theoretically explored negative refraction in one-dimensional photonic crystals (1D PCs) [11]. By using the dispersion of the photonic bands, he inferred negative refraction zones from frequency regions where PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e17188 1 PLoS ONE \| www.plosone.org Negative Refraction in 1D PCs resistivity of 0.001–0.005 Ohm-cm (room temperature = 25uC, humidity = 30%). On one side of the c-Si wafer, an aluminum film was deposited and then heated at 550uC during 15 minutes in nitrogen atmosphere to produce a good electrical contact. In order to have flat interfaces, an aqueous electrolyte composed of HF/ ethanol/glycerol was used to anodize the silicon substrate. It is well known that the Psi refractive index increases by decreasing the electrical current applied during the electrochemical etching. However, reducing the porosity too much might stop the electrolyte flow through the porous and limit the subsequent high porosity layer that makes the contrast. One way to allow the electrolyte to flow is by increasing the ethanol fraction in the solution. For this reason, an electrolyte composition of 3:7:1 was used. In addition, the HF concentration was maintained constant during the etching process using a peristaltic pump to circulate the electrolyte within the TeflonTM cell. Anodization begins when a constant current is applied between the c-Si wafer and the electrolyte by means of an electronic circuit controlling the anodization process. To produce the multilayers, current density applied during the electrochemical dissolution was alternated from 3 mA/cm2 (layer a) to 40 mA/cm2 (layer b) and eighty periods (160 layers) were made. Psi samples were partially oxidized at 350uC for 10 minutes. The best refractive index values we found that fit the experimental photonic bandgap structure studied here are na~1:1 and nb~2 [12]. We have experimentally measured the refractive indices of single Psi layers made with the same electrochemical conditions as the multilayers [18] and we found that na~1:4060.07 and na~2:2060.11. The refractive indices were measured by using interference fringes from reflectance measurements [18–19]. Nevertheless, it is known that the refractive index and etching rate for a single layer are modified in the presence of a multilayer structure up to approximately 14%, a phenomenon that has been systematically observed [19]. This result might have the consequence of compromising the mechanical stability of the structure. Indeed, in certain regions seen in Fig. Introduction We also theoretically verified the correctness condition near the second bandgap edge (1350 nm) and found that it is fulfilled up to an incidence angle of 20 degrees. In order to compare the experiments with theory we developed an approximation that accounts for the positive refraction that the negative refraction beam suffers at the structure output. The correction uses Snell’s law and an effective refractive index, based on two effective dielectric constants [17]. We found good agreement between experimental observations and the theory developed here for the whole incidence angle interval explored. The agreement between current theory and experimental results was good for incidence angles smaller than 15 degrees because the effective mass approximation begins to fail for incidence angles larger than 15 degrees and so does its consequent correction approximation. Since both theories and experimental results agreed well in the negative refraction region, given by the correctness condition, we can use both negative refraction theories with the addition of the output correction given herein to predict negative refraction angles. Once the samples were ready, we investigated the relationship between the negative refraction angle and the incidence angle at 633 nm (TE polarization) for the 1D PC structure. Figure 2 shows the experimental setup we used. The apparatus consists of a plate on which we find a curved support with a sliding base (4), a turning bar (7) and a turning platform (8). There is a light source of 633 nm (5) that can slide on the curved support (4) that points towards the turning Figure 1. 1D PC structure. SEM picture showing the layers a and b, angle of incidence a and negative refraction angle b9 inside the structure and corrected negative refraction angle b at the output, which can be measured experimentally. The light impinges at the right interface (The white line on the left represents 1 micron). doi:10.1371/journal.pone.0017188.g001 Sample preparation and negative refraction angle for the output measurement Figure 1. 1D PC structure. SEM picture showing the layers a and b, angle of incidence a and negative refraction angle b9 inside the structure and corrected negative refraction angle b at the output, which can be measured experimentally. The light impinges at the right interface (The white line on the left represents 1 micron). doi:10.1371/journal.pone.0017188.g001 Porous silicon (Psi) multilayers (Fig. 1) were prepared by electrochemical anodization of crystalline silicon (c-Si) [18]. Porous silicon was fabricated by wet electrochemical etching of highly boron-doped c-Si substrates with orientation (100) and electrical PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e17188 2 Negative Refraction in 1D PCs center. On top of the turning bar (7) we find a xyz platform (2) that holds the 1D PC (1) under test. The turning platform (8), which holds the video camera (10), has two movement axes (9). These materials were placed on a standard optical table. We illuminated the sample edge with a light source at the desired incidence angle a and, by exploring the sample side with the video camera; we found the output refracted beam (corrected negative refraction angle b). Once the beam was detected, its direction was confirmed by means of the beam spot luminance on the image monitor (not shown) that was measured with a luminance meter. As the refracted beam gets weaker for higher incidence angles, we explored angles up to 25 degrees in order to have enough discrimination of the spot luminance in reference to the monitor image background luminance. More details are given in the methods section. to discuss negative refraction theory that allows us to calculate negative refraction angles b’ as a function of incidence angles a (see Fig. 1). Negative refraction theory for 1D PCs has been presented in reference [11], where the condition for negative refraction uses the notion that, if in a given direction the effective mass is negative, the corresponding components of group and phase velocities of light have different signs. This seems to be true for 1D PCs because they are strongly anisotropic, so that the effective masses have different signs in on-plane and normal-to-plane directions [11]. However, in order to fully warrant the occurrence of negative refraction the correctness condition needs to be fulfilled [16]. It is well known that there are significant differences between the properties of 1D PCs and 2D PCs. Sample preparation and negative refraction angle for the output measurement In 2D PCs, when the plane of incidence is chosen to be the periodic plane, the entire wave vector is confined in the first Brillouin zone. In the theory of wave propagation through a crystal lattice, the Brillouin zone is a fundamental region of wavevectors; every vector outside this region is tantamount to some other vector inside it. In contrast, in the 1D PC, only the component of the wave vector along the direction of Negative refraction theory In order to compare the experimental corrected negative refraction angles at the output (angle b) with theory, first we need Figure 2. Experimental set up. The eleven components of the experimental setup for negative refraction observation. doi:10.1371/journal.pone.0017188.g002 April 2011 \| Volume 6 \| Issue 4 \| e17188 3 Negative Refraction in 1D PCs Figure 3. Correctness condition. Correctness condition generaliza- tion from 1D PCs to 2D PCs. doi:10.1371/journal.pone.0017188.g003 periodicity is restricted within the first BZ. This has a very important implication. In 2D PCs, a band with negative slope corresponds to a negative refraction beam. However, this is not true for 1DPCs. We have chosen x to represent the direction of the periodicity (Fig. 1). The slope of a certain band will then be given by Vgperkper, where Vgper and kper are the group velocity and wavevector components in the normal-to-plane direction respectively. Since Vgparkpar is always positive, where Vgpar and kpar are the group velocity and wavevector components in the on-plane direction respectively, and for a band with positive slope Vgperkperw0, then ~S:~kw0, where ~S and ~k are the Poynting vector and the wavevector respectively. For a band with negative slope Vgperkperv0, then the correctness condition for negative refraction to occurs ~S:~kv0 gives Vgparkgparv Vgperkper : ð1Þ ð1Þ Where we have used the fact that the Poynting vector is proportional to the group velocity. The group velocity components Vgper and Vgpar can be obtained from the photonic bands’ dispersion relationship as outlined in [13] as: Vgper~ 1 Lkper Lv , ð2Þ Vgpar~{ Lkper Lkpar Lkper Lv : ð3Þ Vgper~ 1 Lkper Lv , ð2Þ Vgpar~{ Lkper Lkpar Lkper Lv : ð3Þ Vgper~ 1 Lkper Lv , ð2Þ Figure 3. Correctness condition. Correctness condition generaliza- tion from 1D PCs to 2D PCs. doi:10.1371/journal.pone.0017188.g003 ð2Þ ~VgparT:~kparT is always positive. Therefore inequality 4 gives the negative refraction correctness condition either for 1D PCs or 2D PCs. The parallel direction represents the direction where there are no periodic dielectric regions to coherently scatter the light. For instance, in a 1D PC is the on-plane direction (known as off- axis as well) and for a 2D PC is the off-plane direction. Negative refraction theory ð3Þ The expression for the negative refraction angle b’ for the geometry showed in figure 1 is obtained in [11] by using the continuity of the electric and magnetic fields at the boundary and the effective mass approximation as: We have verified condition 1 (see the methods section), for our proposed 1D PC structure, close to the second (1350 nm) and fourth (633 nm) low frequency band edges (TE Polarization) and used refractive index values and layer thickness described in the experimental section and with n0 equals one. Condition 1 is fulfilled for incidence angles up to 20 degrees and 15 degrees respectively. For 633 nm light, traveling in the structure, one should expect that for angles of incidence larger than 15 degrees there will be more than one beam travelling inside the structure. For the normal-to- plane direction the second and fourth allowed band ends at 1345 nm and 630 nm respectively and they are characterized by a negative parabolicity close to the band edge. On the other hand, for the on-plane direction it is also parabolic close to band edge but it is characterized by a positive effective mass. In the case where the relevant bands have different band slope signs, one can observe the simultaneous propagation of beams [16]. Condition 1 can be generalized as sin2 b’ ð Þ~ n2 0 sin2 a ð Þ C2 , ð5Þ ð5Þ where n0 is the air refractive index, a is the incidence angle, c is the speed of light, h is Planck’s constant, l is the working light wavelength, l0 is the wavelength associated with the top of the fourth subband, for instance and lTl0. The effective mass approximation works fine only if the condition l{l0 ð Þ=lSS1 is fulfilled. The parameter C2 is expressed as C2~ 1{ mper mpar n2 0sin2 a ð Þz 2m2 percl hmpar 1{ l l0 : ð6Þ ð6Þ The effective masses of light in the normal-to-plane direction mper and on-plane direction mpar are calculated by using the expressions given in the methods section. ~VgparT:~kparTv ~VgperT:~kperT : ð4Þ ð4Þ We can also calculate negative refractive angles by using the group velocity, which represents the direction of propagation inside the medium as follows: Where~kparT and ~VgparT are the total wavector and group velocity in the parallel direction. Comparison between experimental results and theory Figure 5, shows the experimental and negative refraction results for the theories (eqs. (5) and (7)) for the behavior between angle of incidence versus angle of refraction. Since the experimental values represent corrected negative refraction angles at the output (angle b) we cannot compare them directly with the theories because they represent negative refraction angles b9 inside the structure. Since the negative refraction beam does not only propagate in the normal-to-plane direction we have to construct an effective medium approximation that takes into account the on-plane propagation direction as well. We can use the normal-to-plane eper~ fan2 azfbn2 b and on-plane epar~ fa n2 a z fb n2 b {1 effective dielectric constants, known to work fine in a multilayer system [17]. Therefore we can construct an effective medium approxi- mation with an effective index of refraction given by p g g b Reference [16] investigated light propagation in a 2D PC that consisted of dielectric rods in air with a hexagonal arrangement for the H-polarization case. They first performed a finite-difference time-domain (FDTD) simulation of light propagation along the C-K interface with an incidence angle of 8 degrees. The C-K interface goes from the center of the Brillouin zone to a vertex that joins two edges. Second, they supposed that their periodic structure could be described with an effective medium having an effective dielectric constant consistent with Maxwell-Garnett theory [16] and, there- fore, an effective refractive index. In such a case, the field inside the PC is a plane wave. Third, by using the plane wave expansion method (PWE) they determined that for low dielectric contrasts between rods and air there is mainly one predominant component contributing to the Floquet-Bloch wave (FB). If the dielectric contrast between rods and air is bigger than 2, mixing between the different components in the FB sum starts to occur. This was corroborated by their FDTD simulations. Fourth, for both treatments, the effective homogeneous medium and the periodic medium with the PWE method gave almost the same angle for the propagating beam. This value is in excellent agreement with the FDTD simulation result. Given this, one might think that it is possible to describe a photonic crystal medium, for low dielectric contrast, as a homogeneous medium with an effective index. However, if you take the same angle of incidence, but choose a different interface such as C-M. Negative refraction theory ~kperT and ~VgperT are the total wavector and group velocity in the perpendicular direction. In the 1D PC case the vectors, according with figure 3, are given by~kparT~(kpar1,kpar2,0), ~VgparT~(Vpar1,Vpar2,0), ~kperT~(0,0,kper1), ~VgperT~(0,0,Vper1) and in the 2D PC case by ~kparT~(kpar1,0,0), ~VgparT~(Vpar1,0,0), ~kperT~(0,kper2,kper1) and ~VgperT~(0,Vper2,Vper1). tan b’ ð Þ~ Vgper Vgpar : ð7Þ ð7Þ Equation (7) is given in the methods section. Figure 4 shows the comparison between Eqs. (5) and (7). We used a working wavelength of 633 nm (TE Polarization) which is close to the fourth low frequency band edge (l0-630 nm), and we used It is clear that if we are in a band with a negative slope where ~VgperT:~kperTv0 is always true and since in the parallel direction PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e17188 4 Negative Refraction in 1D PCs Negative Refraction in 1D PCs refractive index values and layer thickness described in the experimental section with n0 equals one. Clearly both curves are similar with angle value differences up to 4 degrees, within the explored incidence angle interval. This result supports the use of the theoretical approach represented by Eq. (5) to predict negative refraction angles. Nonetheless, the fact the effective medium approach fails to generally describe beam propagation in some cases, this does not preclude the use such approximation to describe beam propagation in a particular direction if there is only one predominant FB wave travelling in that direction. We have done a finite element simulation of our structure (figure 6-top) where we can observe a single negative refraction beam (beam with angle b9) that impinges towards the normal-to-plane interface where it is positively refracted as a single beam. This implies that we can use an effective medium approximation in such direction but we have to bare in mind that the effective refractive index does not represent the refractive index of the structure as if it were a homogeneous medium in all directions. Comparison between experimental results and theory doi:10.1371/journal.pone.0017188.g005 Equation (10) is valid for 0vaminƒa, where amin is given by correctness condition and showed that for the experimental conditions we used, the correctness condition is fulfilled up to an incidence angle of 15 degrees. We also theoretically verified the correctness condition near the second bandgap edge (1350 nm) and found that it is fulfilled up to an incidence angle of 20 degrees. Finally, we corroborated the angular experimental values with negative refracted angular values obtained with both negative refraction theories by applying an output correction that uses Snell’s law and an effective refractive index, based on the two effective dielectric constants. We found good agreement between experimental results and our theory in the entire incidence angle interval explored. The agreement between current theory and experimental results was good up to an incidence angle of 15 degrees because the effective mass approximation begins to fail for incidence angles larger than 15 degrees and the same is true for its consequent correction approximation. Since both theories and the experimental observations agreed well in the negative refraction region given by the correctness condition, we can use the combination of theory and output correction to predict negative refraction angles. This is very useful from a practical point of view. For instance, it could be useful for space filtering applications [20] such as a photonic demultiplexer or for sensing applications. A demultiplexer could be based on the fact that it is possible to have different wavelengths light impinging on the same incidence angle, since b9 depends on the wavelength, light with different wavelengths is dispersed in different directions at the output. Equations (10) and (12) will consequently be useful to estimate the output angles. A (Bio)chemical sensor could instead exploit the fact that the multilayers are porous and we can change their refractive indices by infiltrating different chemical or biological compounds that again would shift the angles b9 and b. Compound concentration should be proportional to this angular shift. 1{ mper mpar n2 0 sin2 amin ð Þ§ 2m2 percl hmpar 1{ l l0 : ð11Þ ð11Þ Inequality (11) assures us that the angle b 0 is real. Inequality (11) assures us that the angle b 0 is real. Comparison between experimental results and theory Notwithstanding, equation (10) is a good approximation to calculate corrected negative refraction angles at the output and it works well in the negative refraction region given by the correctness condition. All the experiments and calculations were done for TE polarization and a similar approach can be used for TM polarization where we expect to find analogous results as it was shown in reference [12]. Comparison between experimental results and theory The C-M interface goes from the center of the Brillouin zone to the middle of an edge. The propagation results are completely different to the precedent case and cannot be described by a homogeneuos medium approximation. From this we can infer that the wave is able to see the periodicity of the medium even when the index contrast is low. noutput~ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b’ j j p 2 fan2 azfbn2 b z p 2 { b’ j j p 2 fa n2 a z fb n2 b {1 v u u u t , ð8Þ ð8Þ where fj represents the fraction volume of each layer (43% for a- layers and 57% for b-layers), and nj is the refractive index of each layer. The two angular prefactors multiplying each dielectric bound are necessary to account for the contribution of each component (normal-to-plane and on-plane). Then we can use Snell’s law and the effective refractive index as: cos(b)~noutput cos(b’): ð9Þ ð9Þ Combining equations (5), (7), (8) and (9) the corrected negative refraction angles at the output can be calculated as: Combining equations (5), (7), (8) and (9) the corrected negative refraction angles at the output can be calculated as: b~cos{1 noutput ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{ n2 0 sin2 a ð Þ C2 s 0 @ 1 A: ð10Þ ð10Þ Figure 4. Negative refraction theories comparison. Angle of refraction b9 vs. angle of incidence for the 1D PC proposed structure. The theory presented in Kavokin [11] is compared against group velocity theory. The light wavelength is 633 nm (TE polarization) and we used refractive index values and layer thickness described in the experimental section. doi:10.1371/journal.pone.0017188.g004 Figure 4. Negative refraction theories comparison. Angle of refraction b9 vs. angle of incidence for the 1D PC proposed structure. The theory presented in Kavokin [11] is compared against group velocity theory. The light wavelength is 633 nm (TE polarization) and we used refractive index values and layer thickness described in the experimental section. doi:10.1371/journal.pone.0017188.g004 April 2011 \| Volume 6 \| Issue 4 \| e17188 PLoS ONE \| www.plosone.org 5 Figure 5. Comparison between negative refraction theories and experiments. Negative refraction experimental values compared against uncorrected theoretical values (Eqs. (5) and (7)). doi:10.1371/journal.pone.0017188.g005 Negative Refraction in 1D PCs Negative Refraction in 1D PCs Figure 5. Comparison between negative refraction theories and experiments. Negative refraction experimental values compared against uncorrected theoretical values (Eqs. (5) and (7)). Comparison between experimental results and theory Now, by combining equations (7), (8), and (9) the corrected negative refraction angles at the output can be calculated as well as: b~cos{1 noutput ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ tan2 b’ ð Þz1 p ! , ð12Þ ð12Þ and the analytical expression for tan(b’) is given in the methods section. Clearly, Eq. (5) values fit well with experimental values up to 15 degrees (fig. 6-middle). The angular difference db’ between values predicted by eqs. (5) and (7) is the reason why the corrected refraction angles at the output, obtained by using eq. (10), differs from the experimental ones for angles of incidence larger than 15 degrees. This is understandable because as the incidence angle increases the effective mass approximation begins to fail. Indeed by increasing the incidence angle, the band edge is pushed towards small wavelengths making the separation l{l0 ð Þ increase. The results for the corrected negative refraction angles obtained with eq. (12) are shown in figure 6, (bottom). Notice that equation (12) predicts values that lie within the experimental accuracy obtained for all the angles of incidence. Notwithstanding, equation (10) is a good approximation to calculate corrected negative refraction angles at the output and it works well in the negative refraction region given by the correctness condition. All the experiments and calculations were done for TE polarization and a similar approach can be used for TM polarization where we expect to find analogous results as it was shown in reference [12]. and the analytical expression for tan(b’) is given in the methods section. Clearly, Eq. (5) values fit well with experimental values up to 15 degrees (fig. 6-middle). The angular difference db’ between values predicted by eqs. (5) and (7) is the reason why the corrected refraction angles at the output, obtained by using eq. (10), differs from the experimental ones for angles of incidence larger than 15 degrees. This is understandable because as the incidence angle increases the effective mass approximation begins to fail. Indeed by increasing the incidence angle, the band edge is pushed towards small wavelengths making the separation l{l0 ð Þ increase. The results for the corrected negative refraction angles obtained with eq. (12) are shown in figure 6, (bottom). Notice that equation (12) predicts values that lie within the experimental accuracy obtained for all the angles of incidence. PLoS ONE \| www.plosone.org Determination of the refracted angle b: step sequence (see Fig. 2) In conclusion, we have experimentally completed the angular characterization of negative refraction in a 1D PC structure, near the low frequency edge of the fourth photonic bandgap and compared it with current theory and theory based on group velocity developed here. We have validated the current negative refraction theory approach with our theory. We found good agreement between both theories with differences within 4 degrees in the explored incidence angle interval. In order to know the negative refraction zone, we have analytically derived the The first step consisted on choosing a convenient position of the light source on the sliding support. That was chosen in function of the free space needed for hand intervention. Once this position was determined, it was kept invariant along all the measurements. The light beam was kept as angular reference for zero degrees. So, through the turning movement v and the linear displacement x, the second step consisted in obtaining a regular tangent light beam PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e17188 6 Negative Refraction in 1D PCs Negative Refraction in 1D PCs Figure 6. 1D PC results at a working wavelength of 633 nm (TE polarization). (Top) finite element negative refraction simulation showing beam propagation inside the structure and input and output interfaces. The angle of incidence a is 15 degrees, angles b9 and b are 272 and 263 degrees respectively. (Middle) Comparison between negative refraction experimental values and corrected theoretical values (Eq. (10)). (Bottom) Comparison between negative refraction experimental values and corrected theoretical values (Eq. (12)). We used refractive index values and layer thickness described in the experimental section. Error bars represent systematic errors plus random errors (two standard deviations). doi:10.1371/journal.pone.0017188.g006 Figure 6. 1D PC results at a working wavelength of 633 nm (TE polarization). (Top) finite element negative refraction simulation showing beam propagation inside the structure and input and output interfaces. The angle of incidence a is 15 degrees, angles b9 and b are 272 and 263 degrees respectively. (Middle) Comparison between negative refraction experimental values and corrected theoretical values (Eq. (10)). (Bottom) Comparison between negative refraction experimental values and corrected theoretical values (Eq. (12)). We used refractive index values and layer thickness described in the experimental section. Error bars represent systematic errors plus random errors (two standard deviations). doi:10.1371/journal.pone.0017188.g006 done by means of the movement x (sixth step). PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e17188 Effective mass approximation expressions The effective mass expressions that only work close to the band- edge can be obtained from reference [11] and are given by: mTE per~mTM per ~ B azb ð Þ2c bnbzaC ð Þsin B0 ð Þcos A0 ð Þz anazbD ð Þsin A0 ð Þcos B0 ð Þ , The transference from the refracted light intensity (that we expect to follow a Gaussian-like distribution according to our simulations [12]) to the monitor emitted light (measured with the luminance meter), cannot be considered proportional because of the energy conversions involved (all with their own non linearities and convergence limits). The narrow intensity per unit of area distribution of the refracted light and the acceptance angle of the camera suggested that the most important systematic error was due to two factors. First, the angular determination error: angular measurement through mechanic goniometers could reach without problems +1 degree error; but the Lmax reading gave us a non discernible reading along 3 degrees around the Lmax b angle. This effect is known as spatial filtering. Second, we explored the negative refracted spot light along a circumference centered in the same spot as if it where the center for the w movement. Unfortunately the real center (for the turning platform) and the refracted spot was several microns away (at least the distance from the spot to the specimen edge). Therefore, a further correction due to the parallax and eccentricity compensation is needed to solve this problem. Once more, as the refracted beam presented a narrow distribution and the Lmax gave us a 3 degree error, this was covered largely other systematic errors involved. We used the same light source and polarizer reported in [12] and the negative refractive transmitted light was captured by a CCD camera (KP-D50, Hitachi)) coupled with a singlet lens (focal length of 8 mm, NT-45114, Edmund Optics) placed at 8 mm from the sample. The signal from the camera was sent to a color analogical monitor and a luminance- meter (CS-100, Minolta) was placed at 50 cm from the monitor. Acknowledgments where The authors thank Dr. J. Antonio del Rio, Dr. Julia Tagu¨en˜a-Martinez for providing porous silicon samples, helpful discussions and comments to this manuscript. Dr. Alejandro Reyes for constructive comments on the manuscript. Dr. Rocio Nava and Dr. Beatriz de la Mora for sample preparation and useful comments. Gildardo Casarrubias and Rene Guardian for the sputtering system and SEM pictures and Patrick Perron for technical assistance. A~ 2pa l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q , B~ 2pb l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q , C~ 1 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q z ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q 0 B @ 1 C A: A~ 2pa l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q , B~ 2pb l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q , C~ 1 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q z ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q 0 B @ 1 C A: The correctness condition can be expressed as The correctness condition can be expressed as Ca2 A z b2 B cos A ð Þsin B ð Þz a2 A z Cb2 B sin A ð Þcos B ð Þ{ 2 C2{1 ab AB sin A ð Þsin B ð Þ 2 666666664 3 777777775 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{ cos A ð Þcos B ð Þ{C sin A ð Þsin B ð Þ ð Þ2 q \| cos{1 cos A ð Þcos B ð Þ{C sin A ð Þsin B ð Þ ð Þ 2 4 3 5 v l2 2pn0 sin a ð Þ ð Þ2 2 C2{1 ab AB sin A ð Þsin B ð Þ 6664 7775 where A~ 2pa l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q , B~ 2pb l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q , C~ 1 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q z ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q 0 B @ 1 C A: Determination of the refracted angle b: step sequence (see Fig. 2) Then we explored the specimen side looking for a negative-refracted beam by means of the video camera, the turning platform and controlling the parallax error (by keeping the refracted light spot centered on the TV monitor and in focus). This seventh step involved the movements: w, p and f. After we found the light spot, we explored a narrow angle dw maintaining the light spot centered in the monitor, as we explained before. Using a luminance-meter (measure of the luminous intensity of light travelling in a given direction) we controlled the light level emitted by the monitor in the portion of the image containing the refracted light spot. With this procedure we found the angle b for observed all along the lateral face of the specimen (parallel to y direction). The third step was to assure that the specimen illuminated edge was placed just over the turning center of the apparatus. This task was performed by acting the movement y. The fourth step was moving the turning bar (7) around to place the 1D- PC in the desired incident angle a in reference to the light beam. To achieve this, we acted the movement v and verified the angular position on the goniometer (6) scale. At this point, it was necessary to place the 1D-PC specimen in a way that assured us that the incident beam was totally contained in the illuminated edge, and without reaching the specimen normal face (x direction). This was PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e17188 7 Negative Refraction in 1D PCs which the luminance-meter gave the highest reading (Lmax), and then we checked the refracted intensity for points five degrees away from this last one, verifying that their intensity was less than 50% of Lmax. This tedious procedure was repeated for incident angles ranging from 1.5 degrees up to 25 degrees. Each angle was measured four times, but we reported the average value as the negative refraction value and two standard deviations errors as random errors. Effective mass approximation expressions Effective mass approximation expressions mTE=TM par ~ azb ð Þ22pmTE=TM per l0 b nb z aC n2 a sin B0 ð Þcos A0 ð Þz a na z bD n2 b sin A0 ð Þcos B0 ð Þ+ n2 azn2 b na{nb ð Þ2l0 4pn3 an3 b sin A0 ð Þsin B0 ð Þ 2 666666664 3 777777775 , where A~ 2pa l0 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q , B~ 2pb l0 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q , C~ n2 azn2 b 2nb , D~ n2 azn2 b 2na : a The signs ‘‘2’’ and ‘‘+’’ in the expression for mTE=TM par correspond to TE and TM polarized light respectively. Equation (7) expression Equation (7) expression Equation (7) expression tan b’ ð Þ~{ azb ð Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{ cos A ð Þcos B ð Þ{C sin A ð Þsin B ð Þ ð Þ2 q 2pn0 sin a ð Þ l Ca2 A z b2 B cos A ð Þsin B ð Þz a2 A z Cb2 B sin A ð Þcos B ð Þ{ 2 C2{1 ab AB sin A ð Þsin B ð Þ 2 666666664 3 777777775 , where A~ 2pa l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q , B~ 2pb l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q , C~ 1 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q z ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 b{n2 0 sin2(a) q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n2 a{n2 0 sin2(a) q 0 B @ 1 C A: tan b’ ð Þ~{ azb ð Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{ cos A ð Þcos B ð Þ{C sin A ð Þsin B ð Þ ð Þ2 q 2pn0 sin a ð Þ l Ca2 A z b2 B cos A ð Þsin B ð Þz a2 A z Cb2 B sin A ð Þcos B ð Þ{ 2 C2{1 ab AB sin A ð Þsin B ð Þ 2 666666664 3 777777775 , References 11. Kavokin AV, Malpuech G, Shelykh I (2005) Negative refraction of light in Bragg mirrors made of porous silicon. Physics Letters A 339: 387–392. 1. Baba T, Asatsuma T, Matsumoto T (2008) Negative refraction in photonic crystals. MRS Bulletin 33: 927–930. y 2. Russell PStJ (1986) Optics of Floquet-Bloch Waves in Dielectric Gratings. Appl 2. Russell PStJ (1986) Optics of Floquet-Bloch Waves in Dielectric Gratings. Appl Phys B: Photophys Laser Chem 39: 23. 12. Lugo JE, de la Mora B, Doti R, Nava R, Taguen˜a J, et al. (2009) Multiband negative refraction in one-dimensional photonic crystals. Optics Express 17: 3036–3041. Phys B: Photophys Laser Chem 39: 23. 3. Russell PStJ, Birks TA, Loyd-Lucas FD (1995) In: E Burstein, C Weisbuch, eds. Confined Electrons and Photons, New Physics and Applications, Vol. 340 of NATO Advanced Studies Institute, Series B: Physics, Plenum, New York. 585 p. 13. Yuan YC, Ming HZ, Long SJ, Fang LC, Wang Q (2007) Frequency bands of negative refraction in finite one-dimensional photonic crystals. Chin Phys 16: 173. 14. Srivastava R, Thapa BK, Pati S, Ojha SP (2008) Negative refraction in 1D photonic crystals. Solid State Communications 147: 157–160. 14. Srivastava R, Thapa BK, Pati S, Ojha SP (2008) Negative ref 4. Russell PStJ, Birks TA (1996) In: Soukoulis M, ed. Photonic Band Gap Materials, Vol. 315 of NATO Advanced Studies Institute, Series E: Applied Sciences, Kluwer, Dordrecht. 71 p. p y 15. Boedecker G, Henkel C (2003) All-frequency effective medium theory of a photonic crystal. Optics Express 11: 1590. 5. Russell PStJ (1986) Interference of integrated Floquet-Bloch waves. Phys Rev A 33: 3232. photonic crystal. Optics Express 11: 1590. 16. Foteinopoulou S, Soukoulis CM (2005) Electromagnetic wave propagation in two-dimensional photonic crystals: A study of anomalous refractive effects. Phys Rev B 72: 165112. 6. Zengerle R (1987) Light propagation in singly and doubly periodic planar wave- guides. J Mod Opt 34: 1589. 17. Joannopoulos JD, Meade RD, Winn JN (1995) Photonic Crystals Molding the Flow of Light. New Jersey: Princenton University Press. 7. Kosaka H, Kawashima T, Tomita A, Notomi M, Tamamura T, et al. (1998) Superprism phenomena in photonic crystals. Phys Rev B 58: R10096. p p p p y y . Kosaka H, Kawashima T, Tomita A, Notomi M, Tamura T, et al. g y y 18. Author Contributions Conceived and designed the experiments: JEL RD. Performed the experiments: JEL RD. Analyzed the data: JEL. Contributed reagents/ materials/analysis tools: JF. Wrote the paper: JEL RD JF. PLoS ONE \| www.plosone.org 8 April 2011 \| Volume 6 \| Issue 4 \| e17188 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e17188 8 Negative Refraction in 1D PCs Negative Refraction in 1D PCs Negative Refraction in 1D PCs PLoS ONE \| www.plosone.org References Nava R, de la Mora MB, Tagu¨en˜a-Martı´nez J, del Rı´o JA (2009) Refractive index contrast in porous silicon multilayers. Phys Status Solidi C 6: 1721. 8. Kosaka H, Kawashima T, Tomita A, Notomi M, Tamura T, et al. (1999) Self- collimating phenomensa in photonic crystal. Appl Phys Lett 74: 1212. index contrast in porous silicon multilayers. Phys Status Solidi C 6: 1 collimating phenomensa in photonic crystal. Appl Phys Lett 74: 12 19. Pavesi L (1997) Porous silicon dielectric multilayers and microcavities. La Rivista del Nuovo Cimento 20: 1. 9. Notomi M (2002) Negative refraction in photonic crystals. Opt Quantum Electron 34: 133. 10. Zeng Y, Fu Y, Chen X, Lu W (2006) Effective dielectric constant of two- dimensional photonic crystals in optical bands. Solid state communications 138: 205–210. 20. Gerken M, Miller DAB (2003) Wavelength demultiplexer using the spatial dispersion of multilayer thin-film structures. IEEE Photon Technol Lett 15: 1097–1099. PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e17188 9
https://openalex.org/W2131621637	https://uscholar.univie.ac.at/detail/o:448441.pdf	English	null	Malleable nature of mRNA-protein compositional complementarity and its functional significance	Nucleic acids research	2,015	cc-by	9,332	ABSTRACT ular biology that are still open. Recently, we have demon- strated a remarkable degree of proteome-wide composi- tional complementarity between mRNAs and their cognate proteins. This has provided not only an important clue as to the code’s origin, but also suggested that the relation- ship between the two biopolymers could extend beyond just unidirectional information transfer (1–3). More specifically, we have uncovered a strong correlation between the pyrim- idine content of individual mRNA coding sequences and the average propensity of their cognate protein sequences to interact with pyrimidine mimetics. The latter property of protein sequences was derived from the so-called polar re- quirement (PR) scale, which captures the propensity of in- dividual amino acids to interact with substituted pyridines, such as dimethylpyridine (4,5). Importantly, we have shown for a number of different proteomes that pyrimidine density profiles of mRNA coding sequences closely match the PR profiles of their cognate protein sequences (1). Briefly put, mRNA regions that are rich in pyrimidines code for protein regions that exhibit high propensity to interact with pyrim- idine mimetics, and vice versa. ular biology that are still open. Recently, we have demon- strated a remarkable degree of proteome-wide composi- tional complementarity between mRNAs and their cognate proteins. This has provided not only an important clue as to the code’s origin, but also suggested that the relation- ship between the two biopolymers could extend beyond just unidirectional information transfer (1–3). More specifically, we have uncovered a strong correlation between the pyrim- idine content of individual mRNA coding sequences and the average propensity of their cognate protein sequences to interact with pyrimidine mimetics. The latter property of protein sequences was derived from the so-called polar re- quirement (PR) scale, which captures the propensity of in- dividual amino acids to interact with substituted pyridines, such as dimethylpyridine (4,5). Importantly, we have shown for a number of different proteomes that pyrimidine density profiles of mRNA coding sequences closely match the PR profiles of their cognate protein sequences (1). Briefly put, mRNA regions that are rich in pyrimidines code for protein regions that exhibit high propensity to interact with pyrim- idine mimetics, and vice versa. It has recently been demonstrated that nucleobase- density proﬁles of typical mRNA coding se- quences exhibit a complementary relationship with nucleobase-interaction propensity proﬁles of their cognate protein sequences. C⃝The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT This ﬁnding supports the idea that the genetic code developed in response to direct binding interactions between amino acids and appropriate nucleobases, but also suggests that present-day mRNAs and their cognate proteins may be physicochemically complementary to each other and bind. Here, we computationally recode com- plete Methanocaldococcus jannaschii, Escherichia coli and Homo sapiens mRNA transcriptomes and analyze how much complementary matching of syn- onymous mRNAs can vary, while keeping protein se- quences ﬁxed. We show that for most proteins there exist cognate mRNAs that improve, but also signif- icantly worsen the level of native matching (e.g. by 1.8 viz. 7.6 standard deviations on average for H. sapi- ens, respectively), with the least malleable proteins in this sense being strongly enriched in nuclear lo- calization and DNA-binding functions. Even so, we show that the majority of recodings for most proteins result in pronounced complementarity. Our results suggest that the genetic code was designed for fa- vorable, yet tunable compositional complementarity between mRNAs and their cognate proteins, support- ing the hypothesis that the interactions between the two were an important deﬁning element behind the code’s origin. We have also derived knowledge-based interaction propensities between individual RNA nucleobases and amino-acid residues from contact statistics in a large set of high-resolution 3D structures of RNA-protein complexes (2,3). Moreover, we have used classical molecular dynam- ics simulations and free energy techniques to provide a de- tailed, physically realistic picture of nucleobase/amino-acid interactions at the atomistic level (6,7). This has allowed us to not only confirm the above findings using orthogonal ap- proaches, but also extend them to the case of purines and especially guanine. Adenine-rich mRNA stretches, interest- ingly, exhibit the opposite behavior in that they tend to code for protein stretches with an aversion for interacting with adenines. Taken together, the above observations provide strong support for the idea that the code originated as a con- sequence of direct binding preferences between amino acids and their cognate codons (4,8–9), especially those which are adenine-poor. Adenine-rich codons, in turn, may have en- tered the code in order to modulate and weaken this binding (2). Importantly, however, our results suggest that apprecia- ble signatures of binding can only be seen if one examines Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030 Vienna, Austria at Institute for Theoretical Chemistry and Str http://nar.oxfordjournals.org/ Downloaded from Received November 03, 2014; Revised January 18, 2015; Accepted February 22, 2015 at Institute for Theoretical Chemistry and Structural Biology on August 9, 2016 http://nar.oxfordjournals.org/ Downloaded from *To whom correspondence should be addressed. Tel: +43 1 4277 9522; Fax: +43 1 4277 9522; Email: bojan.zagrovic@univie.ac.at Published online 08 March 2015 Published online 08 March 2015 3012–3021 Nucleic Acids Research, 2015, Vol. 43, No. 6 doi: 10.1093/nar/gkv166 Malleable nature of mRNA-protein compositional complementarity and its functional signiﬁcance Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030 Vienna, Austria Profile-matching calculations For sequence-profile correlations, a sliding window- averaging procedure was used (window of 21 for proteins and 63 for mRNAs), with all protein sequences shorter than or equal to twice the window size (42 residues) excluded. We have already shown that correlations exhibit only a weak dependence on the size of the averaging window––here, we have chosen the window size that was used in previous studies (1–3,6–7,10). Our final sets con- tained 1666, 4091 and 14413 protein-mRNA pairs for M. jannaschii, E. coli and H. sapiens, respectively. Protein PR profiles were calculated using the computationally derived PR scale (5), as previously described (1), while the level of complementarity between window-averaged mRNA pyrimidine content profiles and protein PR profiles was quantified using Pearson correlation coefficients R. p p g p q Here, we address these questions by focusing on the re- lationship between the pyrimidine content of mRNA se- quences and the PR of their cognate protein sequences. There are nine amino acids encoded by codons with vary- ing pyrimidine content (Leu, Ile, Val, Pro, Thr, Ala, Gly, Ser and Arg), which together occupy a major fraction of the ge- netic code table (41 out of a total of 61 non-stop codons). In the present analysis, we recode mRNA sequences by vary- ing the pyrimidine content of the codons corresponding to these nine amino acids and evaluate the extent to which this modulates the observed correlations with protein PR, which in turn does not change. Effectively, for three differ- ent organisms representing each of the three domains of life (Methanocaldococcus jannaschii for Archea, Escherichia coli for Bacteria and Homo sapiens for Eukarya), we explore the influence of codon usage on the relationship between pyrimidine content of mRNAs and PR profiles of their cog- nate protein sequences. In this way, we probe the limits of mRNA-protein compositional complementarity levels as set by a combination of the genetic code and various codon- usage patterns in recoded mRNAs. Finally, we should em- phasize that the present study is primarily aimed at analyz- ing the properties of mRNA-protein compositional comple- mentarity and not finding evidence concerning the hypoth- esis that such complementarity reflects an intrinsic poten- tial of the two biopolymers to bind (1–3). Profile-matching calculations While our results, as discussed below, do provide support for this still largely untested proposal, our principal focus here is the mRNA- protein compositional complementarity, which in contrast is a robust, easily reproducible fact. INTRODUCTION The relationship between mRNAs and their cognate pro- teins, as defined by the universal genetic code, is a corner- stone of all known biological systems. The origin of the code, however, remains largely unexplained, representing one of the most important foundational problems in molec- Nucleic Acids Research, 2015, Vol. 43, No. 6 3013 Data sets Complete proteomes of M. jannaschii, E. coli and H. sapi- ens were extracted from the UniProtKB database (13) (June 2013 release), excluding proteins designated as ‘uncertain’ and retaining only the reviewed Swiss-Prot entries for down- stream analysis. The starting sets contained 1782, 4157 and 19618 proteins for M. jannaschii, E. coli and H. sapiens, re- spectively. Where available, the coding sequences (CDS) of their corresponding mRNAs were downloaded from The European Nucleotide Archive (ENA) (14) using the ‘Cross- references’ section of each UniProtKB entry, while ensur- ing that ENA-translated mRNAs match the corresponding UniProtKB canonical protein sequences perfectly. Protein and mRNA sequences with non-canonical amino acids or nucleotides were excluded from the analysis. This resulted in 1667, 4149 and 14419 protein-mRNA pairs for M. jan- naschii, E. coli and H. sapiens proteomes, respectively. p y p g ( ) An important question to be addressed in this context concerns the extent to which the observed compositional complementarity can be modulated (i.e. its malleability), while still observing the prescriptions of the universal ge- netic code. In other words, does the code allow for markedly different levels of complementarity between a given pro- tein sequence and various synonymous mRNA sequences that could potentially code for it? Moreover, how optimal is the level of complementarity exhibited by native mRNA sequences? The fact that an amino acid can be encoded by more than one codon essentially allows one to rewrite the mRNA coding sequence, corresponding to one and the same protein sequence, in many different ways. Preferential use of particular codons as opposed to their expected occur- rences based on the universal genetic code, the codon-usage bias, generally differs between different organisms and dif- ferent genes (11,12). How does the fact that some amino acids are encoded by multiple codons affect the relation- ship between mRNA nucleobase content and nucleobase- interaction propensities of their cognate protein sequences? at Institute for Theoretical Chemistr http://nar.oxfordjournals.org/ Downloaded from MATERIALS AND METHODS relatively long, unstructured mRNA and protein stretches (1–3,6–7). Even more significantly, our results give rise to a novel, potentially far-reaching hypothesis that even present- day mRNAs and their cognate proteins might in general be physicochemically complementary to each other and bind, especially in adenine-poor mRNA regions (1–3,6–7,10). Codon-usage distances As a measure of the distance between codon-usage patterns in different contexts, we used root-mean-square deviation between codon occurrences defined as: RMSD = 20 i xi, j −xi,k 2 20 20 at Institute for Theoretical Chemistry and Str http://nar.oxfordjournals.org/ Downloaded from where j and k represent different contexts in which frac- tions of a given codon i are being compared. For example, these could include codon occurrences in the standard ge- netic code or in the native or the best-matched mRNA tran- scriptome for a given species. Here, codon occurrences in the standard genetic code (or genetic-code-based patterns of codon usage) refer to the expected occurrences of indi- vidual codons as dictated by the universal genetic code ta- ble. Note that in the reduced PUR/PYR alphabet the ge- netic code shrinks to only 20 codons when considering only those amino acids which have degenerate codons in terms of PYR content (Leu, Ile, Val, Thr, Ala, Pro, Gly, Ser or Arg). For example, instead of six codons for serine (AGU, AGC, UCA, UCG, UCU, UCC), the reduced representation re- sults in only three codons with either 1 (RRY), 2 (RYY) or 3 (YYY) pyrimidines, denoted here as Ser1, Ser2 and Ser3. Both randomization procedures were performed using a reduced alphabet (PUR or PYR) with codon selection pro- portional to the occurrence of a given codon in the stan- dard genetic code. Leucine, for example, is specified by six codons in the genetic code table with four of them having the pyrimidine content of 66.7% and two of them having the pyrimidine content of 100%. This means that the probabil- ity of choosing these two codon types during recoding was 4/6 = 66.7% in the first case or 2/6 = 33.3% in the second. In general, depending on the relative frequencies of differ- ent codon types for a given amino acid, the probabilities of occurrence of individual codon types varied. yp In the standard genetic code GUG, UUG and AUU en- code Val, Leu and Ile, respectively, which are all among amino acids targeted by our recoding approaches. On the other hand, many non-eukaryotes, including E. coli, are known to use GUG, UUG or AUU codons for initiator Met (15,16), which in principle could be a source of noise in our analysis. RESULTS The complete list of species-specific Gene Ontology (GO) annotation terms was obtained from the UniProt-GOA (17,18) and GO databases (19,20) (Dec 2013 release). For each organism, we first evaluated the distribution of differ- ences (R) between Pearson correlation coefficients corre- sponding to the worst- and the best-matched mRNAs of every protein in the proteome (i.e. its malleability as de- fined by R = Rworst – Rbest). Subsequently, we compared the top and the bottom 5% of such a distribution against the full background proteome of the same organism. Here, we used the EASE modification (21,22) where for each ob- tained protein count corresponding to a given GO term in the subset, we subtracted 1 in the case of enrichment or added 1 in the case of depletion. This procedure makes the downstream statistical analysis more stringent by penaliz- ing the significance of those GO terms with low protein counts (21). In this way, for each GO term present in a given organism, we obtained protein counts for the analyzed sub- set and the background proteome, allowing us to assess the significance of the observed enrichment or depletion of each GO term via Fisher’s exact test (22–24). Finally, we derived the false discovery rate (FDR)-corrected one-sided P-values To what extent can one alter the level of matching between native mRNA pyrimidine-content profiles and their cognate proteins’ PR profiles if mRNAs are recoded using synony- mous codons that vary in their pyrimidine content? In or- der to address this question, we have recoded each mRNA using a steered Monte Carlo procedure (see Methods for details) in which at each step we reassign a codon at a ran- domly chosen position to one of its synonymous codons at random, with the newly recoded mRNA serving as input for the next recoding step, as illustrated in Figure 1 (left). In one variant of this process, we only accept those reassign- ments for which the matching strictly improves, resulting ultimately in an optimally matched mRNA/protein pair. In another variant, we only accept those changes for which the matching strictly deteriorates, resulting ultimately in an op- timally anti-matched mRNA/protein pair. Importantly, in all of mRNA recoding attempts, protein sequences remain unchanged. Multidimensional scaling (MDS) MDS in 3D was performed using a built-in R (version 2.14.1) function cmdscale on a pairwise distance matrix (with RMSD as defined above) of a set of 10 different codon-usage patterns (standard genetic code, 3 native, 3 best- and 3 worst-matched mRNA transcriptomes corre- sponding to each of the analyzed organisms). Codon-usage distances In order to account for these alternative encodings systematically, all start codons were excluded from the re- coding procedure, and were simply kept fixed. mRNA recoding Two types of recoding procedures were used for sampling the mRNA sequence space: steered and non-steered. In steered recoding, each mRNA sequence went through 10 000 steps of a Monte Carlo-type procedure in which at each step a single, randomly chosen codon was reassigned to an- other synonymous codon. Reassignments were carried out only at those positions at which choosing a synonymous codon could lead to a change in pyrimidine content, and were attempted following the frequency of a given codon type in the standard genetic code. In this way, each step of the Monte Carlo procedure resulted in a newly recoded mRNA, which then served as the input for the next cycle of recoding. Since in steered recoding the aim was to ei- ther increase or decrease the level of matching between a protein’s PR profile and its cognate mRNA PYR profile as compared to the native mRNA, after each codon reassign- ment step the two profiles were compared by calculating the Pearson R between them. If the goal was to optimize matching and a given codon change resulted in improved matching, the change was accepted, and if not, the codon 3014 Nucleic Acids Research, 2015, Vol. 43, No. 6 (24) for the enrichment or depletion using a significance cut- off of 0.01. selection was repeated. Conversely, if the goal was to op- timize mismatching, changes which increased the level of mismatching were selected and others rejected. In this way, steered recoding progressively either increased or decreased the level of profile-matching between an mRNA and its cog- nate protein, and resulted in a single best- or worst-matched mRNA for each protein in a given proteome (Figure 1, left). In non-steered recoding, on the other hand, each mRNA was recoded independently 10 000 times by each time ran- domly changing all degenerate codons that could lead to a change in pyrimidine content. In this type of recoding, the same native mRNA served as input for a new recoding cy- cle, finally resulting in a set of 10 000 independent, recoded mRNA variants per each native mRNA in the transcrip- tome (Figure 1, right). RESULTS In Figure 2, we show the com- parison between the protein and its native mRNA as well as the same for the recoded best- and worst-matched syn- onymous mRNAs (Rbest = −0.97, top panel, and Rworst = 0.62, bottom panel, respectively). As can be seen, rewriting of the mRNA for this particular protein can result in an improvement in the level of matching of about 0.2 Pearson units (Figure 2, top) or its deterioration of well over 1 Pear- son unit, effectively changing the sign of the correlation in the direction of anti-matching (Figure 2, bottom). What do these changes look like at the level of com- plete transcriptomes? By collecting all of the best-matched and all of the worst-matched mRNAs obtained as a re- sult of steered recoding, we can recreate full mRNA tran- scriptomes and examine their profile-matching distribu- tions (Figure 3). Not unexpectedly, the distributions of Pearson Rs, capturing the degree of matching, shift left- ward for the best-matched recoded mRNAs and rightward for their worst-matched recoded counterparts. Importantly, however, the magnitude of this shift is substantial in both di- rections and for all organisms analyzed (Figure 3). For ex- ample, for the human proteome the median of the distribu- tion decreases from its native value of −0.75 by 0.22 Pear- son units in the case of the best-matching distribution or increases by a remarkable 1.03 Pearson units in the case of the worst-matching distribution, with similar changes seen for M. jannaschii and E. coli proteomes (Figure 3). In fact, the median of the E. coli worst-matching distribution shifts to 0.46, indicating not only a loss of matching, but actu- ally a high level of anti-matching even for an average pro- p g pp y g How malleable are different mRNAs when it comes to their potential to yield different levels of matching upon recoding? Depending on the composition of their cognate proteins, native mRNAs explore different ranges of pro- file matching with their cognate proteins when recoded. For each protein, we have analyzed the difference in Pear- son R between its worst-matched and its best-matched re- coded mRNA variant (R = Rworst – Rbest) as a quanti- tative measure of malleability in complementary matching. RESULTS In Figure 2, we show the results of this type of optimiza- tion for a representative human protein (HLA class I histo- compatibility antigen B-59 -chain), whose level of match- ing between its PR sequence profile and its native mRNA’s Nucleic Acids Research, 2015, Vol. 43, No. 6 3015 Figure 1. A schematic of the recoding procedure. We recode native mRNA sequences by varying the pyrimidine content of the codons corresponding to Leu, Ile, Val, Thr, Ala, Pro, Gly, Ser or Arg, while protein sequences do not change. Amino acids with codons whose pyrimidine content can vary are shown in blue, and their corresponding codons in green when native, or red when recoded. In steered recoding (left), we randomly change one codon in each out of 10 000 cycles, evaluate the complementarity by using the Pearson correlation coefficient (Rnew) and accept the change if it goes in the desired direction or reject it if not, finally optimizing mRNA-protein matching (Rbest) or mismatching (Rworst). In non-steered recoding (right), we recode native mRNAs independently 10 000 times without optimizing the native level of matching (Rnative), resulting in a range of Rs (R1, R2, ..., R10000) for each native mRNA. Figure 1. A schematic of the recoding procedure. We recode native mRNA sequences by varying the pyrimidine content of the codons corresponding to Leu, Ile, Val, Thr, Ala, Pro, Gly, Ser or Arg, while protein sequences do not change. Amino acids with codons whose pyrimidine content can vary are shown in blue, and their corresponding codons in green when native, or red when recoded. In steered recoding (left), we randomly change one codon in each out of 10 000 cycles, evaluate the complementarity by using the Pearson correlation coefficient (Rnew) and accept the change if it goes in the desired direction or reject it if not, finally optimizing mRNA-protein matching (Rbest) or mismatching (Rworst). In non-steered recoding (right), we recode native mRNAs independently 10 000 times without optimizing the native level of matching (Rnative), resulting in a range of Rs (R1, R2, ..., R10000) for each native mRNA. tein. In other words, regardless of the organism examined, it is possible to recode the set of native mRNAs to obtain a transcriptome that as a whole gives a significantly worse, but also significantly better level of profile matching as com- pared to the one observed for native mRNA sequences (Fig- ure 3). RESULTS In general, however, given the high level of matching with native sequences to begin with, the shifts in the direc- tion of worse matching are significantly greater than those in the direction of better matching (e.g. 7.6 viz. 1.8 standard deviations on average for H. sapiens, respectively). Here it should also be mentioned that the level of sampling used in steered recoding appears to be sufficient to capture the main features of the resulting profile-matching distributions and in particular those with worst-matched transcripts: in Supplementary Figure S1, we show the average Pearson R as a function of the number of recoding steps for the M. jannaschii transcriptome where the distribution mean shifts from 0.19 to 0.22 upon an increase in the extent of recoding from 104 to 105 steps i.e. the improvement upon a 10-fold increase in sampling appears to be only marginal. PYR profile corresponds to the median over the complete human proteome (Rnative = −0.75). Note that the PR scale is defined such that the lower the number, the higher the propensity to interact with pyrimidine mimetics. Therefore, negative values of the Pearson correlation coefficient corre- spond to direct matching between the mRNA PYR content and its cognate protein’s propensity to interact with pyrimi- dine mimetics and vice versa. In Figure 2, we show the com- parison between the protein and its native mRNA as well as the same for the recoded best- and worst-matched syn- onymous mRNAs (Rbest = −0.97, top panel, and Rworst = 0.62, bottom panel, respectively). As can be seen, rewriting of the mRNA for this particular protein can result in an improvement in the level of matching of about 0.2 Pearson units (Figure 2, top) or its deterioration of well over 1 Pear- son unit, effectively changing the sign of the correlation in the direction of anti-matching (Figure 2, bottom). PYR profile corresponds to the median over the complete human proteome (Rnative = −0.75). Note that the PR scale is defined such that the lower the number, the higher the propensity to interact with pyrimidine mimetics. Therefore, negative values of the Pearson correlation coefficient corre- spond to direct matching between the mRNA PYR content and its cognate protein’s propensity to interact with pyrimi- dine mimetics and vice versa. RESULTS Indeed, while the matching of a typical human protein with its cognate mRNAs covers a range of approximately 1.2 Pearson units, for some human proteins this is close to 0 (no malleability in matching) and for others close to 2 (full malleability, ranging from perfect matching to perfect anti- matching) as shown in Figure 4A. Could this potential to yield different levels of matching, or lack thereof, be bio- logically and functionally relevant? To address this ques- 3016 Nucleic Acids Research, 2015, Vol. 43, No. 6 Figure 2. mRNA pyrimidine content (%PYR) and protein polar require- ment (PR) profiles for human HLA class I histocompatibility antigen B-59 chain (UniProt AC: Q29940). Shown are profiles for the best (top), native (middle) and worst (bottom) mRNA in terms of matching with the protein PR, as obtained by steered recoding. ribosome-related functions are also strongly enriched in the group of highly malleable mRNAs in M. jannaschii and E. coli proteomes (Supplementary Tables S1 and S2). On the other hand, these two organisms exhibit no statistically sig- nificant enrichment when it comes to least malleable mR- NAs. We have also calculated the average R for each GO term in H. sapiens associated with 100 or more annotated pro- teins (Supplementary Table S3, left) and found that GO terms enriched among the bottom 5% (Figure 4A and C) exhibit systematically lower malleabilities (R < 1.2) than those enriched among the top 5% (R ∼1.3 or greater), even over all proteins. We next analyzed the distributions of Rs for two exemplary GO molecular functions: ‘chro- matin binding’ and ‘structural constituent of ribosome’, which are enriched in bottom and top 5%, respectively (Figure 4C), and showed they are significantly different (Wilcoxon rank-sum test, P < 2.2e-16, Supplementary Fig- ure S2). Taken together, we conclude that even individual GO terms (Supplementary Table S3) can be distinguished based on their average Rs, further reinforcing the obser- vation that malleability of complementary matching is as- sociated with biological function. Interestingly, there ap- pears to be a peculiar relationship between the malleability in matching of a given mRNA and its native level of match- ing (Figure 4B for H. sapiens and Supplementary Figure S3A and SB for M. jannaschii and E. coli transcriptomes, respectively). RESULTS Overall, the lower the malleability, the higher the native level of matching: in fact, all mRNAs with R < 0.5 have native values of R < −0.8. The converse, how- ever, is not true: mRNAs with high native matching cover the whole range of Rs. On the other hand, the higher the malleability, the wider the range of native levels of match- ing, while the lower the native matching, the higher the mal- leability (Figure 4B, Supplementary Figure S3A and SB). at Institute for Theoretical Chemistry and St http://nar.oxfordjournals.org/ Downloaded from y ( g pp y g ) We have next focused on matching-optimized transcrip- tomes obtained via steered recoding, but this time from the perspective of their codon-usage patterns. How different are codon-usage patterns of the best-matched and the worst- matched transcriptomes when compared to either the na- tive patterns or those based on the genetic code alone? With this in mind, we have calculated the RMSD between differ- ent codon-usage patterns involving the three organisms (na- tive, best-matched, worst-matched and genetic-code-based, see Methods for details) and obtained a pairwise distance matrix with 45 mutual distances in total (Figure 5A). For vi- sualization purposes, we have performed multidimensional scaling of these distances to 3D as illustrated in Figure 5B and C. Importantly, embedding in 3D results in a highly representative picture and captures over 90% of variance among all of the distances (Figure 5B). There are several im- portant observations one can glean from this analysis. First, all three native transcriptomes (Figure 5C, blue) are biased when compared to codon frequencies based on the genetic code alone (Figure 5C, orange), with the human transcrip- tome being least biased, at least when it comes to the 20 codons which were considered in this analysis (see Methods for details). Second, all recoded transcriptomes (Figure 5C, red and green) also show significant bias when compared to the genetic-code baseline. Third, recoded transcriptomes of all three organisms group together such that all the best- Figure 2. mRNA pyrimidine content (%PYR) and protein polar require- ment (PR) profiles for human HLA class I histocompatibility antigen B-59 chain (UniProt AC: Q29940). Shown are profiles for the best (top), native (middle) and worst (bottom) mRNA in terms of matching with the protein PR, as obtained by steered recoding. RESULTS tion, we have focused on the bottom and the top 5% of the distribution of R, that is, the extremes that exhibit the smallest and the largest changes in the level of matching upon recoding, respectively, and analyzed their GO finger- prints (Figure 4A and C). Among the least malleable hu- man mRNAs, we observe a significant enrichment of nu- clear proteins and functions related to DNA and chromatin binding, transcription and RNA splicing, while the most malleable mRNAs are completely distinct from this and encompass cytoskeletal, mitochondrial, extracellular and ribosome-related functions and processes. Interestingly, the Nucleic Acids Research, 2015, Vol. 43, No. 6 3017 Figure 3. Profile-matching distributions for native transcriptomes and transcriptomes constructed from the best and the worst mRNAs in terms of profile matching. Shown are distributions obtained via steered recoding for M. jannaschii, E. coli and H. sapiens. Inset: distribution parameters (mean, sigma and median). Figure 3. Profile-matching distributions for native transcriptomes and transcriptomes constructed from the best and the worst mRNAs in terms of profile matching. Shown are distributions obtained via steered recoding for M. jannaschii, E. coli and H. sapiens. Inset: distribution parameters (mean, sigma and median). Figure 4. Functional significance of mRNA recoding malleability. (A) Distribution of the difference in Pearson R between the worst and the best recodings of human mRNAs as obtained by steered recoding. Regions shaded in red correspond to the bottom and the top 5% of the distribution. (B) Relationship between malleability (R = Rworst – Rbest) and the native level of matching R for the human proteome quantified using Spearman correlation coefficient . (C) A heatmap showing functional enrichment and depletion for the subset of the least (bottom 5%, left column) and the most (top 5%, right column) malleable human mRNAs, as indicated by smaller or larger Rs, respectively. The heatmap is colored based on the significance of FDR-corrected P-values, with red denoting enrichment and blue depletion. Numbers in the parentheses denote the average R values per GO term over all proteins. Figure 4. Functional significance of mRNA recoding malleability. (A) Distribution of the difference in Pearson R between the worst and the best recodings of human mRNAs as obtained by steered recoding. Regions shaded in red correspond to the bottom and the top 5% of the distribution. RESULTS (B) Fraction of variance captured by multidimensional scaling as a function of the number of dimensions. (C) Multidimensional scaling of RMSD values between different codon-usage patterns. Figure 5. Codon-usage pattern for native and recoded human transcripts. (A) RMSD values between codon usage patterns corresponding to the universal genetic code (UGC) as well as native (subscript native), best-matched (subscript best) and worst-matched (subscript worst) M. jannaschii (M), E. coli (E) and H. sapiens (H) transcriptomes. (B) Fraction of variance captured by multidimensional scaling as a function of the number of dimensions. (C) Multidimensional scaling of RMSD values between different codon-usage patterns. Figure 6. Top 5 individual codons that exhibit the largest change in their frequencies upon worst-to-best transition together with the associated rel- ative change. The number next to the amino-acid abbreviation denotes the number of pyrimidines in the codon (e.g. Ile2 – codon for isoleucine with 2 pyrimidines etc.). amino-acid scale used for scoring protein sequences (e.g. the PR scale as used herein) also contribute significantly (1– 3,6–7,10). Steered recoding by design results in highly biased mRNA sequences for each given set of proteins and it is not a priori clear what part of the accessible mRNA se- quence space is covered by such extreme sequences. In fact, it is not even clear how large the fraction of the mRNA sequence space is, which yields native-like levels of match- ing. A highly pertinent question in this regard is what levels of cognate mRNA-protein matching would be obtained by unbiased recoding on average. In other words, what is the level of matching between a given protein and a typical ran- dom mRNA sequence belonging to it? We have performed transcriptome-wide non-steered recoding of mRNAs (see Methods for details), in which each cycle begins with the same native mRNA sequence, and finally results in 10 000 independently recoded mRNAs per native mRNA, each of them exploring the codon-usage landscape in a non-steered fashion (Figure 1, right). In this way, we have obtained an approximate, albeit undersampled picture of the com- plete mRNA sequence landscape, allowing us to study its global features. Our analysis reveals that mRNA sequences recoded in a non-steered fashion largely overlap with the native ones when it comes to their level of matching with cognate protein sequences (Figure 7A). RESULTS (B) Relationship between malleability (R = Rworst – Rbest) and the native level of matching R for the human proteome quantified using Spearman correlation coefficient . (C) A heatmap showing functional enrichment and depletion for the subset of the least (bottom 5%, left column) and the most (top 5%, right column) malleable human mRNAs, as indicated by smaller or larger Rs, respectively. The heatmap is colored based on the significance of FDR-corrected P-values, with red denoting enrichment and blue depletion. Numbers in the parentheses denote the average R values per GO term over all proteins. matched transcriptomes (Figure 5C, red) populate the same region of the codon-usage space, and the same is true of all the worst-matched transcriptomes (Figure 5C, green). The best-matched and the worst-matched groups are, how- ever, significantly distant from each other. Finally, codon- usage patterns do not appear to be related to the level of matching in native transcriptomes, as all three native tran- scriptomes exhibit similar levels of matching, but highly distinct codon-usage patterns. The best-matched transcrip- tomes, on the other hand, exhibit both similar levels of matching as well as codon-usage patterns, and the same holds for worst-matched transcriptomes. In other words, even though recoding is initiated from different starting po- sitions (i.e. different codon-usage patterns for the three na- tive transcriptomes), the resulting extremes all exhibit very similar codon-usage patterns, which differ between different types of extremes (i.e. best-matched versus worst-matched).i The finding that extreme transcriptomes have similar, yet highly distorted codon-usage patterns when compared to native transcriptomes, allows one to examine which partic- ular codons exhibit the biggest change in usage upon tran- sition from the worst- to the best-matched transcriptomes. Indeed, we observe a fairly consistent picture in all three organisms when it comes to the contribution of individ- ual codons/amino acids to this transition (Figure 6). The top five amino acids that most significantly contribute to the shift of the profile-matching distribution upon worst-to- best transition are, starting with the most important con- 3018 Nucleic Acids Research, 2015, Vol. 43, No. 6 Figure 5. Codon-usage pattern for native and recoded human transcripts. (A) RMSD values between codon usage patterns corresponding to the universal genetic code (UGC) as well as native (subscript native), best-matched (subscript best) and worst-matched (subscript worst) M. jannaschii (M), E. coli (E) and H. sapiens (H) transcriptomes. RESULTS Transcriptome-wide non-steered recoding of mRNAs using codon frequencies as defined by the genetic code table. (A) For each organism, the initial distribution (red) captures the level of profile-matching of native transcriptomes from which non-steered recording was initiated resulting ultimately in 10 000 recoded mRNAs for each native mRNA. A representative random subsample of 1000 transcriptomes for each organism is shown in the final distribution (blue). (B) Distributions of profile-matching for the native human transcriptome (black) and 10 000 recoded mRNA variants of human - synuclein (blue). Red arrow denotes the native R for human -synuclein. Inset: mRNA PYR and protein PR profiles for native human -synuclein. other biological purpose. Recently, Tartaglia and cowork- ers have used computational methods to predict that - synuclein may strongly interact with its cognate transcript, especially in the 5′ UTR (28). It will be exciting to explore a potential connection between these two not necessar- ily contradictory findings. The above possibilities notwith- standing, our preliminary GO analysis of mRNA/protein pairs with native sequences that appear particularly well- optimized for or against matching (outliers in the list given in Supplementary Table S4) did not reveal any statistically significant enrichment of particular functions or processes among these two groups (data not shown). genetic code alone, the same conclusion is reached: native levels of matching do not require exclusive optimization to be reached, but are shared by the large majority of different realizations of synonymous transcriptomes as obtained in a non-steered manner (Supplementary Figure S4). Non-steered recoding also allowed us to analyze how much native matching of a given mRNA/protein pair de- viates from the matching of typical recoded variants of the same mRNA. For some proteins, for example, the mRNAs recoded in the non-steered fashion are on average signifi- cantly better-matched and for some they are significantly worse-matched than the native mRNA (the full list is given in Supplementary Table S4). Such signals of potential evo- lutionary optimization may be particularly relevant in those cases where the native level of matching is in absolute terms a significant outlier from the behavior of average proteins in a given organism. Human -synuclein, a protein involved in the pathophysiology of Parkinson’s disease, is a prime ex- ample in this sense (Figure 7B). RESULTS For example, the average level of matching of native human sequences cor- responds to a Pearson R of −0.73, while the grand aver- age over 10 000 variants of the human transcriptome, re- coded in non-steered fashion as described above, is −0.71 (and a similar situation is seen for M. jannaschii and E. coli). What is more, if one performs non-steered recoding of the type described above, but this time uses organism- specific codon frequencies instead of those dictated by the Figure 6. Top 5 individual codons that exhibit the largest change in their frequencies upon worst-to-best transition together with the associated rel- ative change. The number next to the amino-acid abbreviation denotes the number of pyrimidines in the codon (e.g. Ile2 – codon for isoleucine with 2 pyrimidines etc.). tributor: Ile, Arg, Ser, Thr and Ala. This ranking is the same for all three organisms, with the exception of M. jan- naschii where Arg and Thr switch places. More specifically, our analysis reveals that in order to most drastically im- prove the level of matching, one should increase the us- age of pyrimidine-rich codons for isoleucine, threonine and alanine (Ile2, Thr2, Ala2––where the number indicates the number of pyrimidines in the codon) at the expense of their pyrimidine poor counterparts (Ile1, Thr1, Ala1). Similarly, usage of intermediate codons in terms of pyrimidine con- tent for Arg and Ser (Arg1 and Ser2, as opposed to Arg0 and Arg2 or Ser1 and Ser3) should also increase (Figure 6). Here, it should be emphasized that codon-usage bias is by no means the only factor influencing the extent of pro- file matching as the amino-acid abundance and the exact Nucleic Acids Research, 2015, Vol. 43, No. 6 3019 Figure 7. Transcriptome-wide non-steered recoding of mRNAs using codon frequencies as defined by the genetic code table. (A) For each organism, the initial distribution (red) captures the level of profile-matching of native transcriptomes from which non-steered recording was initiated resulting ultimately in 10 000 recoded mRNAs for each native mRNA. A representative random subsample of 1000 transcriptomes for each organism is shown in the final distribution (blue). (B) Distributions of profile-matching for the native human transcriptome (black) and 10 000 recoded mRNA variants of human - synuclein (blue). Red arrow denotes the native R for human -synuclein. Inset: mRNA PYR and protein PR profiles for native human -synuclein. Figure 7. RESULTS At the Pearson R of 0.37, the native -synuclein and its mRNA exhibit the second- worst level of matching among all human proteins (the only worse R belonging to the relatively short mitochondrial cy- tochrome C oxidase subunit 8, Supplementary Table S4). In addition, the native mRNA of -synuclein is by almost three standard deviations worse-matching than its average synonymous variant recoded in a non-steered fashion (Fig- ure 7B, Supplementary Table S4). In other words, the native mRNA of -synuclein appears to be significantly optimized to weaken compositional complementarity with its cognate protein, and one may speculate that the purpose of this may be to reduce the binding between the two, whatever its po- tential biological function may be. What makes this partic- ularly attractive is the fact that -synuclein exhibits a strong propensity to self-aggregate in the cell (25–27). It is possi- ble that complementary interactions with their cognate mR- NAs help solubilize proteins, a feature which may be re- duced in the case of -synuclein and its mRNA for some DISCUSSION One possibility, already discussed before (1–3,6–7,10), is that putative complemen- tary binding manifests itself only in those situations where both polymers are largely unstructured, such as, for exam- ple, during translation, but we do not exclude the possibility of such interactions also being relevant for folded biopoly- mers (10). at Institute for Theoretical Chemistry and St http://nar.oxfordjournals.org/ Downloaded from y The ability to recode entire mRNA transcriptomes to ex- hibit a much stronger or a much weaker level of matching when compared to native ones (Figure 3) comes at a price of distorting the original, organism-specific codon-usage bias (Figure 4C). This may provide a part of the explanation for why one does not necessarily observe the levels of match- ing that are as high or low as in the extreme transcriptomes obtained via steered recoding. Namely, organism-specific codon-usage bias represents an evolutionarily optimal solu- tion to a set of different requirements and constraints. For example, it is known that codon-usage bias in a given or- ganism mostly reflects mutational biases (i.e. biases in point mutations, or biases during base repair) or the adaptation to cellular tRNA abundances (i.e. translational optimiza- tion) to various extents (11–12,30–31). In addition, it may also directly influence mRNA secondary structure (32,33), or even the efficiency of transcription factor binding to cod- ing regions of DNA (34). The fact that different organisms exhibit distinct codon-usage signatures actually reflects the complexity of meeting various specific requirements when it comes to gene regulation and expression in an organism- specific manner. On the other hand, the level of mRNA- protein complementarity appears to be largely insensitive to details of organism-specific codon-usage bias; in fact, the large majority of different mRNA encodings for most pro- teins lead to significant levels of complementary matching (Figure 7A). We would like to interpret this as evidence that such complementarity is or was indeed functionally relevant to such a degree that it was actually embedded in the struc- ture of the genetic code. As discussed before (1–3,6–7,10), one such function in primordial systems could have been translation via direct templating of proteins from their cog- nate mRNAs, which as a corollary could have led to comple- mentarity between the two polymers. DISCUSSION Although both our conver- gence analysis (Supplementary Figure S1) as well as our analysis of codon-usage bias among the worst sequences (Figure 5) speak against this possibility, one cannot fully discount it. However, we should again emphasize that the primary goal of the present study was to test if significant shifts of matching distributions are at all possible, and not to determine them exactly. variants for a given protein exhibit similar binding poten- tial, the genetic code provides enough flexibility to steer it in any direction needed. in any direction needed. There are several caveats concerning our analysis that should be discussed. First, we have used the standard genetic code throughout our randomization experiments, which may have biased the results in the case of eukaryotic proteins encoded by the mitochondrial genome as these are known to be translated via a modified genetic code. How- ever, given that there are only 13 such proteins in the hu- man mitochondrial genome (29), this has not affected our main conclusions to any appreciable extent. Second, there is no guarantee that the PR scale (5), which was in an in- variant form used in all of the above analysis, optimally re- flects the physical interaction propensity of different amino acids for real codons. This scale, based on pyridine inter- action propensity of individual amino acids (1,4–5), is only a proxy for their interaction propensity with uracils and cy- tosines. It is possible that the malleability in terms of codon- usage patterns as discussed above is biased by the properties of the PR scale used. On the other hand, the general con- sistency between the PR scale and knowledge-based poten- tials capturing the affinity of amino-acid residues for bio- logically relevant pyrimidines (3) suggests that the bulk of the effect is still captured by the PR scale. In any case, it will be important to perform similar analyses using other nucleobase/amino-acid interaction propensity scales (3,6– 7). Our expectation, however, is that one would see general behavior similar to that of the PR scale. Finally, all of our analysis was carried out at the primary sequence level and it is not a priori clear how this relates to the case of folded 3D structures of mRNAs and proteins. DISCUSSION The level of compositional sequence complementarity be- tween a given mRNA and its cognate protein depends, in principle, on three key factors: (1) the structure of the ge- netic code, (2) the specific composition of the mRNA and (3) the specific composition of the protein. Changes in any of these three factors could affect the level of complemen- tary mRNA/protein matching. Here, we have asked how synonymous mRNA recoding affects the level of matching with its cognate protein, provided that the universal genetic code is used for encoding and that protein sequence remains fixed. Remarkably, we have shown that it is indeed possible to recode the mRNA pools of different organisms such that the level of matching with their cognate proteins is not only nullified, but actually moved in the direction of significant anti-matching on average (Figures 2 and 3). Importantly, however, our results show that the great majority of possible mRNA encodings still retain the native-like level of match- ing, with only a small subset of mRNA sequences having the potential to either strongly improve or worsen the na- tive matching (Figure 7A). In light of the hypothesis that compositional complementarity between mRNAs and their cognate proteins is indicative of their binding potential (1– 3,6–7,10), our results suggest that although most mRNA 3020 Nucleic Acids Research, 2015, Vol. 43, No. 6 sons. First, these differences reflect the features of pro- teome composition and codon usage bias, which differ be- tween different organisms. For example, a lower abundance of residues whose codon pyrimidine content can at all be changed means less dramatic shifts no matter how good the sampling. In fact, the extent of the shifts in the worst di- rection upon recoding (i.e. Rworst −Rnative which equals 0.9, 0.99 and 1.06 for M. jannaschii, H. sapiens and E. coli, re- spectively, Figure 3) follows the same order as the fraction of residues in those proteomes whose codon PYR content can at all be changed (i.e. fractions of Ile, Leu, Val, Thr, Ala, Ser, Pro, Arg, Gly combined, which are 54.3%, 59.3% and 61.9% for M. jannaschii, H. sapiens and E. coli, respec- tively). Second, the above difference may to some extent be a consequence of incomplete sampling: it is possible that the worst-matching distributions are not fully converged, unlike the best-matching distributions. REFERENCES 22. Huang,D.W., Sherman,B.T. and Lempicki,R.A. (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc, 4, 44–57. 1. Hlevnjak,M., Polyansky,A.A. and Zagrovic,B. 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DISCUSSION However, from struc- tural stabilization of mRNAs to translational feedback con- trol to protein and mRNA chaperone activity (2), cognate mRNA/protein interactions could be functionally relevant ( ) Our analysis has shown that there is a strong statistical association between the malleability of mRNAs and cer- tain biological functions and processes, and it is tempting to speculate that this may indicate functional significance of profile matching for these groups of mRNAs and pro- teins. Indeed, low malleability is strongly associated with a high level of native matching (Figure 4B), and it is pos- sible that in those cases, direct binding between mRNAs and their cognate proteins may play an important func- tional role. mRNA sequences with low malleability are in- deed enriched with nuclear functions and processes, many of which involve direct interactions between nucleic acids and proteins, including cognate ones (Figure 4C). Alterna- tively, it is possible that the observed association between malleability and function simply reflects either composi- tional biases or insufficient sampling due to length differ- ences of these particular subsets of proteins/mRNAs. It is, however, generally hard to separate and evaluate these effects independently since they are simply inherent prop- erties of proteins/mRNAs. Larger differences between the worst-matching sets than between the native or the best- matching sets belonging to the three organisms studied herein (Figure 3) are there probably because of similar rea- Nucleic Acids Research, 2015, Vol. 43, No. 6 3021 even in present-day systems and even to a degree that is still not fully appreciated. Future work will shed light on this exciting possibility. 15. Sacerdot,C., Fayat,G., Dessen,P., Springer,M., Plumbridge,J.A., Grunberg-Manago,M. and Blanquet,S. 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https://openalex.org/W4385073993	https://www.nature.com/articles/s41598-023-38847-7.pdf	English	null	CCND1-associated ceRNA network reveal the critical pathway of TPRG1-AS1-hsa-miR-363-3p-MYO1B as a prognostic marker for head and neck squamous cell carcinoma	Scientific reports	2,023	cc-by	8,715	CCND1‑associated ceRNA network reveal the critical pathway of TPRG 1‑AS1‑hsa‑miR‑363‑3p‑MYO1B as a prognostic marker for head and neck squamous cell carcinoma OPEN Head and neck squamous cell carcinoma (HNSC) is one of the leading causes of cancer death globally, yet there are few useful biomarkers for early identification and prognostic prediction. Previous studies have confirmed that CCND1 amplification is closely associated with head and neck oncogenesis, and the present study explored the ceRNA network associated with CCND1. Gene expression profiling of the Head and Neck Squamous Cell Carcinoma (HNSC) project of The Cancer Genome Atlas (TCGA) program identified the TPRG1-AS1-hsa-miR-363-3P-MYO1B gene regulatory axis associated with CCND1. Further analysis of the database showed that MYOB was regulated by methylation in head and neck tumors, and functional enrichment analysis showed that MYO1B was involved in "actin filament organization" and "cadherin binding ". Immune infiltration analysis suggested that MYO1B may influence tumorigenesis and prognosis by regulating the immune microenvironment of HNSC. MYO1B enhanced tumor spread through the EMT approach, according to epithelial mesenchymal transition (EMT) characterisation. We analyzed both herbal and GSCALite databases and found that CCND1 and MYO1B have the potential as predictive biomarkers for the treatment of HNSC patients. RT-qPCR validated bioinformatic predictions of gene expression in vitro cell lines. In conclusion, we found a CCND1-related ceRNA network and identified the novel TPRG1-AS1-hsa-miR-363-3p-MYO1B pathway as a possible HNSC diagnostic biomarker and therapeutic target. Abbreviations HNSC Head and neck squamous cell carcinoma EMT Epithelial mesenchymal transition ceRNA Competitive endogenous RNA TCGA The cancer genome atlas DElncRNAs, DEmiRNAs, DEmRNAs Differentially expressed lncRNAs/miRNAs/mRNAs GO Gene ontology KEGG Kyoto encyclopedia of genes and genomes Every year, over 830,000 people worldwide are diagnosed with head and neck Squamous cell carcinoma, includ- ing over 43,000 die as a result of this disease1. The most commonly malignancies of the head and neck are head and neck squamous cell carcinomas (HNSC), which arise from the mucosal epithelium of the oral cavity, throat, and larynx. Squamous cell carcinomas of the head and neck are frequently linked to tobacco-derived carcinogen exposure and heavy alcohol usage2, and tumors of the oropharynx are linked to infection with oncogenic strains of human papillomavirus (HPV), primarily HPV-16, but also HPV-18 and other strains to a lesser extent3,4. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results i h The CCND1 expression was separated into group with poor expressiveness of CCND1 (n = 233) and group with high expressiveness of CCND1 (n = 233) according to the median of CCND1 expression, and the log2Fold change and FDR values of the distinct genes were measured using the R package "edgeR" to construct CCND1-related ceRNA network. By setting the lncRNA threshold\|log2FC\|> 0.3, miRNA threshold\|log2FC\|> 0.3 and mRNA threshold\|log2FC\|> 0.3, FDR values < 0.05 was declared statistically significant. A total of 902 lncRNAs, 116 miRNAs, and 3591 mRNAs were filtrated. Differential analysis Volcano plots were drawn using the R package "ggVolcano" (Fig. 3A–C). To determine the DERNAs’ regulatory interactions, the ceRNA network was established and displayed using Cytoscape in the light of miRcode, miRTarBase, TargetScan, and miRDB databases using 28 DElncRNAs, 7 DEmiRNAs, and 40 DEmRNAs (Fig. 3D). The R package "clusterProfiler" enrichment analysis reflected that the DEmRNAs were linked with "structural constituent of ribosome", "collagen-containing extracellular RNAs", and "collagen-containing extracellular RNAs". (Fig. 3E, Fig. S2A–C). setting the lncRNA threshold\|log2FC\|> 0.3, miRNA threshold\|log2FC\|> 0.3 and mRNA threshold\|log2FC\|> 0.3, FDR values < 0.05 was declared statistically significant. A total of 902 lncRNAs, 116 miRNAs, and 3591 mRNAs were filtrated. Differential analysis Volcano plots were drawn using the R package "ggVolcano" (Fig. 3A–C). To determine the DERNAs’ regulatory interactions, the ceRNA network was established and displayed using Cytoscape in the light of miRcode, miRTarBase, TargetScan, and miRDB databases using 28 DElncRNAs, 7 DEmiRNAs, and 40 DEmRNAs (Fig. 3D). The R package "clusterProfiler" enrichment analysis reflected that the DEmRNAs were linked with "structural constituent of ribosome", "collagen-containing extracellular RNAs", and "collagen-containing extracellular RNAs". (Fig. 3E, Fig. S2A–C). if To determine the DERNAs’ regulatory interactions, the ceRNA network was established and displayed using Cytoscape in the light of miRcode, miRTarBase, TargetScan, and miRDB databases using 28 DElncRNAs, 7 DEmiRNAs, and 40 DEmRNAs (Fig. 3D). The R package "clusterProfiler" enrichment analysis reflected that the DEmRNAs were linked with "structural constituent of ribosome", "collagen-containing extracellular RNAs", and "collagen-containing extracellular RNAs". (Fig. 3E, Fig. S2A–C). Validation analysis of CCND1 related ceRNAs. To construct CCND1 expression-related ceRNAs, we scrutinized DERNAs expression levels in CCND1 low expression group and CCND1 high expression group, and FDR < 0.05 was defined as statistical significance (Fig. 4A–C, Fig. S3A,B). DERNAs expression levels in tumor tissues and matched paracancerous tissues from HNSC patients were also evaluated (Fig. 4D–F, Fig. S3C,D). www.nature.com/scientificreports/ carcinoma to 25% in patients with hypopharyngeal squamous cell carcinoma5. Currently, the main treatments for HNSC are surgery, radiotherapy, chemotherapy, immunotherapy, and targeted therapy. The major treatment options for early restricted HNSC include surgical resection and radiation, and if cancer advances and metas- tasizes substantially to lymph nodes and other systemic organ tissues, chemotherapy and immunotherapy are effective systemic treatments for HNSC. Commonly used antitumor medications include cisplatin and docetaxel, along with PD-1 checkpoint inhibitors6,7. However, pharmacological therapy frequently fails due to the cancer cells’ drug resistance. The current clinical treatment of progressive head and neck Squamous cell carcinoma is not promising, therefore, the pathophysiology of HNSC must be understood in order to develop novel diagnostic, prognostic, and therapeutic resistance biomarkers for HNSC research. p g p Long non-coding RNAs (LncRNAs) safeguard chromosome integrity and are engaged in transcription, trans- lation, and epigenetic modification8–10, while the imbalance in LncRNAs have yet been credited to ailments which include the cancer11. MicroRNAs (MiRNAs) can target and regulate mRNAs to govern biological processes and modulate gene expression, and dysregulation of miRNA expression is strongly associated with cancer-related dysfunction12. In recent years, the network of competitive endogenous RNAs (ceRNAs) has revealed a fresh device for RNA interactivity13, and the ceRNAs mechanism has been demonstrated in a number of different types of malignancies, for instance gastric cancer, liver cancer, and lung cancer14–16. yp g g g Cancer cell proliferation is allied to an imbalance in cell cycle protein expression17, and cyclin D1 protein cell cycle protein D1 (CCND1) acts synergistically with CDK4 and CDK6 to play a key role in the transition from G1 to S phase of cells18. In head and neck Squamous cell carcinoma, CCND1 amplification is allied to the progression of heterogeneous proliferative lesions to carcinoma in situ and is associated with a unfavorable clini- cal outcome19. Meanwhile, abnormal expression of CCND1 is allied to the development and poor prognosis of hepatocellular carcinoma, breast cancer, lung cancer, and lymphoma20–23. www.nature.com/scientificreports/ It has been reported that CCND1 is involved in the mechanism associated with CDK4/6 cell cycle kinase inhibitor resistance in estrogen receptor- positive breast cancer24 while silencing CCND1 was found to increase the ovarian cancer cells’ susceptibility to olaparib medicines25.hi p Therefore in research work, gene expression profiles and clinical information from HNSC patients were gath- ered and thoroughly analyzed from the Cancer Genome Atlas database in developing a ceRNA network coupled with CCND1. The TPRG1-AS1-has-miR-363-3p-MYO1B regulatory network was distinguished by analysis for expression, survival, and correlation. Our finding adds to the understanding of the molecular mechanism of HNSC and comes up with new targets for the treating of HNSC. CCND1‑associated ceRNA network reveal the critical pathway of TPRG 1‑AS1‑hsa‑miR‑363‑3p‑MYO1B as a prognostic marker for head and neck squamous cell carcinoma OPEN Due to the lack of early HNSC screening indicators, two-thirds of patients are diagnosed with intermediate to advanced disease, and the survival rate of patients with locally advanced disease is significantly lower than that of patients with early disease, ranging from a 5-year survival rate of 49% in patients with laryngeal squamous cell 1Department of Pharyngolaryngology Head and Neck Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China. 2Department of Thyroid Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China. email: qxg0914@126.com \| https://doi.org/10.1038/s41598-023-38847-7 Scientific Reports \| (2023) 13:11831 www.nature.com/scientificreports/ Results i h High expression of CCND1 in HNSC and its relationship with patient prognosis. The general workflow of this study is displayed in Fig. 1. The HPA database confirmed that CCND1 was significantly upregu- lated in terms of protein level in HNSC (Fig. 2A). The GEPIA2 database analysis revealed that elevated CCND1 expression reduced OS in HNSC, leading to a worse prognosis (Fig. 2B). To further explore the theorem of CCND1 high expression in HNSC, we went into the genome and copy number of CCND1 at the cBioPortal web- site, and the OncoPrint plot showed the amplification of CCND1 in the HNSC dataset (Fig. 2C). As depicted in Fig. 2D, the mRNA expression of CCND1 amplified HNSC samples was significantly higher than that of HNSC samples showing diploid CCND1 and CCND1 deletion. In addition, the copy number of CCND1 was discov- ered to be positively associated to mRNA expression in HNSC (Fig. 2E). In combination, the data suggest that CCND1 expression is elevated in HNSC, also the increase in CCND1 copy number may be the main mechanism leading to the upregulation of CCND1 expression in HNSC patients. In addition, combined with the results of clinical data analysis, CCND1 expression was significantly higher in smokers than non-smokers (Fig. S1A), CCND1 expression was greatly higher in drinkers than non-drinkers (Fig. S1B), and high CCND1 expression correlated with lymph node metastasis staging of HNSC (Fig. S1C). In conclusion, evidence suggest that CCND1 expression is upregulated in HNSC and is associated with tumor development. Construction of DERNAs and ceRNA networks of CCND1‑related HNSC. The CCND1 expression was separated into group with poor expressiveness of CCND1 (n = 233) and group with high expressiveness of CCND1 (n = 233) according to the median of CCND1 expression, and the log2Fold change and FDR values of the distinct genes were measured using the R package "edgeR" to construct CCND1-related ceRNA network. By setting the lncRNA threshold\|log2FC\|> 0.3, miRNA threshold\|log2FC\|> 0.3 and mRNA threshold\|log2FC\|> 0.3, FDR values < 0.05 was declared statistically significant. A total of 902 lncRNAs, 116 miRNAs, and 3591 mRNAs were filtrated. Differential analysis Volcano plots were drawn using the R package "ggVolcano" (Fig. 3A–C). Construction of DERNAs and ceRNA networks of CCND1 related HNSC. Results i h And according to the one-way Kaplan–Meier regression analysis and LncRNA-miRNA-mRNA inter-matching, there were three DElncRNAs, one DEmiRNAs, and seven DEmRNAs to compound a ceRNA network closely connected to survival (Fig. 4H, Fig. S3E–G). After correlation analysis, the correlation between C2orf27A https://doi.org/10.1038/s41598-023-38847-7 Scientific Reports \| (2023) 13:11831 \| www.nature.com/scientificreports/ and hsa-miR-363-3P was not statistically significant (Fig. 5J), and the negative correlation between hsa-m Figure 1. The flowchart of data collection and analysis. Figure 1. The flowchart of data collection and analysis. and hsa-miR-363-3P was not statistically significant (Fig. 5J), and the negative correlation between hsa-miR- 363-3P and DLEU1 was statistically significant (Fig. 5I). The final correlation verified that hsa-miR-363-3p was inversely interacted with the expression of TPRG1-AS1, meanwhile TPRG1-AS1 was positively correlated with the mRNAs of MYO1B. Subcellular localization of DLEU1 and TPRG1-AS1 was analyzed at LncLocator online website, and the outcomes indicated that TPRG1-AS1 was localized in "Cytoplasm" (Fig. 4G) and DLEU1 was localized to "Cytosol" (Fig. S3H). The above results suggest that TPRG1-AS1 can increase the level of MYO1B through hsa-miR-363-3p sponge. Expression pairwise analysis of DERNAs and CCND1 showed that hsa-miR- 363-3p was negatively correlated with CCND1. In contrast, TPRG1-AS1, MYOB, and CCND1 were positively correlated (Fig. 5C–H). ceRNAs base pairing results are shown in (Fig. 5A,B). and hsa-miR-363-3P was not statistically significant (Fig. 5J), and the negative correlation between hsa-miR- 363-3P and DLEU1 was statistically significant (Fig. 5I). The final correlation verified that hsa-miR-363-3p was inversely interacted with the expression of TPRG1-AS1, meanwhile TPRG1-AS1 was positively correlated with the mRNAs of MYO1B. Subcellular localization of DLEU1 and TPRG1-AS1 was analyzed at LncLocator online website, and the outcomes indicated that TPRG1-AS1 was localized in "Cytoplasm" (Fig. 4G) and DLEU1 was localized to "Cytosol" (Fig. S3H). The above results suggest that TPRG1-AS1 can increase the level of MYO1B through hsa-miR-363-3p sponge. Expression pairwise analysis of DERNAs and CCND1 showed that hsa-miR- 363-3p was negatively correlated with CCND1. In contrast, TPRG1-AS1, MYOB, and CCND1 were positively correlated (Fig. 5C–H). ceRNAs base pairing results are shown in (Fig. 5A,B). https://doi.org/10.1038/s41598-023-38847-7 Scientific Reports \| (2023) 13:11831 \| www.nature.com/scientificreports/ w.nature.com/scientificreports/ Figure 2. (A) CCND1 was expressed at higher levels in HNSC than in normal tissues by immunohistochemical analysis of the HPA database. (B) GEPIA2 database survival analysis data CCND1 overall survival rate. (C) The distribution of CCND1 genomic alterations in TCGA HNSC is shown on a cBioPortal OncoPrint plot. Results i h (D,E) The association between CCND1 copy number and mRNA expression are shown in the dot plot (D) and correlation plot (E) by cBioPortal. Figure 2. (A) CCND1 was expressed at higher levels in HNSC than in normal tissues by immunohistochemical analysis of the HPA database. (B) GEPIA2 database survival analysis data CCND1 overall survival rate. (C) The distribution of CCND1 genomic alterations in TCGA HNSC is shown on a cBioPortal OncoPrint plot. (D,E) The association between CCND1 copy number and mRNA expression are shown in the dot plot (D) and correlation plot (E) by cBioPortal. Expression of ceRNAs in pan‑cancerous tissues. To comprehend how prognostic models are expressed in pan-cancer, expression analysis of DERNAs was performed on TCGA and GTEx databases using the online database Xena Shiny (https://shiny.hiplot.com.cn/ucsc-xena-shiny/). As shown in Fig. S4, TPRG1- AS1 was upregulated in more than 1/3 of cancers, MYO1B in more than 2/3 of cancers, and hsa-miR-338-3p was downregulated in more than 1/3 of tumors. Methylation and protein expression levels of MYO1B. Genetic and environmental influences on human gene methylation levels26. To investigate the mode of MYO1B gene aberrant upregulation in HNSC, the UALCAN database was employed to detect the methylation levels. The outcomes demonstrated that the methylation level of MYO1B in HNSC was considerably lower than that in normal cells (Fig. S5A). DNA methylation is an influential DNA modification that governs gene silence27. Methylation sites (cg18731811, Scientific Reports \| (2023) 13:11831 \| https://doi.org/10.1038/s41598-023-38847-7 www.nature.com/scientificreports/ Figure 3. (A) The volcano map of 1438 upregulated and 2153 downregulated mRNAs. (B) The volcano map of 207 upregulated and 695 downregulated mRNAs. (C) The volcano map of 69 upregulated and 47 downregulated mRNAs. (D) A ceRNA network includes of 28 DElncRNAs, 7 DEmiRNAs and 40 DEmRNAs. (E) GO enrichment analysis of DEmRNAs. Figure 3. (A) The volcano map of 1438 upregulated and 2153 downregulated mRNAs. (B) The volcano map of 207 upregulated and 695 downregulated mRNAs. (C) The volcano map of 69 upregulated and 47 downregulated mRNAs. (D) A ceRNA network includes of 28 DElncRNAs, 7 DEmiRNAs and 40 DEmRNAs. (E) GO i h t l i f DE RNA Figure 3. (A) The volcano map of 1438 upregulated and 2153 downregulated mRNAs. (B) The volcano map of 207 upregulated and 695 downregulated mRNAs. (C) The volcano map of 69 upregulated and 47 downregulated mRNAs. (D) A ceRNA network includes of 28 DElncRNAs, 7 DEmiRNAs and 40 DEmRNAs. Results i h (E) GO enrichment analysis of DEmRNAs. cg20700762, cg09973663, cg22768676, cg24376339, cg02119693, cg03134230, cg22885000, cg09760210) were positively correlated with MYO1B expression, and methylation sites (cg03796659, cg15096140, cg25475516, cg17167076, cg27659841, cg20391764, cg12404831, cg12738764, cg06844526) were negatively correlated with MYO1B expression. It was also found that most of the methylation sites negatively associated with MYO1B expression were located in the promoter region, and those positively associated with MYO1B expression were located within the gene body (e.g., Fig. S5C,D). The above results deduce that methylation may actively engage in the regulation of MYO1B expression, which then affects the development of HNSC. g p ,f p Based on the UALCAN database, we found that MYO1B protein levels were upregulated in HNSC (Fig. S5B). g pf p Based on the UALCAN database, we found that MYO1B protein levels were upregulated in HNSC (Fig. S5B). Immune infiltration analysis. In order to investigate the immune infiltration level of MYO1B in tumors, the association with both gene copy number and immune cell infiltration level was first evaluated using the "SCNA" module (see Fig. 6A). The "Gene" module showed that MYO1B expression was observed to be nega- tively connected with B cells and CD8 + T cells, but favorably correlated with CD4 + T cell and neutrophil infil- tration (Fig. 6B). The "Survival" module was combined to detect the prognostic correlation between the level of MYO1B gene-related B-cell and CD8 + T-cell infiltration in HNSC patients (Fig. 6C). Finally, the "Correlation" module was employed to investigate the link between MYO1B and 16 immune cell markers (Table S1), and the consequences inferred that MYO1B expression was adversely affected to some markers of CD8 + T cells, T cells, B cells, NK cells, and also with M2 Macrophage, Treg cell partial markers were positively correlated. https://doi.org/10.1038/s41598-023-38847-7 Scientific Reports \| (2023) 13:11831 \| w.nature.com/scientificreports/ Epithelial mesenchymal transition (EMT) analysis. EMT is a key procedure in tumor advance, tis- sue repair and gains an aggressive phenotype and has a significant impact on tumor invasion migration and Figure 4. Expression level of DERNAs in CCND1low expression group and CCND1high expression group. (A) DElncRNA: TPRG1-AS1. (B) DEmiRNA: miR-363-3p (C) DEmRNA: MYO1B. Pairing of gene expression patterns between tumor tissue and normal tissue in patients with HNSC. (D) DElncRNA: TPRG1-AS1 (E) DEmiRNA: miR-363-3p (F) DEmRNA: MYO1B. (G) Subcellular localization of TPRG1-AS1. Results i h (H) The high- and low-expression values of DERNAs were compared by a Kaplan–Meier survival curve for TCGA HNSC patient cohort. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. www.nature.com/scientificreports/ Figure 4. Expression level of DERNAs in CCND1low expression group and CCND1high expression group. (A) DElncRNA: TPRG1-AS1. (B) DEmiRNA: miR-363-3p (C) DEmRNA: MYO1B. Pairing of gene expression patterns between tumor tissue and normal tissue in patients with HNSC. (D) DElncRNA: TPRG1-AS1 (E) DEmiRNA: miR-363-3p (F) DEmRNA: MYO1B. (G) Subcellular localization of TPRG1-AS1. (H) The high- and low-expression values of DERNAs were compared by a Kaplan–Meier survival curve for TCGA HNSC patient cohort. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Epithelial mesenchymal transition (EMT) analysis. EMT is a key procedure in tumor advance, tis- sue repair, and gains an aggressive phenotype and has a significant impact on tumor invasion, migration, and metastasis28. The loss of epithelial markers and the development of mesenchymal markers are considered signs that cells undergo EMT. We checked MYO1B levels with EMT markers across this study (Fig. S6A–C). MYO1B was shown to be inversely associated to the epithelial marker mucin-1 (MUC1) and positively tied to the mes- Epithelial mesenchymal transition (EMT) analysis. EMT is a key procedure in tumor advance, tis- sue repair, and gains an aggressive phenotype and has a significant impact on tumor invasion, migration, and metastasis28. The loss of epithelial markers and the development of mesenchymal markers are considered signs that cells undergo EMT. We checked MYO1B levels with EMT markers across this study (Fig. S6A–C). MYO1B was shown to be inversely associated to the epithelial marker mucin-1 (MUC1) and positively tied to the mes- https://doi.org/10.1038/s41598-023-38847-7 Scientific Reports \| (2023) 13:11831 \| www.nature.com/scientificreports/ Figure 5. (A) Schematic model of ceRNA. Blue indicates downregulated; red indicates upregulated. (B) Base pairing between miR-363-3p and the target site in the TPRG1-AS1 and MYO1B predicted by miRanda and TargetScan, respectively. (C–H) Correlation analysis between these three predictive RNAs and CCND1 in HNSC, log2(RPM + 0.01) was used for miR-363-3p expression and log2(TPM + 0.01) was used for TPRG1-AS1, MYO1B, CCND1 expression. Figure 5. (A) Schematic model of ceRNA. Blue indicates downregulated; red indicates upregulated. (B) Base pairing between miR-363-3p and the target site in the TPRG1-AS1 and MYO1B predicted by miRanda and TargetScan, respectively. Results i h (C–H) Correlation analysis between these three predictive RNAs and CCND1 in HNSC, log2(RPM + 0.01) was used for miR-363-3p expression and log2(TPM + 0.01) was used for TPRG1-AS1, MYO1B, CCND1 expression. enchymal indicators N-calcineurin (CDH2) and fibronectin (FN1). It is suggested that MYO1B may accele tumor growth by triggering EMT. MYO1B‑associated gene enrichment analysis and interaction network. To further investigate the possible functions of MYO1B in HNSC, we analyzed the top 200 MYO1B-related genes in HNSC using GO and KEGG pathways (Fig. S6D–G). The enrichment associated with MYO1B was "regulation of the actin cytoskeleton". In addition, GO enrichment analysis related to MYO1B, including biological process (BP), cel- lular component (CC), and molecular function (MF), focused on "actin filament organization", "cadherin bind- ing", "actin binding" and "actin filament binding". g , gi g The functional relationship network of CCND1 and MYO1B was estimated by GeneMINIA, which observed 20 anticipate target genes (Fig. 7A). Meanwhile 20 potential candidate proteins were also displayed in the pro- tein–protein interaction (PPI) network maps produced by STRING (Fig. 7B). Targeted herbal medicines and drug sensitivity analysis of CCND1 and MTO1B. Drug resist- ance remains a major limiting factor in the cure of cancer patients, and evidence suggests that despite tumors going into remission quickly, they develop drug resistance leading to disease recurrence29. HERB is a high- throughput database of experimental and reference guide herbal medicines for identifying 265 latent object herbs for CCND1, and 4 potential target herbs for MYO1B (Fig. 7C,D). In addition, the GSCALite database was explored based on the tumor treatment response portal (CTRP) and GDSC to assess the correlation of drug sen- Scientific Reports \| (2023) 13:11831 \| https://doi.org/10.1038/s41598-023-38847-7 www.nature.com/scientificreports/ ww.nature.com/scientificreports/ Figure 6. (A) Association between MYO1B gene copy number and immune cell infiltration levels in HNSC cohorts. (B) Correlation of CCND1 expression with immune infiltration level in HNSC. (C) Kaplan–Meier plots were used to analyze the immune infiltration and overall survival rate of HNSC. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Figure 6. (A) Association between MYO1B gene copy number and immune cell infiltration levels in HNSC cohorts. (B) Correlation of CCND1 expression with immune infiltration level in HNSC. (C) Kaplan–Meier plots were used to analyze the immune infiltration and overall survival rate of HNSC. p < 0.05, *p < 0.01, Figure 6. Results i h (A) Association between MYO1B gene copy number and immune cell infiltration levels in HNSC cohorts. (B) Correlation of CCND1 expression with immune infiltration level in HNSC. (C) Kaplan–Meier plots were used to analyze the immune infiltration and overall survival rate of HNSC. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. sitivity with CCND1 and MYO1B. As shown in Fig. 7E,F, both CCND1 and MYO1B were resistant to "5-Fluo- rouracil" and "paclitaxel". In addition, high levels of expression of CCND1 and MYOB genes were sensitive to 11 drugs. Among them, "Cetuximab" targeted drugs have played an important role in the treatment of HNSC30. In vitro validation of CCND1‑related regulatory genes. The bioinformatically predicted CCND1- related ceRNA regulatory genes were selected for RT-qPCR validation. The results revealed that the expression levels of lncRNA TPRG1-AS1 and mRNA MYO1B were significantly higher in HNSC cell lines than in HaCat cell (Fig. 8A–F), and the expression levels of miRNA miR-363-3p were significantly lower in HNSC cell lines than in HaCat cell (Fig. 8G–I). The consistent with the bioinformatic expression pattern, implying some reli- ability of the bioinformatic analysis results. Discussion Advances in medical technology have led to significant advances in diagnostic and therapeutic techniques for HNSC, nevertheless relevant researches have declared that the overall survival rate of HNSC has not released31. For HNSC monitoring and therapy, it is essential to explore the molecular mechanisms of development. CeRNAs have a contribution to the activation of gastric, hepatocellular, and lung cancers14–16. To identify novel tumor biosignatures expressed in HNSC, we established CCND1-related ceRNAs. cyclin D1 protein cell cycle protein D1 (CCND1) synergizes with CDK4 and CDK6 to play a crucial part in the transition of cells from the G1 to the S phase18. In head and neck cancer, amplification of CCND1 is linked to the appearance of heterogeneous proliferative lesions to cancer in situ, as well as a poor clinical outcome19.i p p In this study, the samples of screened HNSC patients were classified into two groups based on CCND1 levels: low and high in the TCGA database, and ceRNA networks associated to CCND1 were predicted using computation, screening, and inter-matching. DEmRNAs were enriched in "structural constituent of ribosome ", "extracellular matrix structural constituent" and "growth factor binding". Therefore, utilizing survival and Scientific Reports \| (2023) 13:11831 \| https://doi.org/10.1038/s41598-023-38847-7 www.nature.com/scientificreports/ Figure 7. (A) Functional association networks of CCND1 and MYO1B. (B) Top 20 protein interaction networks of CCND1 and MYO1B; line thickness indicates the strength of data support. (C) The five most relevant herbs targeting CCND1. (D) Herbs targeting MYO1B. (E,F) Analysis of the correlation between CCND1, MYO1B and drug sensitivity based on (E) CTRP database and (F) GDSC database. Positive correlation implies that high expression of a gene is resistant to the drug, whereas negative correlation indicates sensitivity to this drug. Figure 7. (A) Functional association networks of CCND1 and MYO1B. (B) Top 20 protein interaction networks of CCND1 and MYO1B; line thickness indicates the strength of data support. (C) The five most relevant herbs targeting CCND1. (D) Herbs targeting MYO1B. (E,F) Analysis of the correlation between CCND1, MYO1B and drug sensitivity based on (E) CTRP database and (F) GDSC database. Positive correlation implies that high expression of a gene is resistant to the drug, whereas negative correlation indicates sensitivity to this drug. Scientific Reports \| (2023) 13:11831 \| https://doi.org/10.1038/s41598-023-38847-7 om/scientificreports/ correlation analyses, we identified a prognostic pattern for HNSC, as in TPRG1-AS1 (up-regulated)- hsa-miR- 363 3p (down regulated) MYO1B (up regulated) Figure 8. www.nature.com/scientificreports/ surgical and histopathological techniques used to identify definitive surgical margins37. MYO1B has also been found to be strongly expressed in cervical cancer and to be tied with HPV infection, lymphatic metastasis, and case grading, while knockdown of MYO1B in vitro in tumor cells significantly reduced MMP1/MMP9 activity and substantially repressed the proliferation, migration and invasive ability of cervical carcinoma tumor cells38. y g y In our study, TPRG1-AS1 and MYO1B expression in HNSC tissues was substantially greater than in normal tissues, and survival analysis revealed that the up-regulation of TPRG1-AS1 and MYO1B was attributed to unfavourable prognosis. In contrast to TPRG1-AS1 and MYO1B, miR-363-3p was down-regulated in HNSC, and also indicated a worse prognosis. p g Hypermethylated standards of DNA in the promoter region are related to transcriptional repression and can inactivate tumor suppressor genes39, although many CpG islands (CGI) are located in gene promoters, they are also located within gene bodies40. The methylation of CPG in the gene body and gene expression are positively correlated, according to related investigates41, in contrast to the negative phase of methylation in the promoter that regulates gene expression42. One study found elevated methylation levels in the MYO1B gene body in colorectal cancer43. This is consistent with the finding that MYO1B up-regulation was inversely connected with methylation in the promoter region and positively linked with methylation in the majority of gene bodies in this study, suggesting that MYO1B may be regulated by methylation levels in HNSC. y gg g y g y y Comprehending the interaction between the tumor and host immune system is essential for discovering forecasting biomarkers, lowering medication resistance, and constructing novel therapeutic approaches44. The immune components of the HNSC microenvironment include tumor-infiltrating lymphocytes (TIL; including T cells, B cells, and natural killer (NK) cells) and myeloid cells (including macrophages, neutrophils, dendritic cells, and myeloid suppressor cells (MDSCs))19. Studies have shown that most HNSC microenvironments are highly immunosuppressive45,46 and that tumor immunity in the tumor microenvironment is mainly mediated by antitumor-active cells (effector T (Teff) cells) and NK cells, whereas immunosuppression and tumor cell growth is mediated by immunosuppressive-active cells (regulatory T (Treg) cells), the MDSCs and M2 macrophages mediated. Conversely, increased numbers of Treg cells, MDSCs, neutrophils, or M2 macrophages were linked to advanced HNSCC or a poor prognosis. In the tumor microenvironment, elevated levels of CD8 + T cells and NK cells were attributed to the greater survival47. www.nature.com/scientificreports/ The results of this study showed that MYO1B was negatively correlated with B-cell and CD8 + T-cell infiltration, and patients with high infiltration levels of B-cells and CD8 + T-cells had a better prognosis, (see Fig. 5B). Meanwhile, MYO1B was negatively correlated with NK cell marker levels and favorable connection with Treg cell and M2 macrophage marker levels (see Table S1). It is suggested that MYOB is associated with immune infiltration of HNSC. ggi Epithelial mesenchymal transition (EMT) plays a part in tumorigenesis and increases tumor metastatic properties through enhanced invasiveness48. To understand whether MYO1B is associated with the EMT pro- cess. We correlated gene expression with epithelial mesenchymal markers to explore the mechanisms associated with MYO1B in HNSC. MYO1B was shown to be inversely connected with epithelial markers and favorably correlated with mesenchymal markers in the study. It is proposed that MYO1B may facilitated the propagation and metastasis of HNSC through the EMT process. Meanwhile, it has been recently reported that silencing of MYO1B alleviated the EMT potential of HNSC49.li p Cetuximab combined with platinum-based fluorouracil as first-line chemotherapy for patients with recur- rent or metastatic squamous cell carcinoma of the head and neck improved overall patient survival compared to platinum plus fluorouracil chemotherapy alone50. This study suggested that up-regulation of CCND1 and MYO1B both showed resistance to "5-Fluorouracil" and "paclitaxel" and sensitivity to "Cetuximab" targeting drugs, indicating the potential of CCND1 and MYO1B as predictive biomarkers for HNSC patients treatment. (Fig. 7E,F). We also identified multiple target herbs associated with CCND1 and MYO1B, which need to be further investigated in the treatment of HNSC. g Although the ceRNA-based TPRG1-AS1-hsa-miR-363-3p-MYO1B axis seems to be a viable predictive bio- marker for medical applications, the study’s shortcomings must be acknowledged. Firstly, the interaction between ceRNA network needs to be further verified through experiments. Secondly, the role and mechanism of TPRG1- AS1-hsa-miR-363-3p-MYO1B axis in HNSC should be experimentally further investigated. p p y g In summary, We established a crucial TPRG1-AS1-hsa-miR-363-3p-MYO1B axis that is linked to the gen tion of HNSC and may provide novel targets for HNSC therapy. Discussion Verification of the three associated regulatory genes in HaCat cell and HNSC cells using quantitative real-time PCR, p < 0.05, p < 0.01, p < 0.001, **p < 0.0001, compared with the HaCat cell. (A–C), TPRG1-AS1. (D–F), hsa-miR-363-3p. (G–I), MYO1B. www.nature.com/scientificreports/ Figure 8. Verification of the three associated regulatory genes in HaCat cell and HNSC cells using quantitative l i PCR 0 05 0 01 * 0 001 **** 0 0001 d i h h H C ll (A C) Figure 8. Verification of the three associated regulatory genes in HaCat cell and HNSC cells using quantitative real-time PCR, p < 0.05, p < 0.01, p < 0.001, **p < 0.0001, compared with the HaCat cell. (A–C), TPRG1-AS1. (D–F), hsa-miR-363-3p. (G–I), MYO1B. orrelation analyses, we identified a prognostic pattern for HNSC, as in TPRG1-AS1 (up-regulated)- hsa-miR- 63-3p (down-regulated)- MYO1B (up-regulated). g g TPRG1-AS1 is upregulated in HNSC and promotes tumorigenesis by upregulating MYO1B through hsa- miR-363-3p sponging. It has been shown that TPRG1-AS1 serves as a potential driver of non-coding RNA to promote hepatocellular carcinoma progression, and its sponging of miR-4691-5p and miR-3659 as ceRNA leads to RBM24 expression, which inhibits hepatocellular carcinoma progression by activating the mechanism of tumor apoptosis32. According to the reports, MiR363 low expression is associated with proliferation and inva- sion of gastric cancer cells33 and miR-363-3p interacts with LncRNA OIP5-AS1 and promotes hepatocellular carcinoma progression by upregulating SOX434. Meanwhile, aberrant expression of MYO1B boosts HNSCC cell migration and cervical lymph node metastasis35. Myosin 1b is associated with early treatment failure in oral tongue squamous cell carcinoma, a subgroup of head and neck cancer36, and myosin 1b can be used as a bio- marker for oral tongue squamous cell carcinoma to help early detection of tumor cells complementing existing Scientific Reports \| (2023) 13:11831 \| https://doi.org/10.1038/s41598-023-38847-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ The z-values indicate the standard deviation of the median between HNSC samples, while the Log2 spectral count rate values for samples were normalized within and between samples.h p p p The BioPortal dataset (https://www.cbioportal.org/) covers a variety of data types, including somatic muta- tions, DNA methylation, and pathway enrichment, to assist in the analysis of multidimensional cancer gene datasets. Bioinformatic approach to observe MYO1B amplification and mutation in tumor tissues. Levels of immune infiltration. Timer (https://cistrome.shinyapps.io/timer/) is an website tool focused on the quantification of cellular immune infiltration. The "SCNA" module provides a comparative exploration of tumor infiltration levels between tumors with different somatic cell copy number variations of a specific gene. The "Survival" module shows the clinical significance of immune cells in tumors. In addition, the "Genes" mod- ule visualizes the relationship between genes of interest and immune penetration levels in various malignancies. Finally, the "Correlation" module predicts the association between the MYO1B and 16 immune cell markers. Functional enrichment analysis and interaction network. To illustrate the biological processes and pathways that may be implicated in the HNSC prognostic model, the top 200 genes connected to CCND1 and MYO1B were gathered from GEPIA2 (gepia2.cancer-pku.cn). We used the R package "ClusterProfiler" to enrich these genes for GO and KEGG pathways and chosen the five most significant pathways, which were visualized by the R package "ggplot2". GeneMINIA (http://genemania.org/) provides information on co-expression, genetic interplay, protein–pro- tein physical interactions, shared protein structural domains, co-localization, and pathways. A distinct scoring algorithm is utilized to forecast functional relationship networks of target genes. STRING (https://string-db.org/) is a database that maps interaction networks based on protein co-promotion activities. Targeted drug analysis. The Herbal Medicines Database (http://herb.ac.cn/) is a natural herbal medicine platform for querying possible target herbs. GSCAlite (http://bioinfo.life.hust.edu.cn/web/GSCALite/) is a gene set tumor analysis platform. The interconnection of 481 drug probes in the CTRB gene with genes was analyzed using Spearman. Furthermore, the same exploration was also carried out on genes and over 100 pharmaceuticals in the Genomics of Drug Sensitivity in Cancer (GDSC) database. In vitro cellular RNA extraction and validation of quantitative real‑time PCR. One human immortalized epidermal cell line (HaCat) and three HNSC cell lines (M4E, CAL27 and TU686) were identified by STR. www.nature.com/scientificreports/ ceRNA network. Based on CCND1 levels, HNSC tumor samples were separated into two groups: low expression (n = 233) and high expression (n = 233). The R package "edgeR" utilized to estimate the log2FC and FDR results for each gene. Thresholds for lncRNA (\|log2FC\|> 0.3), miRNA (\|log2FC\|> 0.3), and mRNA (\|log2FC\|> 0.3) were set, and the levels of these RNAs were judged statistically different when the FDR values were < 0.05. The R pack- age "ggplot2" performed volcano mapping of differentially expressed RNAs (DERNAs). g gg gf y Mircode (http://www.mircode.org/) was employed to forecast the interregulation between DElncRNAs and DEmiRNAs, at that time TargetScan (http://www.targetscan.org/), miRDB (http://www.mircode.org/) and miR- TarBase (https://mirtarbase.cuhk.edu.cn/) databases to anticipate DEmiRNAs target genes and detect intersection with DEmRNAs. Cytoscape version 3.8.0 shows the CCND1-related ceRNA network. Construction of ceRNA network. Patients’ clinical information was retrieved from the TCGA database. Then, the R package "survival" was utilized to commit a univariate Kaplan–Meier analysis of the data to assess the association in both genes and OS. Survival-associated DElncRNAs, DEmiRNAs, and DEmRNAs were uti- lized to produce the ceRNA network.h p The expression levels of DERNAs in paired HNSC patients and normal tissues along with in CCND1 low and CCND1 high expression groups were analyzed to screen the ceRNA network associated with CCND1. As well, pairwise analysis of lncRNA-miRNA, miRNA-mRNA, lncRNA-mRNA, DEmRNAs-CCND1, lncRNA-CCND1, and miRNA-CCND1 carried out to reveal the expression correlation between DERNAs-CCND1. The potential mechanism is proven by the LncRNAs’ cellular localization; LncLocator analyzed the subcellular localization of lncRNAs (www.csbio.sjtu.edu.cn/bioinf/lncLocator). Base pairing between TPRG1-AS1-hsa-miR-363-3p- MYO1B was predicted using miRanda and TargetScan. Methylation and protein expression and mutation analysis. One of the earliest modification pro- cesses found was DNA methylation. The methylation levels of target genes were examined using the UALCAN database (http://ualcan.path.uab.edu/) to elucidate the mechanism of aberrant overexpression of MYO1B in HNSC. Beta values indicated that DNA methylation levels were in the range of 0 (unmethylated) to 1 (fully methylated). Additionally, we explored for methylation sites and related differentially methylated areas using the MEXPRESS (http://mexpress.be) and METHURV (https://biit.cs.ut.ee/Methsurv/) databases. Dataset CPTAC from the UALCAN database (http://ualcan.path.uab.edu/) was used to analyze the target gene protein expres- sion levels. Materials and methods Expression of CCND1 in HNSC. The GEPIA2 database (http://gepia2.cancer-pku.cn/) "survival analysis" module was used for CCND1 gene survival analysis. Human Protein Atlas (HPA, http://www.proteinatlas.org/) was employed to analyze the protein expression level of CCND1 in head and neck cancer. The amplification sta- tus of CCND1 and the amplification and mutation status of MYO1B were obtained through the Cancer Genom- ics cBioPortal website (http: //www.cbioportal.org/). Data preparation for HNSC. RNAseq and miRNAseq data for HNSC were acquired from the TCGA database (https://portal.gdc.cancer.gov/). The criteria for exclusion of samples were ⑴ radiotherapy prior to histological diagnosis of HNSC, ⑵existence of tumor other than HNSC, ⑶absence of the any clinical data, and ⑷ unavailability of samples to match with RNA and miRNA samples in the database. In this investigation, 466 HNSC tissue samples were collected. Patients’ clinical data were also collected from the TCGA database, and no ethics committee permission was required. This work followed the TCGA publishing guidelines. Gene expression analysis. In light of the preceding description, the advancement of HNSC was associ- ated with enhanced CCND1 expression, as a possible predictive model for HNSC, we designed a CCND1-related Scientific Reports \| (2023) 13:11831 \| https://doi.org/10.1038/s41598-023-38847-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Total RNA Extraction Kit (Vazyme, Nanjing, Jiangsu, China). Reverse transcription was performed according to the instructions of HiScript® II Reverse Transcriptase Reagent (Vazyme, Nanjing, Jiangsu, China). Quantitative polymerase chain reaction (Q-PCR) was performed with ChamQ Universal SYBR qPCR Master Mix reagent (Vazyme, Nanjing, Jiangsu, China) according to the instructions. GAPDH and U6 were used as internal refer- ence genes. Relative expression was calculated using the ΔΔ-Ct method. The primer sequences are shown in Table S2. Total RNA Extraction Kit (Vazyme, Nanjing, Jiangsu, China). Reverse transcription was performed according to the instructions of HiScript® II Reverse Transcriptase Reagent (Vazyme, Nanjing, Jiangsu, China). Quantitative polymerase chain reaction (Q-PCR) was performed with ChamQ Universal SYBR qPCR Master Mix reagent (Vazyme, Nanjing, Jiangsu, China) according to the instructions. GAPDH and U6 were used as internal refer- ence genes. Relative expression was calculated using the ΔΔ-Ct method. The primer sequences are shown in Table S2. Statistical analysis. Statistical analyses were performed using R software (https://www.r-project.org/, ver- sion 4.2.0). Cytoscape version 3.8.0 visualized the data using univariate Kaplan–Meier analysis to reveal the con- nection between gene expression and patient OS. p < 0.05 was judged statistically significant. The edgeR package was applied for the variance analysis and FDR < 0.05 was declared statistically significant. Data availability All data generated or analyzed during this study are included in this article. I uploaded the dataset and related code at the following URL: https://www.jianguoyun.com/p/DUtHc6gQg7uLCxisiOUEIAA Xena Shiny (https:// shiny.hiplot.com.cn/ucsc-xena-shiny/), GEPIA2 (http://gepia2.cancer-pku.cn/), BioPortal (https://www.cbiop ortal.org/), TCGA (https://portal.gdc.cancer.gov/), Mircode (http://www.mircode.org/), TargetScan (http://www. targetscan.org/), miRDB (https://mirdb.org/), miRTarBase (https://mirtarbase.cuhk.edu.cn/), LncLocator (www. csbio.sjtu.edu.cn/bioinf/lncLocator), UALCAN (http://ualcan.path.uab.edu/), MEXPRESS (http://mexpress.be), Timer (https://cistrome.shinyapps.io/timer/), GeneMINIA (http://genemania.org/), STRING (https://string-db. org/), Herbal Medicines (http://herb.ac.cn/), GSCAlite (http://bioinfo.life.hust.edu.cn/web/GSCALite/). Received: 8 May 2023; Accepted: 16 July 2023 Received: 8 May 2023; Accepted: 16 July 2023 Received: 8 May 2023; Accepted: 16 July 2023 References A ceRNA hypothesis: The rosetta stone of h 4. Sun, Q. et al. LncRNA LOXL1-AS1 facilitates the tumorigenesis and stemness of gastric carcinoma via regulation of miR-708-5p/ USF1 pathway. Cell Prolif. 52(6), e12687 (2019). 5. Wang, H. et al. STAT3-mediated upregulation of lncRNA HOXD-AS1 as a ceRNA facilitates liver cancer metastasis by regulating SOX4. Mol. Cancer. 16(1), 136 (2017). 16. Hua, Q. et al. LINC01123, a c-Myc-activated long non-coding RNA, promotes proliferation and aerobic glycolysis of non-small cell lung cancer through miR-199a-5p/c-Myc axis. J. Hematol. Oncol. 12(1), 91 (2019).i 16. Hua, Q. et al. LINC01123, a c-Myc-activated long non-coding RNA, promotes proliferation and aerobic glycolysis of n cell lung cancer through miR 199a 5p/c Myc axis J Hematol Oncol 12(1) 91 (2019) 7. Chen, Y. et al. 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Fundingh g This work was supported by a grant from Thermal Ablation of Thyroid Nodules International Joint Laboratory [Henan Province; YUKEWAI (2016), number 11]. [Henan Province; YUKEWAI (2016), number 11]. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-38847-7. Correspondence and requests for materials should be addressed to X.Q. 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. Competing interests The authors declare no competing interests. Correspondence and requests for materials should be addressed to X.Q. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W2914026446	https://europepmc.org/articles/pmc6393361?pdf=render	English	null	Beyond Pathway Analysis: Identification of Active Subnetworks in Rett Syndrome	Frontiers in genetics	2,019	cc-by	7,480	Beyond Pathway Analysis: Identiﬁcation of Active Subnetworks in Rett Syndrome Ryan A. Miller 1†, Friederike Ehrhart 1,2†, Lars M. T. Eijssen 1,3, Denise N. Slenter 1, Leopold M. G. Curfs 2, Chris T. Evelo 1,2,4, Egon L. Willighagen 1 and Martina Kutmon 1,4* 1 Department of Bioinformatics - BiGCaT, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, Netherlands, 2 GKC-Rett Expertise Centre, MUMC+, Maastricht, Netherlands, 3 Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, Netherlands, 4 Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, Maastricht, Netherlands Pathway and network approaches are valuable tools in analysis and interpretation of large complex omics data. Even in the ﬁeld of rare diseases, like Rett syndrome, omics data are available, and the maximum use of such data requires sophisticated tools for comprehensive analysis and visualization of the results. Pathway analysis with differential gene expression data has proven to be extremely successful in identifying affected processes in disease conditions. In this type of analysis, pathways from different databases like WikiPathways and Reactome are used as separate, independent entities. Pathway and network approaches are valuable tools in analysis and interpretation of large complex omics data. Even in the ﬁeld of rare diseases, like Rett syndrome, omics data are available, and the maximum use of such data requires sophisticated tools for comprehensive analysis and visualization of the results. Pathway analysis with differential gene expression data has proven to be extremely successful in identifying affected processes in disease conditions. In this type of analysis, pathways from different databases like WikiPathways and Reactome are used as separate, independent entities. Here, we show for the ﬁrst time how these pathway models can be used and integrated into one large network using the WikiPathways RDF containing all human WikiPathways and Reactome pathways, to perform network analysis on transcriptomics data. This network was imported into the network analysis tool Cytoscape to perform active submodule analysis. Using a publicly available Rett syndrome gene expression dataset from frontal and temporal cortex, classical enrichment analysis, including pathway and Gene Ontology analysis, revealed mainly immune response, neuron speciﬁc and extracellular matrix processes. Our active module analysis provided a valuable extension of the analysis prominently showing the regulatory mechanism of MECP2, especially on DNA maintenance, cell cycle, transcription, and translation. In conclusion, using pathway models for classical enrichment and more advanced network analysis enables a more comprehensive analysis of gene expression data and provides novel results. Keywords: pathway analysis, WikiPathways, Reactome, Rett syndrome, network analysis, RDF, topology, active subnetworks ORIGINAL RESEARCH published: 21 February 2019 doi: 10.3389/fgene.2019.00059 Edited by: Marco Antoniotti, Università degli studi di Milano Bicocca, Italy Reviewed by: Zhi-Ping Liu, Shandong University, China Daniela Albrecht-Eckardt, BioControl Jena GmbH, Germany Reviewed by: Zhi-Ping Liu, Shandong University, China Daniela Albrecht-Eckardt, BioControl Jena GmbH, Germany *Correspondence: Martina Kutmon martina.kutmon@maastrichtuniversity.nl Specialty section: This article was submitted to Bioinformatics and Computational Biology, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Bioinformatics and Computational Biology, Received: 11 November 2018 Accepted: 24 January 2019 Published: 21 February 2019 Dataset The publicly available dataset studying the transcriptome in human brain tissue of Rett syndrome patients and healthy controls from the Gene Expression Omnibus (GEO) was used (GEO:GSE75303). The original study was published by Lin et al. (2016). The dataset contains transcriptome data obtained with Illumina HumanHT-12 V4.0 expression beadchips. The samples were taken postmortem from human frontal and temporal cortex of three Rett syndrome patients (MECP2 mutations c.378-2A>G, c.763C>T, c.451G>T) and three age-, gender-, and ethnicity- matched controls. Raw and normalized data as well as study metadata were obtained (GEO:GSE75303) and subjected to quality control, including signal distribution and sample grouping analyses, using plotting functions from ArrayAnalysis.org (Eijssen et al., 2013). No samples were excluded for further analysis. The provided normalized data on GEO was ﬁltered to remove all probes with a detection p-value of 1 for all samples, indicating overall absence of expression. Thereafter, the limma package for R (version 3.30.13, Ritchie et al., 2015) was used to compute diﬀerential expression between Rett patients and controls for the frontal and temporal cortex samples separately. For each probe, this results in estimates of the fold change and p-value signiﬁcance between the patient and control groups. Probes were re-annotated with Ensembl gene identiﬁers based on Ensembl build 91 using the BridgeDbR package (version 1.16.0, Leemans et al., 2018) with the Hs_Derby_Ensembl_91.bridge database (van Iersel et al., 2010). g y Rett syndrome (MIM: 312750, Rett, 1966) is a rare genetic disorder, caused in most patients by a loss of function mutation in the MECP2 gene (Amir et al., 1999). The accompanying MECP2 protein is multifunctional and acts as an epigenetic repressor, transcriptional repressor, and transcriptional activator. MECP2 binds DNA on methylated CpG islands and is involved in several regulatory activities: attracting histone deacetylases (HDAC1), increasing packing density of DNA, repressing and in speciﬁc genes also activating gene expression, and due to its phosphorylation sites, MECP2 activity is sensitive to intracellular signaling (Chunshu et al., 2006; Ehrhart et al., 2016). Due to its regulatory role, many downstream genes are aﬀected in case of loss of function, resulting in a broad range of symptoms including moderate to severe intellectual disability, gait problems, stereotypic movements, dystonia, scoliosis, epileptic seizures, and sleep problems (Hagberg et al., 2002; Neul et al., 2010). 1. INTRODUCTION Miller RA, Ehrhart F, Eijssen LMT, Slenter DN, Curfs LMG, Evelo CT, Willighagen EL and Kutmon M (2019) Beyond Pathway Analysis: Identiﬁcation of Active Subnetworks in Rett Syndrome. Front. Genet. 10:59. doi: 10.3389/fgene.2019.00059 In a diseased state, many molecular processes in the human body are aﬀected and dysregulated. Performing pathway analysis on molecular data sets comparing healthy vs. diseased subjects is immensely eﬀective in ﬁnding aﬀected pathways and it enables researchers to study the underlying processes in detail, to reveal possible disease mechanisms. While standard enrichment methods February 2019 \| Volume 10 \| Article 59 Frontiers in Genetics \| www.frontiersin.org Miller et al. Active Subnetworks in Rett Syndrome have limitations and pathways are analyzed independently with their arbitrary process boundaries (Khatri et al., 2012), the pathway models themselves are very interesting from a network science perspective. These models contain detailed information about biological molecules and their interactions with one another, which can be visualized and analyzed using network biology tools (Kutmon et al., 2014). The detailed models of these biological processes are collected in online pathway databases like WikiPathways (Slenter et al., 2017) and Reactome (Fabregat et al., 2017). The availability of pathway models in the structured and semantic Resource Description Framework format (RDF) creates the possibility to integrate all pathway models into one large network and therefore incorporate the relations and overlap between them (Waagmeester et al., 2016). By removing artiﬁcial boundaries, this will enable us to study the systemic eﬀects of diseases, such as Rett syndrome, using network biology methods. Speciﬁcally, we can look for subnetworks, even if not present in pathways as found in pathway databases, which reﬂect modules of diﬀerential biological activity. also contain interactions in pathways not found through those methods. Dataset In the past 10 years, omics data analysis on the level of genome, transcriptome, or proteome saw an increase in importance, to analyse and understand the holistic impact of MECP2, respectively, the impact of an impaired MECP2. Shovlin and Tropea (2018) recently reviewed the available transcriptomics studies on Rett syndrome and came to the conclusion that the most researched impact of MECP2 dysfunction lies with dendritic connectivity and synapse maturation, mitochondrial dysfunction, and glial cell activity. Recent pathway analysis results of single and integrated studies identiﬁed changes in intracellular signaling, including EIF2 (eukaryotic translation initiation) signaling, cytoskeleton, and cell metabolism including mitochondrial function (Bedogni et al., 2014; Ehrhart et al., 2018) . Frontiers in Genetics \| www.frontiersin.org Active Module Analysis The constructed network was loaded into Cytoscape (version 3.7.0), a network analysis and visualization tool (Shannon et al., 2003). Diﬀerential expression analysis data (log2 fold changes and p-values) for both frontal and temporal cortex were added as node attributes to the network. We used the WikiPathways RDF from October 2018 release (Waagmeester et al., 2016) to retrieve information about all interactions in the pathway models of two major pathway databases, WikiPathways and Reactome. With this network approach, the pathway models are not treated as independent modules, but they are integrated on an interaction level, which enables linking information from diﬀerent pathways based on their shared participants and thus bringing related interactions closer to each other. As shown in Figure 1, each interaction is represented by an interaction node in the network with edges to all participant nodes (either source, target, or participant). For each interaction, it is recorded in which pathway or pathways the interaction is present. By connecting all the retrieved interactions, a large network representing all human pathway models was created. The SPARQL query language was used to retrieve the relevant data. The scripts to generate the constructed network are available on GitHub (https://github.com/wikipathways/wprdf2cytoscape). Interactions with at least two annotated interaction participants (gene product, metabolite, complex) are included. Gene The Cytoscape app jActiveModules (version 3.2.1, Ideker et al., 2002) was used to identify active submodules in the large network that show signiﬁcant changes in expression. These subnetworks are freed from the artiﬁcial pathway boundaries of conventional pathway models found in WikiPathways and Reactome. The following parameters were used to ﬁnd active submodules: p-value as the node attribute, number of modules was set to ﬁve, overlap threshold of 0.8, and search strategy with a search depth of two. Pathway-Based Network Construction Biological pathway models are small sub-networks describing speciﬁc biological processes. Connecting and integrating pathway models in one large network enables us to use network biology tools and approaches to study and investigate the network. Enrichment Analysis We performed pathway analysis with PathVisio (version 3.3.0, Kutmon et al., 2015) and Gene Ontology (GO) analysis with GO-Elite (version 1.2, Zambon et al., 2012). For GO analysis with GO-Elite, the input gene lists for frontal and temporal cortex contained all signiﬁcantly changed genes (p-value < 0.05) with an absolute fold change cutoﬀ of 1.5. Ensembl identiﬁers of all measured genes in the datasets were provided as the background list. Number of permutations was set to 2,000. Pruned GO-term results (i.e., GO terms for which genes in subterms that were found to be signiﬁcant were removed) were ﬁltered based on Z-score (> 1.96), permuted p-value (< 0.05) and a minimum number of changed genes of ﬁve. Pathway analysis was performed on a combined human pathway collection from all curated WikiPathways pathways including the Reactome pathway set (in total 903 pathways, October 2018 release). Diﬀerential gene expression was mapped to genes on the pathway diagrams using the Hs_Derby_Ensembl_91.bridge identiﬁer mapping database. Thereafter, pathway statistics was performed on diﬀerential gene expression for temporal and frontal cortex using the following criteria to select only signiﬁcantly diﬀerentially expressed genes (absolute fold change cutoﬀof 1.5 and p-value < 0.05): In this study, we aim to investigate the molecular changes in Rett syndrome patients using a network-based approach by integrating existing pathway models from WikiPathways and Reactome into one large network and identifying disease- aﬀected submodules that show diﬀerential gene expression. We will compare the results with standard enrichment analysis methods, including pathway and Gene Ontology analysis, and expect that the identiﬁed disease modules will February 2019 \| Volume 10 \| Article 59 2 Active Subnetworks in Rett Syndrome Miller et al. products have uniﬁed Ensembl (Zerbino et al., 2017) identiﬁers, metabolites have either Wikidata (Mietchen et al., 2015), ChEBI (Hastings et al., 2015) or HMDB identiﬁers (Wishart et al., 2017), and complexes have Reactome identiﬁers. A list of frequently occurring small molecules (Supplementary Table 1), e.g., H+, H20, ATP, were removed from the network to prevent inclusion of paths with no speciﬁc biological relevance. Such small molecules tend to create artiﬁcial hub nodes simply because e.g., ATP is used/produced in a lot of metabolic reactions. (log2FC < -0.58 OR log2FC > 0.58) AND p-value < 0.05. The resulting ranked pathway list was ﬁltered based on Z-score (> 1.96), permuted p-value (< 0.05), and minimum number of changes (positive) genes of ﬁve. Pathway Analysis Pathway Analysis Pathway analysis was performed in PathVisio for both brain regions separately. Overrepresentation analysis revealed 18 and 21 pathways altered in the datasets for frontal and temporal cortex, respectively (Z-score > 1.96, minimum ﬁve changed genes), see Figure 2. Interestingly, eight pathways were altered in both frontal and temporal cortex. Similar to the results of the GO analysis, several immune system-related and extracellular matrix/cytoskeleton-related pathways were found to be enriched. Additionally, calcium channel related processes including muscle contraction pathways were found in both brain regions. Although muscle contraction pathways are not expected in brain tissue samples, the overlapping diﬀerentially expressed genes were mostly ion channels and signaling cascade proteins also highly relevant for neurons. Supplementary Figure 1 shows the heatmap with a more lenient ﬁlter (Z-score > 1.96, minimum three changed genes). Figure 3 is an example pathway visualization for a pathway that has a high Z-score in both tissue types, Microglia Pathogen Phagocytosis Pathway (Hanspers and Slenter, 2017). • Pathway-based network construction: Script available on Github • Pathway-based network construction: Script available on Github (https://github.com/wikipathways/wprdf2cytoscape) (https://github.com/wikipathways/wprdf2cytoscape) • Active module analysis: Cytoscape (version 3.7.0), jActiveModules app (version 3.2.1) • Active module analysis: Cytoscape (version 3.7.0), jActiveModules app (version 3.2.1) • Active module analysis: Cytoscape (version 3.7.0), jActiveModules app (version 3.2.1) Gene Expression The total number of probes measured was 37,707 from which 29,024 could be linked to Ensembl identiﬁers. After merging multiple probe identiﬁers for the same Ensembl identiﬁer, 19,023 unique gene identiﬁers remained. Diﬀerential gene expression analysis revealed 1,953 in the frontal cortex and 2,436 signiﬁcantly changed genes in the temporal cortex samples of RETT syndrome patients vs. controls. Only 221 in frontal and 341 of the signiﬁcantly changed genes in temporal cortex had a more than 1.5-fold increase or decrease in expression (\|log2 fold change\| > 0.58). In both brain regions, more genes were down-regulated in Rett syndrome patients than up-regulated, see Table 1, which matches with ﬁndings from the original publication (Lin et al., 2016). Pathway-Based Network Construction From the 903 pathway models in the WikiPathways and Reactome collection, 860 pathways contained 27,410 unique interactions. On average, a pathway contained 35 interactions (min = 1, max = 510, median = 22). Interestingly, 3,264 interactions occur multiple times but only 2,103 interactions are present in more than one pathway. As an example, one of the highest occurring interactions is the complex binding of the three subunits of the IκB kinase complex which plays an important role in the propagation of cellular response to inﬂammation (Häcker and Karin, 2006) and is present in 25 diﬀerent pathways. Tools and Settings • Dataset: Normalized data from GEO, plotting functions from ArrayAnalysis.org, limma package for R (version 3.30.13), BridgeDbR package (version 1.16.0) with Hs_Derby_Ensembl_91.bridge database. FIGURE 1 \| WikiPathways network structure. Every interaction is represented as a node in the network with links to all participants. If the interaction is directed, the information about source and target nodes is added as an edge attribute. The nodes represented as small, red rounded rectangles are interactions, blue circles represent gene products and green diamonds embody metabolites. Interactions that share certain participants, such as GeneProduct 1, are brought close together in the resulting network even if they are from different pathways, such as Pathway 1 and 3. FIGURE 1 \| WikiPathways network structure. Every interaction is represented as a node in the network with links to all participants. If the interaction is directed, the information about source and target nodes is added as an edge attribute. The nodes represented as small, red rounded rectangles are interactions, blue circles represent gene products and green diamonds embody metabolites. Interactions that share certain participants, such as GeneProduct 1, are brought close together in the resulting network even if they are from different pathways, such as Pathway 1 and 3. February 2019 \| Volume 10 \| Article 59 3 Frontiers in Genetics \| www.frontiersin.org Miller et al. Active Subnetworks in Rett Syndrome • Enrichment analysis: PathVisio (version 3.3.0) and GO-Elite (version 1.2). • Enrichment analysis: PathVisio (version 3.3.0) and GO-Elite (version 1.2). Active Module Analysis diverse roles. The genes identiﬁed as hubs in the active module network of frontal cortex are two gene products which are not measured in the dataset, RPS27A and UBA52. Both are involved in protein degradation via 26S proteasome, ubiquitination, translation, and DNA excision repair. In the central part of the network, the ribosomal proteins including RPL14, RPL29, and RPL3 form a cluster. This cluster is connected via PPP2CA and PPP2R1A, two phosphatases involved in cell cycle, DNA replication and transcription, to a cluster of centrosomal proteins including CEP78, CEP57, and CEP131. The module combines interactions from 47 unique pathways (Supplementary Table 4) including class I MHC mediated antigen processing and presentation (WP3577), non-sense-mediated decay (WP2710), cell-cycle related pathways (WP1859, WP1775, WP1858, WP2772), and eukaryotic translation elongation and initiation (WP1811, WP1812). Active modules were calculated using the jActiveModules app. The top ﬁve modules with the highest active paths scores were identiﬁed for both comparisons, frontal and temporal cortex. The modules for frontal cortex contained between 300–350 nodes and 560–1,020 edges. The top modules for temporal cortex tended to be smaller ranging from 230–290 nodes and 450–1,000 edges. Figures 4, 5 show the highest-ranked module for frontal and temporal cortex, respectively. Gene expression changes are visualized as node color and signiﬁcance is indicated by the node border color. All modules only contained gene products; no metabolites were found. The complete submodule analysis results for both datasets can be found in Supplementary Data 1 (zip ﬁle containing two Cytoscape session ﬁles). The highest ranked active module for frontal cortex contains 303 nodes (79 interactions and 224 gene products) and 568 edges, see Figure 4. Two hundred and ten of the gene products are measured in the dataset and 112 are changed signiﬁcantly (p-value < 0.05). Twelve gene products have an absolute log2 fold change > 0.58. The subnetwork contains eight signiﬁcantly down-regulated genes (blue rounded rectangles) including two F-Box genes, FBOX32 and FBXO9, involved in phosphorylation- dependent ubiquitination. The subnetwork contains ﬁve signiﬁcantly up-regulated genes (red rounded rectangles) with The highest ranked active module for temporal cortex contains 238 nodes (84 interactions and 154 gene products) and 457 edges, see Figure 5. The module partially overlaps with the module found for frontal cortex. One hundred and fourty three of the gene products are measured in the dataset and 137 are changed signiﬁcantly (p-value < 0.05). Gene Ontology Analysis Pathways with a high Z-score (>1.96) contain signiﬁcantly more changed genes than expected and are considered pathways of interest. An asterisk next to the Z-score value indicates pathways with a signiﬁcant Z-score (>1.96) but less than ﬁve changed genes. FIGURE 2 \| Pathway analysis results for frontal and temporal cortex data. Pathways are clustered in this heatmap based on their Z-scores. Pathways with a high Z-score (>1.96) contain signiﬁcantly more changed genes than expected and are considered pathways of interest. An asterisk next to the Z-score value indicates pathways with a signiﬁcant Z-score (>1.96) but less than ﬁve changed genes. FIGURE 2 \| Pathway analysis results for frontal and temporal cortex data. Pathways are clustered in this heatmap based on their Z-scores. Pathways with a high Z-score (>1.96) contain signiﬁcantly more changed genes than expected and are considered pathways of interest. An asterisk next to the Z-score value indicates pathways with a signiﬁcant Z-score (>1.96) but less than ﬁve changed genes. FIGURE 2 \| Pathway analysis results for frontal and temporal cortex data. Pathways are clustered in this heatmap based on their Z-scores. Pathways with a high Z-score (>1.96) contain signiﬁcantly more changed genes than expected and are considered pathways of interest. An asterisk next to the Z-score value indicates pathways with a signiﬁcant Z-score (>1.96) but less than ﬁve changed genes. Frontiers in Genetics \| www.frontiersin.org Gene Ontology Analysis Gene Ontology overrepresentation analysis identiﬁed 39 and 50 biological processes as altered in frontal and temporal cortex, respectively (Supplementary Tables 2, 3). Summarizing, neuron speciﬁc and immune system-related processes were found to be enriched in both brain regions for Rett syndrome patients. In temporal cortex, additionally, regulation of translational initiation (GO:0006446) and an extracellular matrix/cytoskeleton-related process (GO:0007229) were found to be enriched. Interestingly, the microglia relevant complement factors C1QB and C1QC were found in the enriched GO classes defense response (GO:0006952) and immune eﬀector process (GO:0002252). The resulting network consists of 48,639 nodes and 106,137 edges. The network consists of one major component (46,756 nodes) and 427 smaller components with each less than twenty nodes. The network contains 8,643 gene products, 2,704 metabolites and 9,882 complex / group nodes. Most common interaction types are directed interaction (13,572), complex / group participation (5,298), catalysis (4,787), inhibition (1,185), and conversions (896). TABLE 1 \| Differentially expressed genes in frontal and temporal cortex. Temporal cortex down-regulated Temporal cortex not changed Temporal cortex up-regulated Frontal cortex 88 44 1 down-regulated - Frontal cortex 171 18,576 55 not changed - - - - Frontal cortex 3 62 23 up-regulated - 133 and 88 genes were signiﬁcantly down- and up-regulated in frontal cortex, respectively. Two hundred sixty-two and 79 genes were signiﬁcantly down- and up-regulated in temporal cortex, respectively. Eighty-eight genes are down-regulated, and 23 genes are up-regulated in both brain regions. Only four genes show different expression patterns. The following ﬁltering criteria were used: p-value < 0.05 and absolute log2 fold change > 0.58. TABLE 1 \| Differentially expressed genes in frontal and temporal cortex. Temporal cortex not changed Temporal cortex down-regulated Temporal cortex up-regulated 133 and 88 genes were signiﬁcantly down- and up-regulated in frontal cortex, respectively. Two hundred sixty-two and 79 genes were signiﬁcantly down- and up-regulated in temporal cortex, respectively. Eighty-eight genes are down-regulated, and 23 genes are up-regulated in both brain regions. Only four genes show different expression patterns. The following ﬁltering criteria were used: p-value < 0.05 and absolute log2 fold change > 0.58. February 2019 \| Volume 10 \| Article 59 Frontiers in Genetics \| www.frontiersin.org 4 Miller et al. Active Subnetworks in Rett Syndrome FIGURE 2 \| Pathway analysis results for frontal and temporal cortex data. Pathways are clustered in this heatmap based on their Z-scores. Active Module Analysis Twenty-nine gene products have an absolute log2 fold change > 0.58. The module contains 24 signiﬁcantly February 2019 \| Volume 10 \| Article 59 5 Miller et al. Active Subnetworks in Rett Syndrome FIGURE 3 \| Visualization of the frontal and temporal cortex gene expression on the Microglia Pathway Phagocytosis Pathway. In the left half of the gene boxes, the gene expression change in the frontal cortex is shown. In the right half of the gene boxes, the gene expression in the temporal cortex is shown. The blue colors represent down-regulation of the gene in Rett syndrome patients (negative log2 fold change), while the red shades visualize the up-regulated genes. The darker the color, the stronger the effect. Green borders indicate signiﬁcance of the change (p-value < 0.05). Gray colored nodes are not annotated or measured in the dataset. FIGURE 3 \| Visualization of the frontal and temporal cortex gene expression on the Microglia Pathway Phagocytosis Pathway. In the left half of the gene boxes, the gene expression change in the frontal cortex is shown. In the right half of the gene boxes, the gene expression in the temporal cortex is shown. The blue colors represent down-regulation of the gene in Rett syndrome patients (negative log2 fold change), while the red shades visualize the up-regulated genes. The darker the color, the stronger the effect. Green borders indicate signiﬁcance of the change (p-value < 0.05). Gray colored nodes are not annotated or measured in the dataset. down-regulated genes (blue rounded rectangles) including several ubiquitin conjugating enzymes (UBE2E1, UBE2E3) and translation initiation factors (EIF4A2, EIF4H, EIF4G2). Only ﬁve signiﬁcantly up-regulated genes are found in the subnetwork (red rounded rectangles) but the distance between them is large. This subnetwork contains similar hub nodes as in the frontal cortex subnetwork including RPS27A, UBA52, and PPP2R1A. Additionally, NCBP2 and NCBP1, proteins involved in RNA processing, play an important role in the subnetwork. The module combines interactions from 51 unique pathways (Supplementary Table 5) including transcription / translation (WP1889, WP1906, WP1812), cell cycle (WP1859, WP1775, WP4109), and immune response (WP3577, WP2658) related processes. Active Module Analysis MECP2 due to a mutation has been found to inﬂuence a variety of pathways and biological processes, including pathways related to not only neuron development and function, but also to the immune system, transcription, and translation related processes (which were identiﬁed mainly by transcriptome analysis, Colantuoni et al., 2001; Bedogni et al., 2014; Ehrhart et al., 2018; Shovlin and Tropea, 2018). The aﬀected pathways identiﬁed with our study closely match the results previously found by Ehrhart et al. (2018), in which human brain tissue data of Rett syndrome patients (published by Deng et al., 2007) was analyzed. The expression of the MECP2 protein itself is not signiﬁcantly aﬀected in this dataset (minor, insigniﬁcant down regulation, log2 fold change of –0.1, in both brain regions). The original study by Lin et al. (2016) from which the dataset analyzed in this paper was acquired, focused on the signiﬁcant down-regulation of certain complement system factors in Rett syndrome (C1QA, C1QB, C1QC). Complement system factors are produced generally in liver, however their expression was also found to be changed in stimulated microglia. Furthermore, there is emerging evidence that C1Q factors are involved in several non-immunogenic activities, such as synaptic pruning in microglia (Kouser et al., 2015). Frontiers in Genetics \| www.frontiersin.org 4. DISCUSSION MECP2 is a multifunctional protein which is involved in several transcriptional inhibitory and activational processes. MECP2 was generally regarded as a repressor, however its role as genetic activator has also been conﬁrmed (Chahrour et al., 2008). In previous studies, a loss of function in February 2019 \| Volume 10 \| Article 59 Frontiers in Genetics \| www.frontiersin.org 6 Miller et al. Active Subnetworks in Rett Syndrome FIGURE 4 \| Top-ranked active module for frontal cortex data. The highest-ranked subnetwork contains 303 nodes and 568 edges. It contains 13 signiﬁcantly changed genes (rounded rectangles) when applying the same cutoff as for enrichment analysis (absolute log2 fold change > 0.58). Other measured gene products are visualized as circular nodes. Blue ﬁll color indicates down-regulation while red indicates up-regulation. The darker the color, the stronger the effect. Gray hexagons are gene products not measured in the data set. The very small, gray nodes represent interaction nodes. These were combined from 47 different pathways, with none of the pathways providing more than six interactions. FIGURE 4 \| Top-ranked active module for frontal cortex data. The highest-ranked subnetwork contains 303 nodes and 568 edges. It contains 13 signiﬁcantly changed genes (rounded rectangles) when applying the same cutoff as for enrichment analysis (absolute log2 fold change > 0.58). Other measured gene products are visualized as circular nodes. Blue ﬁll color indicates down-regulation while red indicates up-regulation. The darker the color, the stronger the effect. Gray hexagons are gene products not measured in the data set. The very small, gray nodes represent interaction nodes. These were combined from 47 different pathways, with none of the pathways providing more than six interactions. As expected, our pathway and GO analysis revealed a substantial number of immune system related pathways to be aﬀected in Rett syndrome frontal and temporal cortex tissue samples. Inﬂammatory processes have been identiﬁed previously in Rett syndrome patients, mouse models and in vitro systems, and are suspected to contribute to the development of Rett syndrome (De Felice et al., 2016; Ehrhart et al., 2018). Figure 2 shows many of aﬀected pathways in both frontal and temporal cortex, with similar results found by GO analysis. Interestingly, no complement system or transcription / translation related pathways show up (except Microglia Pathogen Phagocytosis Pathway, which includes C1Q factors). Frontiers in Genetics \| www.frontiersin.org 4. DISCUSSION Only seven of the 31 pathways found through pathway analysis contribute interactions to the active modules identiﬁed for frontal and temporal cortex. The modules mainly contained interactions from transcription / translation and cell cycle related pathways, which were not found with the classical enrichment analysis. These processes were also previously found in transcriptome pathway analysis of Rett syndrome (Bedogni et al., 2014; Ehrhart et al., 2018). Not surprisingly, the subnetworks do not contain metabolic reactions. Only metabolites connecting at least two genes aﬀected by MECP2 would be present in an active subnetwork. The enrichment analysis did not show any metabolic processes that are aﬀected, which is in line with the manifestation of Rett syndrome. Overall, the regulatory eﬀects of MECP2, especially on DNA maintenance, cell cycle, transcription, and translation, is more prominently shown in the active modules, while immune system related responses are more present in pathway analysis. Importantly, the active module approach does not replace analyses like classical enrichment analysis but augments it. When running the active module analysis on the same February 2019 \| Volume 10 \| Article 59 7 Miller et al. Active Subnetworks in Rett Syndrome FIGURE 5 \| Top-ranked active module for temporal cortex data. The subnetwork contains 238 nodes and 457 edges. It contains 29 signiﬁcantly changed genes (rounded rectangles) when applying the same cutoff as for enrichment analysis (absolute log2 fold change > 0.58). Other measured gene products are visualized as circular nodes. Blue ﬁll color indicates down-regulation while red indicates up-regulation. The darker the color, the stronger the effect. Gray hexagons are gene products not measured in the data set. The very small, gray nodes represent interaction nodes. These were combined from 51 different pathways, with none of the pathways providing more than six interactions. FIGURE 5 \| Top-ranked active module for temporal cortex data. The subnetwork contains 238 nodes and 457 edges. It contains 29 signiﬁcantly changed genes (rounded rectangles) when applying the same cutoff as for enrichment analysis (absolute log2 fold change > 0.58). Other measured gene products are visualized as circular nodes. Blue ﬁll color indicates down-regulation while red indicates up-regulation. The darker the color, the stronger the effect. Gray hexagons are gene products not measured in the data set. The very small, gray nodes represent interaction nodes. These were combined from 51 different pathways, with none of the pathways providing more than six interactions. Frontiers in Genetics \| www.frontiersin.org 4. DISCUSSION network using the dataset with permuted gene labels, the resulting subnetworks are very diﬀerent from the identiﬁed Rett subnetworks. This basic computational validation further strengthens our conﬁdence that we indeed have subnetworks speciﬁc and strongly aﬀected in Rett syndrome patients. The results of the permutation analysis are summarized in Supplementary Data 2. predeﬁne a certain cutoﬀ, since genes are ranked based on their signiﬁcance. Some considerations arose when constructing and analyzing the network. For instance, some common metabolites like ATP, ADP, or NADH, while biologically necessary, were excluded from the network, since their involvement in a multitude of interactions created links between almost every node. Additionally, this approach is strongly depending on the a priori input of pathway data in terms of coverage and quality. Currently, human pathway databases contain a little over 50% of the protein coding genes (Slenter et al., 2017), which is also a probable number for the coverage of metabolites and interactions. Pathway models generally contain information about directionality of the interactions. However, available active subnetwork analysis methods only take topology but not directionality into account. This could strongly aﬀect the identiﬁcation of active submodules and would be an important extension of existing algorithms. This was the ﬁrst time the entirety of the WikiPathways knowledgebase, including Reactome pathways, has been used to create a comprehensive human pathway-based network for network analysis of transcriptomics data. While the pathway content of both databases overlaps, both resources also contain unique information. By building a network out of pathways from a combination of pathway databases, a more complete biological (and therefore genome) coverage is enabled. Identifying active modules from a large network has some major beneﬁts, such as the easy applicability to any gene expression dataset, ignoring predeﬁned boundaries used in traditional pathway diagrams, and incorporating the relations and overlap between the pathways. Additionally, this method does not require researchers to The active module discovery approach should be considered as an additional step after classical enrichment analysis. In this February 2019 \| Volume 10 \| Article 59 8 Miller et al. Active Subnetworks in Rett Syndrome Conclusion The authors would like to thank the authors and curators of WikiPathways and Reactome for the provision of content. Special thanks to Lin et al., the authors of the original publication, for generating the data and making it available for re-use. Thanks to Irina Voineagu for responding to our inquiries and answering our questions regarding the dataset. Pathway models have proven themselves as powerful tools for biologists to describe and analyse biological processes. The collaboration between the widely-adopted pathway databases WikiPathways and Reactome and the availability of their data in RDF format allowed us to integrate a large number of pathways from both databases into one large network. This enables us to perform advanced network analyses like active submodule identiﬁcation. By comparing classical enrichment methods with the active submodule identiﬁcation on a Rett syndrome dataset in two diﬀerent brain regions, we found that both approaches provided valuable insights into the disease. Importantly, they were strongly complementary and did not show the same results. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene. 2019.00059/full#supplementary-material FUNDING FE, DS, CE, and EW received funding from ELIXIR, the European research infrastructure for life-science data for development of interoperability approaches used in this study. FE is funded by Stichting Terre, the Dutch Rett syndrome fonds. This project was partly funded by the Dutch Province of Limburg (MK). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N◦825575, EJP-RD (FE, CE). AUTHOR CONTRIBUTIONS RM and FE: data analysis, literature search; LE: data preprocessing and statistical analysis; DS: data analysis; LC: literature search; CE and EW: study design; MK: data analysis, study design, literature search. All authors contributed in writing and editing of the manuscript. DATA AVAILABILITY STATEMENT Importantly, the complete interaction network of WikiPathways with 48,639 nodes and 106,137 edges can be opened and analyzed in Cytoscape, despite of the network to be too large to be visualized. The use of graph databases like Neo4j, which already have connections available to Cytoscape (cyNeo4j app, Summer et al., 2015), could be a useful addition to the approach. Importantly, as part of the systems biology cycle, advanced computational analyses like the one reported in this manuscript lead to new hypotheses and ideas for experiments, which then need to be tested and validated in a laboratory. The dataset analyzed for this study can be found in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE75303). DATA AVAILABILITY STATEMENT study, we used human brain transcriptomics data from a study with Rett syndrome patients, however our approach is not unique to this application or rare diseases. These diseases are by deﬁnition less common and often less extensively studied, which may result in lower availability of speciﬁc pathway models. Nonetheless, the active module approach succeeds and shows great power for additional discoveries. While rare genetic diseases have the advantage that the causative gene is (usually) known, the resulting downstream consequences can be diverse and aﬀect a variety of pathways. By using pathway models in an integrative network approach, further use of the invaluable resources present in pathway databases is enabled and subnetworks of interest can be retrieved based on the entire body of pathways available. Using Cytoscape allows using all available apps such as the jActiveModules app to analyse our network. Importantly, the complete interaction network of WikiPathways with 48,639 nodes and 106,137 edges can be opened and analyzed in Cytoscape, despite of the network to be too large to be visualized. The use of graph databases like Neo4j, which already have connections available to Cytoscape (cyNeo4j app, Summer et al., 2015), could be a useful addition to the approach. Importantly, as part of the systems biology cycle, advanced computational analyses like the one reported in this manuscript lead to new hypotheses and ideas for experiments, which then need to be tested and validated in a laboratory. study, we used human brain transcriptomics data from a study with Rett syndrome patients, however our approach is not unique to this application or rare diseases. These diseases are by deﬁnition less common and often less extensively studied, which may result in lower availability of speciﬁc pathway models. Nonetheless, the active module approach succeeds and shows great power for additional discoveries. While rare genetic diseases have the advantage that the causative gene is (usually) known, the resulting downstream consequences can be diverse and aﬀect a variety of pathways. By using pathway models in an integrative network approach, further use of the invaluable resources present in pathway databases is enabled and subnetworks of interest can be retrieved based on the entire body of pathways available. Using Cytoscape allows using all available apps such as the jActiveModules app to analyse our network. REFERENCES and pre-processing on ArrayAnalysis.org. Nucleic Acids Res. 41, W71–W76. doi: 10.1093/nar/gkt293 and pre-processing on ArrayAnalysis.org. 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BMC Genomics 17:427. doi: 10.1186/s12864-016-2746-7 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 © 2019 Miller, Ehrhart, Eijssen, Slenter, Curfs, Evelo, Willighagen and Kutmon. 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. Frontiers in Genetics \| www.frontiersin.org REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Mietchen, D., Hagedorn, G., Willighagen, E., Rico, M., Gómez-Pérez, A., Aibar, E., et al. (2015). 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https://openalex.org/W4252984163	https://www.qeios.com/read/VRX5LK/pdf	English	null	Other Health Care Professional	Definitions	2,020	cc-by	74	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Other Health Care Professional National Cancer Institute National Cancer Institute Qeios ID: VRX5LK · https://doi.org/10.32388/VRX5LK Source National Cancer Institute. Other Health Care Professional. NCI Thesaurus. Code C53289. National Cancer Institute. Other Health Care Professional. NCI Thesaurus. Code C53289 Health care professional other than listed in section D5 of FDA MedWatch form 3500A. Qeios ID: VRX5LK · https://doi.org/10.32388/VRX5LK 1/1
https://openalex.org/W4382937505	https://www.researchsquare.com/article/rs-3123743/latest.pdf	English	null	Efficacy of probiotics or synbiotics in critically ill patients: A systematic review and meta- analysis	Research Square (Research Square)	2,023	cc-by	10,441	Efficacy of probiotics or synbiotics in critically ill patients: A systematic review and meta- analysis Efficacy of probiotics or synbiotics in critically ill patients: A systematic review and meta- analysis Jiaqi Lou Ningbo No. 2 Hospital Shengyong Cui Ningbo No. 2 Hospital Neng Huang Ningbo No. 2 Hospital Guoying Jin Ningbo No. 2 Hospital Cui Chen Ningbo No. 2 Hospital Youfen Fan Ningbo No. 2 Hospital Chun Zhang Ningbo No. 2 Hospital Jiliang Li (  397272330@qq.com ) Ningbo No. 2 Hospital Jiaqi Lou Ningbo No. 2 Hospital Shengyong Cui Ningbo No. 2 Hospital Neng Huang Ningbo No. 2 Hospital Guoying Jin Ningbo No. 2 Hospital Cui Chen Ningbo No. 2 Hospital Youfen Fan Ningbo No. 2 Hospital Chun Zhang Ningbo No. 2 Hospital Jiliang Li (  397272330@qq.com ) Ningbo No. 2 Hospital Jiliang Li (  397272330@qq.com ) Ningbo No. 2 Hospital Efficacy of probiotics or synbiotics in critically ill patients: A systematic review and meta- analysis Jiaqi Lou Efficacy of probiotics or synbiotics in critically ill patients: A systematic review and meta- analysis Jiaqi Lou 1. Background Mechanical ventilation is the most important life support in the intensive care unit (ICU)[1] and has saved countless lives. Ventilator-associated pneumonia (VAP) is a common nosocomial infection in patients under mechanical ventilation in the ICU, and its development is associated with prolonged duration of mechanical ventilation, length of stay in hospital, hospitalization costs, and increased mortality[2]. Recent studies have demonstrated that the main infection route of VAP[3] includes oropharyngeal secretions entering the lower respiratory tract of patients along the tracheal cannula, and regurgitation of bacteria from the stomach into the lower respiratory tract due to the relaxation of the esophageal sphincter caused by prolonged nasal feeding. Due to injuries (infection and trauma), weakened gastrointestinal motility, and disturbed hormone levels, ICU patients have a disrupted intestinal microbiota characterized by an overgrowth of pathogens, which can consequently lead to VAP. Studies have shown that VAP can be prevented by eliminating the pathogen itself or blocking the bacterial infection route using antibiotics, selective intestinal disinfection, semi-reclining position breathing, tracheal intubation, chlorhexidine mouthwash, or drainage of subglottic secretions. The prevention of VAP is critical for improving the success rate of rescue in patients requiring chronic mechanical ventilation. The gastrointestinal tract is the largest immune organ in the body, and the integrity of the intestinal barrier is key to the functional homeostasis of the intestinal mucosa and resistance to various diseases. It was reported that probiotic strains can protect intestinal barrier integrity[4] and treat or prevent certain diseases in the host[5]. In addition, probiotics can induce the release of antimicrobial factors from host cells, inhibit immune cell proliferation, stimulate IgA production, and promote antioxidant activity[6]. Synbiotics are inactive oligosaccharides that serve as nourishment for probiotics and have been shown to promote the growth and reproduction of probiotics. Probiotics and synbiotics are increasingly being used in the clinical setting. A growing body of work has reported that the addition of exogenous probiotics or synbiotics can lower the incidence of VAP and improve the prognosis of patients in ICU. However, whether oral probiotics or synbiotics can help prevent VAP is still controversial, and studies on the clinical efficacy of probiotics in VAP prevention have been inconsistent[7]. Therefore, this systematic review is aimed to assess the efficacy and safety of probiotics or synbiotics in preventing VAP in critically ill patients in the ICU. Research Article eywords: Critically ill patients, Probiotics, Synbiotics, Meta-analysis, Ventilator-associated pneumonia, Mortality DOI: https://doi.org/10.21203/rs.3.rs-3123743/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/28 Page 1/28 2. Methods The research protocol, outcomes, and relevant items in this systematic review were reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Statement[8]. The protocol for this meta-analysis has been registered with PROSPERO (CRD42023398511). Abstract Background This latest systematic review and meta-analysis aim to examine the effects of probiotic and synbiotic supplementation in critically ill patients. Methods Relevant articles were retrieved from PubMed, Embase, the Cochrane Database, and the Web of Science. The primary output measure was the incident of vendor associated pneumonia, and the secondary outputs were diarra, Clostridium diffusion infection (CDI), incident of sepsis, incident of hospital acquired pneumonia, incident of any infection, duration of mechanical exploitation, in hospital morality, length of hospital stay, ICU morality rate, And length of ICU stay. Data were pooled and expressed as relative risk (RR) and standardized mean difference (SMD) with a 95% confidence interval (CI). Results Third three studies were included in this systematic review and meta-analysis, with 4065 patients who received probiotics or synbiotics (treatment group) and 3821 patients who received standard care or placebo (control group). The pooled data from all included studies demonstrated that the treatment group has significantly reduced incidence of vessel associated pneumonia (VAP) (RR = 0.80; 95% CI: 0.67–0.96; p = 0.021, I2 = 52.5%) and sepsis (RR = 0.97; 95% CI: 0.66–1.42; p = 0.032, I2 = 54.4%), As well as significantly increased duration of mechanical exploitation (SMD=-0.47; 95% CI: -0.74–0.20, p = 0.012, I2 = 63.4%), length of hospital stay (SMD=-0.33; 95% CI: -0.57–0.08, p = 0.000, I2 = 74.2%), ICU mobility (RR = 0.95; 95% CI: 0.71–1.27; p = 0.004, I2 = 62.8%), And length of ICU stay [SMD=-0.29; 95% CI: -0.58-0.01; p = 0.000, I2 = 82.3%)] than the control group There were no significant differences in diarra, CDI, incidence of hospital acquired pneumonia, and in hospital morality between the two groups. Conclusion Our meta-analysis showed that probiotic and synbiotic supplements are beneficial for critically ill patients as they significantly reduce the incidence of vendor associated pneumonia and sepsis, as well as the duration of mechanical exploitation, length of hospital stay, length of ICU stay, and ICU morality How, this intervention has minimal impact on diarra, CDI, incidence of hospital acquired pneumonia, and in hospital morality in critically ill patients. 2.4 Statistical analysis Data were extracted independently by two authors (Jiaqi Lou and Guoying Jin) from the full texts of the studies and compiled into shared sheets. The following information was collected from the included studies: first author, year of publication, duration of treatment, study design, inclusion and exclusion criteria, intervention and number of subjects, and primary and secondary outcomes. Data were validated by a third author (Cui Chen) using a standardized method. The methodological quality assessment shown in Table S1 was based on the Cochrane Reviewers’ Handbook[11]. Discrete numerical variables were reported in risk ratio (RR) and 95% confidence intervals (CIs). Continuous variables are expressed as mean difference (MD). The standardized mean difference (SMD) was used as a summary statistic in this meta-analysis due to the application of different methods to assess the same outcome. When the standard deviations (SDs) of mean differences were not provided in the study, SDs for the changes from baseline were calculated by: SD2 change = SD2 baseline + SD2 final − (2 × correlation coefficient × SD baseline × SD final, assuming that the correlation coefficient (R) was 0.5[9]. The I2 statistic was used to quantify heterogeneity, and forest plots were generated and double-checked by two authors (Jiaqi Lou and Jiliang Li). If I2 < 50%, the pooled outcomes were considered to have low statistical heterogeneity, and if I2 > 50%, the pooled outcomes were considered to have high statistical heterogeneity[12]. Each low heterogeneity analysis was estimated using a fixed-effects model, otherwise, the random-effects model was applied. The possible sources of heterogeneity were analyzed using one or more of the following methods: (i) Subgroup analyses based on the characteristics of age, type of intervention, research design, follow-up duration, and type of intervention; and (ii) leave-one-out sensitivity analysis by omitting the studies one by one to identify which RCT(s) caused the heterogeneity and how each contributed to the overall analysis. The potential publication bias in each analysis was assessed using funnel plots, Egger’s regression test and sensitivity analysis, in which p < 0.05 was considered statistically significant. When there was evidence of funnel plot asymmetry or the Egger’s regression test result was statistically different, potentially missing studies were substituted using the “trim and fill” method to estimate the effect of publication bias on the observed pooled effect size[13]. 2.1 Data source and search strategy 2.1 Data source and search strategy Page 2/28 Relevant articles were searched in PubMed, Embase, the Cochrane Library, and the Web of Science using subject headings and keywords containing “critical ill”, “critically ill”, “Critically illnesses”, “Illness, Critical”, “Critically Ill”, “probiotic”, “probiotics”, “synbiotic” and “synbiotics”. In addition, the references of the included studies and relevant review articles were screened to identify eligible clinical trials that were not found through the database searches. The identified articles were imported, stored, and managed by EndNote 20. Each search result was independently reviewed for eligibility by two authors (Shengyong Cui, Jiaqi Lou), and any disagreement was resolved by the corresponding author (Jiliang Li). l b l Relevant articles were searched in PubMed, Embase, the Cochrane Library, and the Web of Science using subject headings and keywords containing “critical ill”, “critically ill”, “Critically illnesses”, “Illness, Critical”, “Critically Ill”, “probiotic”, “probiotics”, “synbiotic” and “synbiotics”. In addition, the references of the included studies and relevant review articles were screened to identify eligible clinical trials that were not found through the database searches. The identified articles were imported, stored, and managed by EndNote 20. Each search result was independently reviewed for eligibility by two authors (Shengyong Cui, Jiaqi Lou), and any disagreement was resolved by the corresponding author (Jiliang Li). 2.2 Eligibility criteria Inclusion criteria: (1) Type of study: Randomized controlled trial (RCT) or retrospective cohort studies; (2) Participants: Critically ill patients of all ages; (3) Intervention: Probiotics alone or in combination with prebiotics (synbiotics) compared with placebo; and (4) Outcomes: Incidences of ventilator-associated pneumonia, diarrhea, Clostridium difficile infection (CDI), sepsis, hospital-acquired pneumonia, and any infection; Duration of mechanical ventilation, in- hospital mortality, length of hospital stay, ICU mortality, and length of ICU stay. Exclusion criteria: (1) Studies that did not meet the inclusion criteria, which included but were not limited to case reports, conference abstracts, letters, responses, short comments, clinical guidelines, animal experiments, cell experiments, research protocols, reviews, systematic reviews or meta-analysis; (2) Studies without clear inclusion and exclusion criteria; (3) Outcomes that had not been clearly stated; (4) Uncontrolled studies; (5) Preclinical studies in animal models. If multiple articles reported the same or overlapping data, the article with the longer duration of the intervention or larger sample size was included in this study. 2.3 Data extraction and quality assessment Articles were independently selected by two authors by screening the titles and/or abstracts, and full-text articles were then evaluated for eligibility and adequacy in information. For each study that fulfilled the eligibility criteria, study sources (author, publication year, and country), characteristics of study population (sample size, study design, type of subjects, male to female ratio, baseline mean age, sequential organ failure assessment score and acute physiology Ⅱ score), characteristics of prebiotic, probiotic, and synbiotic species (type, dosage, duration of intervention), and outcomes (including at least one of the following: Incidence of ventilator-associated pneumonia, diarrhea, CDI, sepsis, hospital-acquired pneumonia, duration of mechanical ventilation, in-hospital mortality, length of hospital stay, ICU mortality and length of ICU stay) were extracted using a standard data extraction template. The methodological quality of each included RCT was assessed using the Cochrane Collaboration’s risk of bias tool[9] based on the Cochrane Handbook for Systematic Reviews of Interventions Version 5.0.1. The risk of bias for each item was judged as low, high, or unclear based on the criteria of the Cochrane Handbook for Systematic Reviews. Data extraction and quality assessment were performed independently by two authors (Shengyong Cui, Jiaqi Lou), and any discrepancy was settled by discussion and consensus. When a consensus was not reached, a third researcher (Jiliang Li) acted as an arbitrator. Data synthesis was performed using Review Manager (RevMan) 5.4. The quality of case-control studies and cohort studies was assessed by the Newcastle-Ottawa Scale (NOS)[10]. 2.4 Statistical analysis In case of a significant result, the number of potentially missing studies required to make the p-value non-significant was estimated using the “fail-safe N” method. All statistical analyses were performed using StataSE 15.1. A P < 0.05 was considered statistically significant for all tests except for the heterogeneity test, in which case a P < 0.10 was used. 3.2 Risk of bias and quality assessment of individual studies The Newcastle-Ottawa Scale (NOS) evaluates cohort studies and case-control studies through three sections (eight items in total), including selection, comparability, exposure or outcome. The full score is 9 points. Our research team evaluated each selected cohort study. 3.2 Risk of bias and quality assessment of individual studies Four cohort studies were assessed by the NOS and 29 RCTs were assessed by the Cochrane Collaboration risk of bias tool. The characteristics of the included studies and their designs are shown in Fig. 2, and Table 1, which summarizes the risk of bias of the included trials based on different quality domains of the risk of bias tool. Among the 4 cohort studies, the study acomplished by Sato[15] was scored 8 points, the study by Fleming et al. 2019 was scored 8 points, (2) the study by Shimizu et al. 2018 was scored 7 points, the study by Kenna et al. 2016 was scored 5 points. The NOS scores of the four cohort studies all exceeded 5 points, and thus all of these studies were included for subsequent meta-analysis. In the 29 RCTs, the outcome assessors were double-blinded in 86% (25/29) of the trials, and adequate randomized sequence generation was reported in 79% (23/29) of the trials but was unclear in the remaining six RCTs. For the remaining 3 RCTs, 1 was single-blinded[27], 1 was non-blinded[28], and 1 was an open-label crossover trial[33]. All of the 29 RCTs, except 9 RCTs, had a low risk of bias in allocation concealment. While most of the RCTs had a low risk of bias in the blinding of participants and key study personnel, r RCTs had high risks of bias, and 4 RCTs had unclear risks of bias. In addition, all of the RCTs showed a low risk of bias for incomplete outcome data and selective outcome reporting. Table 1 Table 1 Quality assessment of 4 retrospective cohort studies using the Newcastle-Ottawa Scale (NOS). Study, year Selection Comparability Outcome NOS Score Adequate definition of cases Representativeness of the cases Selection of controls Definition of controls Control for important factor Ascertainment of exposure Same method of ascertainment for cases and controls Nonresponse rate Sato et al. 2022 1 1 1 1 2 1 0 1 8 Fleming et al. 2019 1 1 1 1 2 1 0 1 8 (2)Shimizu et al. 2018 1 1 1 1 1 1 0 1 7 Kenna et al. 2016 1 1 1 0 1 0 1 0 5 The Newcastle-Ottawa Scale (NOS) evaluates cohort studies and case-control studies through three sections (eight items in total), including selection, comparability, exposure or outcome. The full score is 9 points. Our research team evaluated each selected cohort study. 3.1 Characteristics of included studies The PRISMA statement flowchart illustrated in Fig. 1 shows the process of study selection. Of the 922 records initially identified from the database search, 271 were duplicates filtered by software and 30 were duplicates filtered by manual screening. Of the 621 remaining articles, 548 were excluded based on Page 3/28 Page 3/28 the articles’ title/abstract, and 103 were reviewed. The detailed full-article review resulted in 33 trials that met our inclusion criteria and were thus included in the present systematic review and meta-analysis. The summarized characteristics of the 29 RCTs and 4 retrospective cohort studies are shown in Table S1[3, 5, 14–44]. A total of 7,886 patients were grouped into the probiotics or synbiotics group (n = 4065) and control group (n = 3821). The majority of the studies were randomized, parallel-group, placebo-controlled trials. All clinical trials were double-blinded except for the studies conducted by reference[3, 27, 28, 33]. Four studies were retrospective cohort studies[15, 20, 22, 26], and 8 were multi-center studies[5, 18, 19, 24, 32, 33, 39, 42]. These trials were published between 2004 and 2022, with sample sizes in individual trials ranging from 19 to 2650. A total of 32 trials were published in English and 1 trial was published in Spanish. Eight trials were carried out in the United States, four trials each were in Iran and China, three trials each in the United Kingdom and India, two each were in Australia, Canada and Greece, and one each in Saudi Arabia, Malaysia, Spain, Thailand, and Netherlands. The participants’ ages at the time of intervention ranged in all-age. The detailed information of the included studies is summarized in Table S1. Interventions in the included trials consisted of different forms of probiotics, synbiotics, or prebiotics. Twenty-five trials investigated the efficacy of probiotics and eight assessed the efficacy of synbiotics. All drugs were given orally or through tube feeding, and the composition of probiotics and synbiotics administered in each group varied among studies. Furthermore, probiotics and synbiotics were administered in different dosage forms, and the duration of the intervention durations also varied among trials. 3.3.4 Duration of mechanical ventilation Meta-analysis of seven trials showed that the duration of mechanical ventilation was significantly shorter in the treatment group than in the control group (SMD = -0.47; 95% CI: -0.74 – -0.20, p = 0.012, I2 = 63.4%) (Figure S5). The I2 statistic indicated a high heterogeneity among the studies. However, funnel plot symmetry and the lack of significant difference in the Egger’s regression test (p = 0.361) indicated that there was no detectable publication bias. We then conducted a subgroup analysis and found that both probiotics and synbiotics can reduce mechanical ventilation time in critically ill patients (Figs. 10–11 and Figures S11-S12). 3.3.3 Incidences of CDI, sepsis, and hospital-acquired pneumonia The risk of CDI was not significantly different between the treatment and control groups in four trials (RR = 1.11; 95% CI: 0.73–1.70, p = 0.384, I2 = 1.7%), but the incidence of sepsis was significantly lower in the treatment group than in the control group in eight trials (RR = 0.97; 95% CI: 0.66–1.42, p = 0.032, I2 = 54.4%). Subgroup analysis of sepsis incidence by trial design showed that probiotics or synbiotics had no effect on the incidence of sepsis in two cohort studies (RR = 1.34; 95% CI: 0.82–2.19, p = 0.396, I2 = 0.00%)[20, 22], but lowered the incidence of sepsis in six RCTs (RR = 0.85; 95% CI: 0.51–1.40, p = 0.023, I2 = 61.8%). There was no difference in the incidence of hospital-acquired pneumonia between patients receiving probiotics or synbiotics and those receiving placebo (p > 0.05 for all). Both the funnel plot and Egger's test showed no publication bias in these analyses (p for Egger's test was 0.861, 0.588, 0.628). There was no significant change in the pooled 95% CI upon removal of each study (Figs. 6–9 and Figures S5-S10). 3.3.2 Diarrhea Complete data on diarrhea were available in 4 studies. Pooled data from these trials demonstrated that there was no significant difference in the incidence of diarrhea between the treatment and control groups (RR = 1.03; 95% CI: 0.98–1.09, p = 0.174, I2 = 39.6%). The funnel plot showed no evidence of publication bias, and Egger’s regression test result was not statistically different (p = 0.310) (Fig. 5, Figure S3 and S4). Sensitivity analysis showed no significant change in the result after excluding each study. 3.3.1 Incidence of VAP Eleven studies[5, 19, 21, 22, 24, 28, 33, 36–39] reported data on the incidence of VAP. Quantitative pooling of data revealed a significantly lower incidence of VAP in patients receiving probiotics or synbiotics compared with controls (RR = 0.80; 95% CI: 0.67–0.96; p = 0.021, I2 = 52.5%) (Fig. 2B). The Egger’s regression test result indicated no significant difference between the two groups (p = 0.310). However, subgroup analysis showed a significantly lower incidence of VAP only in patients receiving probiotics (RR = 0.79, 95% CI: 0.64–0.98, p = 0.008, I2 = 61.3%) and not in those receiving only synbiotics (RR = 0.85, 95% CI: 0.55–1.33, p = 0.531, I2 = 0.0%). Subgroup analysis of 11 research designs of the included studies also ascertained that the use of probiotics or synbiotics in 10 RCTs reduced the incidence of VAP (RR = 0.82, 95% CI: 0.68–0.98; p = 0.023, I2 = 53.4%), but the same conclusion could not be drawn in the cohort studies (Fig. 3, Figures S1 and S2). Considering that the patient populations included adults, children, and infants, we decided to conduct a subgroup analysis of VAP stratified by age. Only one study included in the subgroup analysis of VAP indicators was targeted at children[28]. The subgroup analysis showed that probiotics or synbiotics Page 4/28 treatment reduced VAP incidence, regardless of adults or children. In addition, we performed a subgroup analysis of VAP stratified by trial designs in which only one study was a retrospective cohort study[22], and obtained the same results as the age-stratified subgroup analysis. In the sensitivity analysis, the removal of individual study did not lead to a significant change in the OR values (Fig. 4). Table 2 summarizes the results of different subgroup analyses of VAP. Table 2 Subgroup analysis of VAP by outcome Subgroup No. of trials No. of participants RR 95% CI I2 (%) p for heterogeneity p for heterogeneity among groups Probiotics or Synbiotics Probiotics 9 4019 0.79 0.64– 0.98 61.3 0.008 0.021 Synbiotics 2 331 0.85 0.55– 1.33 0.0 0.531 Adult or Children Adult 10 4200 0.82 0.68-1.00 52.0 0.027 0.021 Children 1 150 0.59 0.35– 0.97 0.0 0.000 Trial design RCT 10 4171 0.82 0.68– 0.98 53.4 0.023 0.021 Retrospective cohort study 1 179 0.49 0.22– 1.13 0.0 0.000 3 3 2 Di h 4. Discussion The intestinal microbiota is a complex microbial community that plays an irreplaceable role in human life. Intestinal dysbiosis is very common in patients with critical illnesses[45, 46]. Since the gut microbiome is critical for maintaining host immunity, disruption of the gut microbiome may result in life- threatening host responses to injuries such as trauma, burns, and infections, which can lead to tissue and organ damage and even death[47]. It was recently reported that ecological dysregulation in ICU patients may affect survival and contribute to organ failure, kidney failure, heart failure, or respiratory failure[48]. Microbe-modulating therapies, in the forms of prebiotics, probiotics, and synbiotics, offer a promising approach for healthcare professionals to manage the progression of serious diseases and associated complications due to their low cost and potentially harmless nature. A study investigating the modulation of microbiota in the treatment of acute respiratory distress syndrome[49] found that selective decontamination of the digestive tract can effectively control the incidence of aspiration pneumonia. Furthermore, probiotics have been shown as a potential approach for restoring the gut microbiome in chronic kidney disease[50]. There is increasing evidence demonstrating that probiotics therapy can reduce systemic inflammation, postoperative infection, and even mortality by modulating the gut microbiome[51–53]. Although some studies have explored the relationships between probiotics or synbiotics supplements and gut barrier function, changes in gut microbiota, and some clinical outcomes in critically ill patients, the results have been inconsistent. A meta-analysis of the pooled data from 25 RCTs affirmed that symbiotic therapy significantly reduced the risk of sepsis complications[54]. However, to the best of our knowledge, there is no meta-analysis that simultaneously assessed the effects of probiotics and synbiotics administration on ventilator- associated pneumonia and included cohort studies and patient populations of all ages. Therefore, in this systematic review and meta-analysis, we examined the efficacy of probiotics and synbiotics on several clinical outcomes including VAP from 33 clinical trials (RCTs and cohort studies) to provide support for the clinical application of these supplementations. Our finding that probiotics or synbiotics can lower the incidence of VAP is consistent with those from a previous meta-analysis by Batra[55], which included nine trials of probiotic intervention in critically ill patients and found a similarly pronounced reduction in the duration of mechanical ventilation, length of ICU stay, and in-hospital mortality in the intervention group. 4. Discussion This conclusion may have been influenced by other factors in the included studies such as study quality, population characteristics, and microbial classification, all of which can lead to varying estimates of the therapeutic efficacy of probiotics or synbiotics. In order to account for these confounding factors, we conducted further subgroup analyses and took into account the presence of outcome bias. Ten RCTs suggested the same result, and only one cohort study did not support the efficacy of enteric microbiotherapy in reducing the incidence of VAP. However, the reduction in the incidence of VAP was only significant in patients receiving probiotics, accompanied by high heterogeneity, but not in those receiving synbiotics. Although the latter group had low heterogeneity, we still believed that there was some clinical heterogeneity since there were only two trials that had a synbiotics group. Similar heterogeneity was observed in subgroup analyses of patients of different ages, with probiotics or synbiotics reducing the incidence of VAP in critically ill adult patients, but no associated benefit in critically ill pediatric patients. Our meta-analysis of seven studies suggested a significant reduction in the number of days of mechanical ventilation between the treatment and control groups, and both probiotics and synbiotics were found to reduce the duration of mechanical ventilation in critically ill patients in the subgroup analyses. Moreover, we found that neither probiotics nor synbiotics reduced the incidence of hospital-acquired pneumonia, which may be attributed to the small number of relevant studies. Some meta-analysis studies have shown that probiotics are beneficial for treating acute diarrhea in pediatric patients[56, 57], but the patient population of these studies did not include pediatric patients in ICU. Other studies[58, 59] also showed that probiotics play a role in preventing antibiotic-associated diarrhea in adults, but they did not examine ICU patients. Our study reported no significant difference in the incidence of diarrhea between the treatment and control groups. We could not perform a subgroup analysis of the effect of age on probiotics in reducing diarrhea due to few trials. According to a review[60], some strains of probiotics and/or prebiotics could be effective in supporting the growth and metabolism of anaerobic bacteria. The Infectious Diseases Society of America also does not recommend the use of probiotics for the prevention of CDI[61]. 3.3.5 ICU mortality and length of ICU stay ICU mortality data was reported in ten studies. There was a significant difference in ICU mortality between the treatment and control groups (RR = 0.95; 95% CI: 0.71–1.27, p = 0.004, I2 = 62.8%). There was no significant publication bias in the selected studies, as indicated by funnel plot symmetry and the lack of significant difference in the Egger’s regression test (p = 0.361). Subgroup analysis also revealed that probiotics or synbiotics could reduce ICU mortality (Figs. 12–13, Figure S13-S14). Probiotics and synbiotics were associated with a significant improvement in the length of ICU stay in eleven studies (SMD = -0.29; 95% CI: -0.58–0.01, p = 0.000, I2 = 82.3%). Neither the funnel plot nor the Egger’s regression test indicated the presence of publication bias (p = 0.241). In addition, subgroup analysis showed that both probiotics and synbiotics alone could reduce the length of ICU stay in critically ill patients. Sensitive analysis showed that no Page 5/28 Page 5/28 single study qualitatively altered the pooled mortality and length of ICU stay, providing evidence for the stability of the meta-analysis (Figs. 14–15 and Figures S15-S16). single study qualitatively altered the pooled mortality and length of ICU stay, providing evidence for the stability of the meta-analysis (Figs. 14–15 and Figures S15-S16). 3.3.6 In-hospital mortality and the length of hospital stay In-hospital mortality was significantly different between the treatment and control groups (RR = 0.94; 95% CI: 0.66–1.03, p = 0.947, I2 = 0.0%). The funnel plot was partially symmetrical, and Egger’s regression for funnel plot asymmetry revealed no risk of publication bias (p = 0.302). Subgroup analysis by intervention type showed that neither synbiotics nor probiotics were effective in reducing in-hospital mortality (Figs. 16–17 and Figures S17-S18). Both the meta-analysis and subgroup analysis by intervention type demonstrated that probiotics or synbiotics can reduce the hospitalization time of critically ill patients (SMD = -0.33; 95% CI: -0.57 – -0.08, p = 0.000, I2 = 74.2%). Visual inspection of the funnel plot and further evaluation by Egger’s test indicated no publication bias (p = 0.307). Sensitivity analysis revealed no substantial change in hospitalization time after omitting each of the sixteen studies, confirming the stability of the results (Figs. 18–19 and Figures S19-20). 4. Discussion In our study, there was no significant change in the risk of CDI with probiotics or synbiotics use compared with placebo among critically ill patients, indicating no benefit from gut microbial agents. Sepsis is a strong host response to infection, and its management remains a major challenge. In recent years, the use of gut microbiomes in the treatment of sepsis has attracted some attention. The maintenance or restoration of the microbiota and its metabolites may be a therapeutic or prophylactic Page 6/28 Page 6/28 approach for treating sepsis[62]. Samara et al.[63] found that supplementation with a probiotic mixture enhanced gut microbiome maturation and reduced intestinal tract inflammation in extremely preterm infants. Another double-blinded placebo-controlled randomized clinical trial[64] also concluded that supplementation with probiotics reduced IL-1β and LPS levels in patients. These findings suggest that probiotics have the potential function of attenuating host inflammatory responses. Our meta-analysis ascertained that probiotics or synbiotics can reduce the incidence of sepsis in critically ill patients, even after the removal of studies with a high risk of bias. approach for treating sepsis[62]. Samara et al.[63] found that supplementation with a probiotic mixture enhanced gut microbiome maturation and reduced intestinal tract inflammation in extremely preterm infants. Another double-blinded placebo-controlled randomized clinical trial[64] also concluded that supplementation with probiotics reduced IL-1β and LPS levels in patients. These findings suggest that probiotics have the potential function of attenuating host inflammatory responses. Our meta-analysis ascertained that probiotics or synbiotics can reduce the incidence of sepsis in critically ill patients, even after the removal of studies with a high risk of bias. We also evaluated the effects of probiotics or synbiotics on the length of hospital and ICU stay and in-hospital and ICU mortality. Except for in-hospital mortality, the other three clinical outcomes were improved with either probiotic or synbiotics treatment. Our results were similar to those from the systematic review and meta-analysis conducted by Manzanares et al.[65] and Alsuwaylihi et al.[66], which were focused on probiotics alone. Interestingly, the benefits of synbiotics in reducing the length of ICU stay, ICU mortality and length of hospital stay were also found in our subgroup analyses, providing new evidence for the clinical application of probiotics or synbiotics in the future. However, limitations of this study include small sample size, personal equation in the process of data extraction, and different composition and dosage of the intervention. 5. Conclusions Probiotics or synbiotics supplementation plays a beneficial role in critically ill patients and presents a novel approach to the management of critical diseases. This systematic review and meta-analysis supports the potential role of probiotics or synbiotics in reducing the incidence of VAP and sepsis, as well as the duration of mechanical ventilation, length of hospital stay, length of ICU stay, and ICU mortality in critically ill patients. 4. Discussion Therefore, our findings should be interpreted with caution. Future high-quality studies of other diseases and various populations in the ICU are warranted to determine whether probiotics or synbiotics can significantly reduce the overall risk of the described clinical outcomes in order to provide more insights into the form, composition, and frequency of this supplementation for optimal outcomes. Abbreviations VAP: Ventilator-Associated Pneumonia; HAP: Hospital Acquired Pneumonia; ICU: Intensive Care Unit; IL-1β: Interleukin-1β; LPS: Lipopolysaccharide. Conflicts of Interest The authors declare that they have no competing interests This work was supported by the Zhejiang Provincial Medical and Health Science and Technology Program Project [grant number 2023RC081]; and the Project of NINGBO Leading Medical & Health Discipline [grant number 2022-F17]. Funders played no role in the study design, execution or manuscript writing. Acknowledgements Not applicable Authors’ contributions JQ Land SY C contributed equally to this work. JQ L, YF F and JL L designed and conceptualized the study. JQ L, SY C, N H and GY J completed the record retrieval and data extraction. Mathematical modeling and meta-analysis were conducted with the help of C C and C Z. The original draft was written by JQ L. All authors have read and agreed to the published version of the manuscript. 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Probiotics for the prevention of antibiotic-associated diarrhoea: a systematic review and meta-analysis. BMJ open. 2021;11(8):e043054. 60. Mounsey A, Lacy Smith K, Reddy VC, Nickolich S. Clostridioides difficile Infection: Update on Management. American family physician. 2020;101(3):168-75. 61. Wilcox MH, Rooney CM. Comparison of the 2010 and 2017 Infectious Diseases Society of America guidelines on the diagnosis and treatment of Clostridium difficile infection. Current opinion in gastroenterology. 2019;35(1):20-4. 62. Niu M, Chen P. Crosstalk between gut microbiota and sepsis. Burns & trauma. 2021;9:tkab036. 62. Niu M, Chen P. Crosstalk between gut microbiota and sepsis. Burns & trauma. 2021;9:tkab036. 63. Samara J, Moossavi S, Alshaikh B, et al. Supplementation with a probiotic mixture accelerates gut microbiome maturation and reduces intestinal inflammation in extremely preterm infants. Cell host & microbe. 2022;30(5):696-711.e5. 64. Moludi J, Kafil HS, Qaisar SA, et al. Effect of probiotic supplementation along with calorie restriction on metabolic endotoxemia, and inflammation markers in coronary artery disease patients: a double blind placebo controlled randomized clinical trial. Nutrition journal. 2021;20(1):47. 64. Moludi J, Kafil HS, Qaisar SA, et al. Effect of probiotic supplementation along with calorie restriction on metabolic endotoxemia, and inflammation markers in coronary artery disease patients: a double blind placebo controlled randomized clinical trial. Nutrition journal. 2021;20(1):47. 65. Manzanares W, Lemieux M, Langlois PL, Wischmeyer PE. Probiotic and synbiotic therapy in critical illness: a systematic review and meta-analysis. Critical care (London, England). 2016;19:262. 65. Manzanares W, Lemieux M, Langlois PL, Wischmeyer PE. Probiotic and synbiotic therapy in critical illness: a systematic review and meta-analysis. Critical care (London, England). 2016;19:262. 65. Manzanares W, Lemieux M, Langlois PL, Wischmeyer PE. Probiotic and synbiotic therapy in critical illness: a systematic review and meta-analysis. Critical care (London, England). 2016;19:262. 66. Alsuwaylihi AS, McCullough F. The safety and efficacy of probiotic supplementation for critically ill adult patients: a systematic review and meta- analysis. Nutrition reviews. 2023;81(3):322-32. 66. Alsuwaylihi AS, McCullough F. The safety and efficacy of probiotic supplementation for critically ill adult patients: a systematic review and meta- analysis. Nutrition reviews. 2023;81(3):322-32. 66. Alsuwaylihi AS, McCullough F. The safety and efficacy of probiotic supplementation for critically ill adult patients: a systematic review and meta- analysis. Nutrition reviews. 2023;81(3):322-32. Figures Figure 1 Figure 1 Page 10/28 PRISMA diagram detailing the literature search and the study selection/exclusion process. PRISMA Preferred Reporting Items for Systematic Reviews and Meta-analyses, RCT randomized controlled trials. PRISMA diagram detailing the literature search and the study selection/exclusion process. PRISMA Preferred Reporting Items for Systematic Reviews and Meta-analyses, RCT randomized controlled trials. PRISMA diagram detailing the literature search and the study selection/exclusion process. PRISMA Preferred Reporting Items for Systematic Reviews and Meta-analyses, RCT randomized controlled trials. Figure 2 Risk of bias charts. (A) Risk of bias in the included studies; (B) Risk of bias summary for all included studies. sk of bias charts. (A) Risk of bias in the included studies; (B) Risk of bias summary for all included studies. Figure 2 Page 11/28 Forest plot of the subgroup analysis of the overall weighted effect size. (A) The overall weighted effect size of probiotics alone or synbiotics alone on the incidence of VAP in critically ill patients; (B) RCTs or retrospective cohort studies alone versus control on the incidence of VAP in critically ill patients; (C) adult or children on the incidence of VAP in patients with critically ill patients. Note: The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 3 Page 13/28 gure 5 orest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of diarrhea in itically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. gure 5 rest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of diarrhea in tically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. ure 5 est plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of diarrhea in cally ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 3 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of ventilator associated pneumonia in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of ventilator associated pneumonia in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 12/28 Page 12/28 Figure 4 Forest plot of the subgroup analysis of the overall weighted effect size. (A) The overall weighted effect size of probiotics alone or synbiotics alone on th incidence of VAP in critically ill patients; (B) RCTs or retrospective cohort studies alone versus control on the incidence of VAP in critically ill patients; (C) adult or children on the incidence of VAP in patients with critically ill patients. Note: The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 4 Forest plot of the subgroup analysis of the overall weigh incidence of VAP in critically ill patients; (B) RCTs or retro adult or children on the incidence of VAP in patients with Figure 4 Forest plot of the subgroup analysis of the overall weighted effect size. (A) The overall weighted effect size of probiotics alone or synbiotics alone on the incidence of VAP in critically ill patients; (B) RCTs or retrospective cohort studies alone versus control on the incidence of VAP in critically ill patients; (C) adult or children on the incidence of VAP in patients with critically ill patients. Note: The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the subgroup analysis of the overall weighted effect size. (A) The overall weighted effect size of probiotics alone or synbiotics alone on the incidence of VAP in critically ill patients; (B) RCTs or retrospective cohort studies alone versus control on the incidence of VAP in critically ill patients; (C) adult or children on the incidence of VAP in patients with critically ill patients. Note: The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 5 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of diarrhea in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of diarrhea in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 14/28 Page 14/28 Page 14/28 igure 6 orest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of any CDI in critica patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 6 Figure 6 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of any CDI in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 15/28 Figure 7 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of sepsis in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the incidence of sepsis in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 16/28 Figure 8 Figure 8 Figure 8 Forest plot of subgroup analysis of the overall weighted effect size of RCTs or retrospective cohort studies on the incidence of sepsis in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of subgroup analysis of the overall weighted effect size of RCTs or retrospective cohort studies on the incidence of sepsis in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 17/28 Page 17/28 Page 17/28 Page 17/28 Figure 9 Figure 9 Forest plot of subgroup analysis of the overall weighted effect size of probiotics or synbiotics versus control on the incidence of nosocomial pneumonia in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of subgroup analysis of the overall weighted effect size of probiotics or synbiotics versus control on the incidence of nosocomial pneumonia in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 18/28 Figure 10 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the duration of mechanical ventilation in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the duration of mechanical ventilation in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 19/28 gure 11 Figure 11 Forest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the duration of mechanical ventilation in critically ill patients in subgroup analysis. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate Forest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the duration of mechanical ventilation in critically ill patients in subgroup analysis. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate Page 20/28 ure 12 est plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the ICU mortality in critically ill ents. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 12 Figure 12 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the ICU mortality in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the ICU mortality in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 21/28 gure 13 rest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the ICU mortality in critically ill patients. Th amond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 13 Figure 13 Forest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the ICU mortality in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 22/28 ure 14 est plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the length of ICU stay in critically ill ients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 14 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the length of ICU stay in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the length of ICU stay in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 23/28 Figure 15 Forest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the length of ICU stay in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the length of ICU stay in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 24/28 gure 16 orest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the mortality in hospital in critica patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 16 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the mortality in hospital in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the mortality in hospital in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 25/28 ure 17 t l t f b l i f th ll i ht d ff t i f bi ti l d bi ti l th i h it l t lit i iti ll ill Figure 17 Figure 17 Forest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the in-hospital mortality in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the in-hospital mortality in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 26/28 Figure 19 Forest plot of subgroup analysis of the overall weighted effect size of probiotics alone and synbiotics alone on the length of hospital stay in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Figure 18 Figure 18 Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the length of hospital stay in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Forest plot of the meta-analysis illustrating the overall weighted effect size of probiotics or synbiotics versus control on the length of hospital stay in critically ill patients. The diamond symbol at the bottom of the forest plot represents the overall weighted estimate. Page 27/28 Page 27/28 Figure 19 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryMaterials.docx Page 28/28
https://openalex.org/W2164094120	http://repository.cshl.edu/id/eprint/28859/1/Schatz%20Genome%20Biology%202013c.pdf	English	null	Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.)	GenomeBiology.com	2,013	cc-by	9,370	Open Access Open Access RESEARCH Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.) Ray Ming1,2†, Robert VanBuren2†, Yanling Liu1†, Mei Yang1†, Yuepeng Han1, Lei-Ting Li2,3, Qiong Zhang1,2, Min-Jeong Kim4, Michael C Schatz5, Michael Campbell6, Jingping Li7, John E Bowers8, Haibao Tang9, Eric Lyons10, Ann A Ferguson11, Giuseppe Narzisi5, David R Nelson12, Crysten E Blaby-Haas13, Andrea R Gschwend2, Yuannian Jiao7,14, Joshua P Der14, Fanchang Zeng2, Jennifer Han2, Xiang Jia Min15, Karen A Hudson16, Ratnesh Singh17, Aleel K Grennan2, Steven J Karpowicz18, Jennifer R Watling19, Kikukatsu Ito20, Sharon A Robinson21, Matthew E Hudson22, Qingyi Yu17, Todd C Mockler23, Andrew Carroll24, Yun Zheng25, Ramanjulu Sunkar26, Ruizong Jia27, Nancy Chen28, Jie Arro2, Ching Man Wai2, Eric Wafula14, Ashley Spence2, Yanni Han1, Liming Xu1, Jisen Zhang29, Rhiannon Peery2, Miranda J Haus2, Wenwei Xiong30, James A Walsh2, Jun Wu3, Ming-Li Wang27, Yun J Zhu27,31, Robert E Paull28, Anne B Britt32, Chunguang Du30, Stephen R Downie2, Mary A Schuler2,33, Todd P Michael34, Steve P Long2, Donald R Ort2,35, J William Schopf36, David R Gang4, Ning Jiang11, Mark Yandell6, Claude W dePamphilis14, Sabeeha S Merchant13, Andrew H Paterson7, Bob B Buchanan37, Shaohua Li1 and Jane Shen-Miller36* Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Abstract Background: Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan. Results: The genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment. Conclusions: The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots. © 2013 Ming 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. Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 2 of 11 Page 2 of 11 Additional file 1). The nine anchored megascaffolds have a combined size of 543.4 Mb, accounting for 67.6% of the genome assembly, and they are mostly proportional to the karyotype of the lotus chromosomes (Figure S2 and S3 in Additional file 1). The high quality of the lotus gen- ome assembly is largely due to the unexpected homozyg- osity of the ‘China Antique’ variety. Repeat content of the sacred lotus genome p g Repetitive sequences account for 57% of the assembled genome, including 47.7% recognizable transposable ele- ments (Table S4 in Additional file 1). Unlike most plants, which exhibit relatively inconsequential non-long terminal repeat retrotransposons (approximately 1% of the genome) [7-9], such non-long terminal repeat retro- transposons contribute 6.4% to the lotus genome. Differ- ing from other plants that usually have more Gypsy-like elements [9,10], Copia and Gypsy-like elements are comparable in copy number and genomic fraction in lotus. Most major DNA transposon families are detected in sacred lotus (occupying 16% of the lotus genome), albeit with more than 10-fold variation in relative abun- dance. An exception, the Tc1/Mariner super-family, is absent from both the lotus and grape genomes [7], sug- gesting the frequent loss of this family of elements. Surprisingly, hAT (Ac/Ds-like) elements contribute to nearly 7% of the lotus genome, represented by more than 100,000 copies, more than in any other sequenced plant genome. Of these, CACTA elements are least abundant (0.4%) while MULE, PIF and Helitron ele- ments have amplified to a moderate degree (2.5%, 2.7% and 3.6%, respectively). The lotus genome further includes 1,447 Pack-mutator-like elements that carry genes or gene fragments [11]. Analysis using expressed sequence tags (ESTs) indicated that at least 10 Pack- mutator-like elements are expressed, suggesting that they may play functional roles. Here, we report the sequencing and analysis of the sacred lotus genome, which descends from the most ancient lineage of angiosperms. We have studied the evolutionary history of the genome and genes involved in relevant processes governing the unique features of this ancient land plant, including its adaptation to aqua- tic environments. Abstract Although lotus is an out-crossing plant, its cultivation and vegetative propaga- tion via rhizomes over the past 7,000 years may have imposed a narrow genetic bottleneck. This could be partly the consequence of its unique feature, seed longev- ity, which might have further reduced the number of generations in its evolutionary history in addition to vegetative propagation. The estimated heterozygosity in ‘China Antique’ is 0.03%, lower than the 0.06% of the sequenced papaya cultivar ‘SunUp’ after 25 generations of inbreeding [6]. The estimated heterozygosity in the American lotus N. lutea ’AL1’ variety is 0.37%, also low. Background Sacred lotus, so named because of its religious signifi- cance in both Buddhism and Hinduism, belongs to the small plant family Nelumbonaceae, with only one genus, Nelumbo, and two species: N. nucifera (Asia, Australia, Russia) and N. lutea (eastern and southern North Amer- ica) [1]. Lotus is in the eudicot order Proteales, which lies outside of the core eudicots (Figure S1 in Additional file 1); its closest relatives are shrubs or trees belonging to the families Proteaceae and Platanaceae. Lotus was a land plant that has adapted to aquatic environments. Used as a food for over 7,000 years in Asia, lotus is cultivated for its edible rhizomes, seeds and leaves. Its buds, flowers, anthers, stamens, fruits, leaves, stalks, rhizomes and roots have been used as herbal medicines for treatment of cancer, depression, diarrhea, heart pro- blems, hypertension and insomnia [2,3]. Its seeds have exceptional longevity, remaining viable for as long as 1,300 years, and its vegetative rhizomes remain healthy for more than 50 years [1,2]. The nanoscopic closely packed protuberances of its self-cleaning leaf surface have been adapted in Europe for the manufacture of a ‘self-cleaning’ industrial paint, Lotusan. The use of this paint results in the so-called lotus effect that is now widely advertised for self-cleaning automobiles, buildings and fabrics. Genome sequencing and assembly We sequenced the genome of the sacred lotus variety ‘China Antique’ with 94.2 Gb (101×) Illumina and 4.8 Gb (5.2×) 454 sequences. The final assembly includes 804 Mb, 86.5% of the estimated 929 Mb lotus genome [4]. The contig N50 is 38.8 kbp and the scaffold N50 is 3.4 Mbp (Table S1 in Additional file 1). The largest 429 scaffolds account for 94.8% of the assembled genome and 98.0% of the annotated genes. Among the 39 plant gen- omes published to date, the median N50 scaffold length is about 1.3 Mb, making lotus the eighth best assembled genome (Table S2 in Additional file 1). We constructed a high-density genetic map using 3,895 sequence-based restriction-associated DNA sequencing markers and 156 simple sequence repeat markers [5]. The former were sorted into 562 co-segregating bins and a total of 698 informative markers were mapped into nine linkage groups for the eight lotus chromosomes, with one gap remaining between two linkage groups (Table S3 in Genome annotation and gene expression Following repeat-masking and annotation, we inferred 26,685 protein-coding genes in lotus, including all 458 core eukaryotic proteins [12]; 82% of the genes have similarity to proteins in SwissProt as identified by Basic Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 3 of 11 Local Alignment Search Tool (E < 0.0001). The average gene length is 6,561 bp with median exon and intron lengths of 153 bp and 283 bp, respectively (Table S1 in Additional file 1). The average gene density is one gene per 30 kb, with genes spread more evenly over the assembled genome than in many other plant genomes (Figure S2 in Additional file 1), which are characterized by gene-rich regions often found at the distal regions of chromosomes arms. A total of 12,344 ESTs were aligned to 11,741 gene models, and 174 alternative splicing events were identified from 164 genes involving 380 EST contigs (Table S5 in Additional file 1). Of the anno- tated genes in lotus, 22,803 (85.5%) show expression in rhizomes, roots, leaves or petioles based on RNAseq data (Figure S4 in Additional file 1). Expression of the remaining genes is likely confined to seeds, flowers and other unsurveyed tissues. Expression of 3,094 protein- coding genes was tissue-specific, including 1,910 genes showing expression only in rhizomes and 841 only in roots; 14,477 genes are expressed across all tissues sur- veyed. Of the 1,910 rhizome-specific genes, we found several AP2-like ethylene-responsive transcription fac- tors, BTB/POZ domain-containing proteins, heat shock proteins, homeobox transcription factors, kinesins and pentatricopeptide repeat-containing proteins (PPRs) (Table S6 in Additional file 1). In lotus, 544 genes were annotated as PPRs, with 201 of these expressed in the four tissues tested, and 199 only expressed in the rhizome. PPRs have been identified as a group of RNA-binding pro- teins involved in RNA processing, stability, editing, maturation and translation in plants. Although the mole- cular mechanism of their function has not yet been eluci- dated, their broad expression in lotus rhizome is notable. We reconstructed the ancestral gene content at key nodes of the evolutionary series, as well as the adapta- tional changes occurring along the branches leading to these nodes: the greatest changes observed in orthogroup presence and absence are specific to terminal lineages (Tables S8 and S9 in Additional file 1 and Figure 1). Synteny and genome evolution l f h A major evolutionary force shaping genome architecture in angiosperms is whole genome duplication (WGD) [14,15]. This process is followed by the ‘diploidization’ of genome organization through rearrangement, and of gene content through ‘fractionation,’ or homeologous gene loss. Intragenomic analysis of lotus indicates that it has experienced at least one WGD (paleotetraploidy, see Figure S6 in Additional file 1), named l, but implies that the Nelumbo lineage did not experience g, the paleo- hexaploidy (triplication) event around 125 million years ago detected in all other sequenced eudicot genomes [6,16-20]. Using lotus as a reference, as many as three post-g grape subgenomic copies are equally evident, the syntenic regions of which show extensive collinearity of homologous genes (Figure 2). Among the 87.1% of the lotus genic regions retained from this duplication, 5,279 (33.3%) are singletons, 8,578 (54.1%) are duplicated, and 2,007 (12.6%) have more than three homeologs, imply- ing there may have been additional paleo-duplications (Table S10 in Additional file 1). Based on three lines of evidence, the lineage nucleotide substitution rate in lotus is about 30% slower than that of grape, widely used in angiosperm comparative genomics due to its basal phylogenetic position in rosids, slow mutation rate, and lack of reduplication. First, while phy- logenetic evidence firmly dates the lotus-grape diver- gence before the pan-eudicot g triplication affecting only grape, synonymous substitution rates (Ks) between gen- ome-wide lotus-grape syntelog pairs (Figure S7 in Addi- tional file 1) are smaller than those among triplicated grape genes. Second, the lotus lineage mutation rate also appears slower (about 29.26% slower) than that of Vitis based on a maximum-likelihood tree of 83 plastid genes [21] and expert dating of the respective speciation events [22] using the r8s program [23] with penalized likelihood. Third, the lotus genome has retained more ancestral loci following its lineage-specific WGD. Lotus is a basal eudi- cot, and its genome is the one from the most ancient lineage of angiosperm sequenced to date (Figure S1 in Additional file 1). Lotus represents an even better model than grape for inferences about the common ancestor of eudicots. Genome annotation and gene expression More than three times as many orthogroup gains occur in the lineage leading to all eudicots, as compared to core eudicots (Figure S5 in Additional file 1), an increase second only to that of the grasses. Ortholog classification and ancestral gene content in eudicots The protein-coding gene sets from lotus and 16 other sequenced angiosperm species were used to identify putative orthologous gene clusters with Proteinortho v4.20 [13]. A total of 529,816 non-redundant genes were classified into 39,649 orthologous gene clusters (orthogroups) containing at least two genes (Table S7 in Additional file 1). Of the 26,685 protein-coding genes in lotus, 21,427 (80.3%) were classified into 10,360 orthogroups, of which 317 contained only lotus genes. From this gene classification, we estimate a minimum gene set of 7,165 genes in 4,585 orthogroups for eudi- cots (Table S7 in Additional file 1). The minimum gene set for core eudicots (7,559 genes in 4,798 orthogroups) is only slightly larger than the eudicot-wide set, suggest- ing that the minimal gene set of the eudicot-monocot ancestor (6,423 genes in 4,095 orthogroups) would add at least 490 orthogroups associated with the eudicots as a whole. Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 4 of 11 Figure 1 Orthogroup dynamics in lotus and other angiosperm genomes. Ancestral gene content and gene family (orthogroup) dynamics in lotus and other eudicot and monocot genomes identify expansion of the number of gene families and gene content associated with the ancestral eudicot. Figure 1 Orthogroup dynamics in lotus and other angiosperm genomes. Ancestral gene content and gene family (orthogroup) dynamics in lotus and other eudicot and monocot genomes identify expansion of the number of gene families and gene content associated with the ancestral eudicot. The remarkably slow mutation rate in lotus compli- cates the dating of the l duplication. l-duplicated lotus genes have a median synonymous substitution rate (Ks) of 0.5428, corresponding to an age of 27 million years ago (MYA) on the basis of average rates in plants [24] or 54 MYA on the basis of the grape lineage rate (Figure S7 in Additional file 1). Because lotus diverged from its clo- sest sister lineage approximately 135 to 125 MYA [21], before the g triplication, this suggests that the mutation rate in lotus is much lower than that in grape, and that the lotus-specific WGD event occurred about 65 MYA with a range between 76 and 54 MYA. This date coin- cides with the Cretaceous-Tertiary mass extinction that led to the loss of roughly 60% of plant species [25]. Ortholog classification and ancestral gene content in eudicots Polyploidization has been associated with increased adap- tation and survivability, and the numerous plant species inferred to have undergone polyploidy within this time- frame suggests a possible advantage to polyploid lineages during the Cretaceous-Paleogene transition, an interpre- tation supported by the l duplication in lotus. By tracing the phylogenetic histories of 688 pairs of grape genes in 528 orthogroups from each of the g dupli- cation blocks [26], we tested the timing of the g paleohexa- ploid event that has been observed in the genomes of Vitis [7], papaya [6], Populus [20] and other core eudicots [14,17]. About 50% of the resolved trees support the Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 5 of 11 Figure 2 High resolution analysis of syntenic regions of Nelumbo nucifera (Nn1/Nm2) and Vitis vinifera (Vv1/Vv2/Vv3). Synteny regions were identified from Figure S5 in Additional file 1. Gene models are arrays in middle of each panel; Colored boxes and lines connect regions of sequence similarity (LastZ) for protein-coding sequences between pair-wise comparisons. Figure 2 High resolution analysis of syntenic regions of Nelumbo nucifera (Nn1/Nm2) and Vitis vinifera (Vv1/Vv2/Vv3). Synteny regions were identified from Figure S5 in Additional file 1. Gene models are arrays in middle of each panel; Colored boxes and lines connect regions of sequence similarity (LastZ) for protein-coding sequences between pair-wise comparisons. Figure 2 High resolution analysis of syntenic regions of Nelumbo nucifera (Nn1/Nm2) and Vitis vinifera (Vv1/Vv2/Vv3). Synteny regions were identified from Figure S5 in Additional file 1. Gene models are arrays in middle of each panel; Colored boxes and lines connect regions of sequence similarity (LastZ) for protein-coding sequences between pair-wise comparisons. of g block duplications were eudicot-wide [26], even though the signal is primarily observed in core eudicots (Figure 3). timing of the g event to have occurred ‘core-eudicot-wide’ after the divergence of lotus, consistent with synteny ana- lysis. By contrast, gene family phylogenies for about half of the g block duplications include lotus genes (Table S11 in Additional file 1), although, in rare cases, duplicated monophyletic groups contain both lotus and eudicot-wide genes. Ortholog classification and ancestral gene content in eudicots (A) Summary of polyploidy events in the history of angiosperm evolution, with a focus on the possible phylogenetic origins of the three subgenomes comprising the gamma paleohexaploidy event in core eudicots. Synteny analysis of the Nelumbo genome indicates that gamma is shared only within the core eudicots; however, phylogenomic analysis suggests a more complex history since around half of the gamma pairs were duplicated core-eudicot-wide and the other half eudicot- wide (See Table S10 in Additional file 1). AA, BB, and CC are three subgenomes of the ancestral hexaploidy. Three possible phylogenetic origins of the ancestral AA genome involved in gamma are denoted by 1, 2 and 3. Lamda is defined as the most recent polyploidy event in the evolutionary history of Nelumbo. All the other Greek symbols are well-known polyploidy events in the evolutionary history of angiosperms. Gamma: genome-triplication (hexaploid) event in core eudicot genomes [7,23]; Sigma and rho: genome duplications detected in grass genomes [8]; Epsilon: angiosperm-wide duplication detected in large-scale gene family phylogenies. Based on gene tree phylogenomics, we hypothesize that the triplication event involved a tetraploid event (BBCC red star) first, then subgenome AA combined with BBCC to form hexaploidy AABBCC (blue dashed line). (B) Predicted gene tree topologies of hypothetical origins of the AA subgenome of the gamma paleohexaploidy. A, B, C indicate surviving genes inherited from AA, BB, CC subgenomes of the AABBCC ancestral hexaploidy. N indicates genes of Nelumbo. comprised of grasses (Poales) and palms (Arecales), was associated with relatively large gains in gene family num- ber and size. proteins in lotus compared to other plants is attributed to expansions in COG2132, a family of multi-copper oxidases. Most plant genomes encode one or two mem- bers of COG2132, whereas lotus has at least 16 members due to WGD and repeated tandem duplications (Figure 4, and see Figure S8 in Additional file 1). The only COG2132 members in Arabidopsis, LPR1 and LPR2, are involved in phosphate starvation signaling in root meristems. Simi- larly, in lotus, expression of COG2132 family members is confined largely to the roots (Figure 4). The lotus-specific expansion appears to form a separate phylogenetic clade from the LPR1 and 2-like proteins, suggesting a novel Ortholog classification and ancestral gene content in eudicots This is consistent with an earlier phylogenomic analysis using data from numerous plant genomes and basal eudicot transcriptomes, suggesting that 18% to 28% Such data suggest that a relatively large amount of genetic novelty is specifically associated with eudicots as a whole, even though the core eudicots shared a genome- triplication after divergence from the basal eudicots. By contrast, in monocots it appears that the evolution of the grass family specifically, rather than the earlier node Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 6 of 11 Figure 3 Polyploidy events in the history of angiosperm evolution. (A) Summary of polyploidy events in the history of angiosperm evolution, with a focus on the possible phylogenetic origins of the three subgenomes comprising the gamma paleohexaploidy event in core eudicots. Synteny analysis of the Nelumbo genome indicates that gamma is shared only within the core eudicots; however, phylogenomic analysis suggests a more complex history since around half of the gamma pairs were duplicated core-eudicot-wide and the other half eudicot- wide (See Table S10 in Additional file 1). AA, BB, and CC are three subgenomes of the ancestral hexaploidy. Three possible phylogenetic origins of the ancestral AA genome involved in gamma are denoted by 1, 2 and 3. Lamda is defined as the most recent polyploidy event in the evolutionary history of Nelumbo. All the other Greek symbols are well-known polyploidy events in the evolutionary history of angiosperms. Gamma: genome-triplication (hexaploid) event in core eudicot genomes [7,23]; Sigma and rho: genome duplications detected in grass genomes [8]; Epsilon: angiosperm-wide duplication detected in large-scale gene family phylogenies. Based on gene tree phylogenomics, we hypothesize that the triplication event involved a tetraploid event (BBCC red star) first, then subgenome AA combined with BBCC to form hexaploidy AABBCC (blue dashed line). (B) Predicted gene tree topologies of hypothetical origins of the AA subgenome of the gamma paleohexaploidy. A, B, C indicate surviving genes inherited from AA, BB, CC subgenomes of the AABBCC ancestral hexaploidy. N indicates genes of Nelumbo. Fi 3 P l l id t i th hi t f i l ti (A) S f l l id t i th hi t f i Figure 3 Polyploidy events in the history of angiosperm evolution. Adaptation to an aquatic environment Figure 4 Lotus-specific expansion in LPR1/LPR2 proteins. (A) The number of LPR1/LPR2 homologs in land plants. Homologs detected by Basic Local Alignment Search Tool against the genomes of land plants are represented by a box. A protein similarity network of those proteins is also shown; lotus proteins are represented as purple nodes, Arabidopsis proteins (LPR1 and LPR2) are represented as green nodes and other land plant proteins are represented as grey nodes. (B) Heatmap of COG2132 gene family member expression in lotus. Reads per kilo base per million (RPKM) values were log2 transformed, where blue correlates to high expression, and yellow to low expression. (C) A maximum-likelihood tree of LPR1/LPR2-like lotus proteins. Branch support was calculated using an Approximate Likelihood-Ratio Test. Lotus homologs are connected with a dashed bracket, whereas proteins whose genes are found in tandem on the genome are connected with a solid bracket. A detailed phylogeny of COG2132 members can be found in Figure S8 in Additional file 1. Figure 4 Lotus-specific expansion in LPR1/LPR2 proteins. (A) The number of LPR1/LPR2 homologs in land plants. Homologs detected by Basic Local Alignment Search Tool against the genomes of land plants are represented by a box. A protein similarity network of those proteins is also shown; lotus proteins are represented as purple nodes, Arabidopsis proteins (LPR1 and LPR2) are represented as green nodes and other land plant proteins are represented as grey nodes. (B) Heatmap of COG2132 gene family member expression in lotus. Reads per kilo base per million (RPKM) values were log2 transformed, where blue correlates to high expression, and yellow to low expression. (C) A maximum-likelihood tree of LPR1/LPR2-like lotus proteins. Branch support was calculated using an Approximate Likelihood-Ratio Test. Lotus homologs are connected with a dashed bracket, whereas proteins whose genes are found in tandem on the genome are connected with a solid bracket. A detailed phylogeny of COG2132 members can be found in Figure S8 in Additional file 1. function not found in Arabidopsis (Figure 4, and see Figure S8 in Additional file 1). Several other gene families also show unusual compo- sitions that may reflect adaptation to aquatic lifestyles. Adaptation to an aquatic environment Submersed plant growth presents unique physiological challenges. Lotus has had to evolve novel features to cope with its aquatic lifestyle. Possible adaptations include an astonishing number of putative copper-dependent pro- teins, of which 63 proteins contain at least one COX2 domain, 55 contain a ‘copper-binding-like’ domain, and 4 contain polyphenol oxidases. The abundance of copper Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 7 of 11 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Figure 4 Lotus-specific expansion in LPR1/LPR2 proteins. (A) The number of LPR1/LPR2 homologs in land plants. Homologs detected by Basic Local Alignment Search Tool against the genomes of land plants are represented by a box. A protein similarity network of those proteins is also shown; lotus proteins are represented as purple nodes, Arabidopsis proteins (LPR1 and LPR2) are represented as green nodes and other land plant proteins are represented as grey nodes. (B) Heatmap of COG2132 gene family member expression in lotus. Reads per kilo base per million (RPKM) values were log2 transformed, where blue correlates to high expression, and yellow to low expression. (C) A maximum-likelihood tree of LPR1/LPR2-like lotus proteins. Branch support was calculated using an Approximate Likelihood-Ratio Test. Lotus homologs are connected with a dashed bracket, whereas proteins whose genes are found in tandem on the genome are connected with a solid bracket. A detailed phylogeny of COG2132 members can be found in Figure S8 in Additional file 1. Figure 4 Lotus-specific expansion in LPR1/LPR2 proteins. (A) The number of LPR1/LPR2 homologs in land plants. Homologs detected by Basic Local Alignment Search Tool against the genomes of land plants are represented by a box. A protein similarity network of those proteins is also shown; lotus proteins are represented as purple nodes, Arabidopsis proteins (LPR1 and LPR2) are represented as green nodes and other land plant proteins are represented as grey nodes. (B) Heatmap of COG2132 gene family member expression in lotus. Reads per kilo base per million (RPKM) values were log2 transformed, where blue correlates to high expression, and yellow to low expression. (C) A maximum-likelihood tree of LPR1/LPR2-like lotus proteins. Branch support was calculated using an Approximate Likelihood-Ratio Test. Lotus homologs are connected with a dashed bracket, whereas proteins whose genes are found in tandem on the genome are connected with a solid bracket. A detailed phylogeny of COG2132 members can be found in Figure S8 in Additional file 1. Adaptation to an aquatic environment The basic helix loop helix (bHLH) family, implicated in light responses including germination, control of flower- ing and de-etiolation, and root and flower development, lacks three of its 20 subfamilies in lotus: Va, implicated in brassinosteroid signaling; VIIIc2, implicated in root hair development; and XIII, implicated in root meristem development [28]. The largest families of bHLH factors in lotus are XII, involved in developmental processes including control of petal size, brassinosteroid signaling Adaptation to phosphate starvation in lotus is also evi- denced by expansion of the UBC24 family and the miR399 family that regulates it (Table S12 in Additional file 1). The miR169 family, implicated in adaptation to drought stress in Arabidopsis [27], also shows expansion in lotus, totaling 22 members. The fact that lotus grows aquatically and may rarely be subjected to drought sug- gests that the miR169 family is involved in other physio- logical processes. Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 8 of 11 and floral initiation, and Ia, implicated in stomatal development and patterning. were detected only rarely in another phylogenomic analy- sis that introduced transcriptome data from the basal eudicots Gunnera and Pachysandra [29]. The 34 uni- genes available from that study were used to populate five MADS box orthogroups with larger taxon sampling in this study. Phylogenies of these orthogroups identify (at boostrap >50%) one eudicot-wide and three core- eudicot-wide duplications (Table S11 in Additional file 1), consistent with the rest of the findings in the pre- sent study. The PRR1/TOC1 circadian clock family, which coordi- nates internal biology with daily light/dark cycles and is highly conserved across many plant species, includes three predicted members in lotus compared to the one or two present in other plant genomes. The fact that PRR proteins have key roles in modulating light and tempera- ture input into the circadian clock suggests that lotus may require more sensitive adjustments to its environ- ment than other plants. Consistent with this, the crypto- chrome (CRY) family of blue light photoreceptors is also increased with five (two CRY1, two CRY2, one CRY3) compared to three in Arabidopsis and four in poplar (Additional file 1, Table S13). Similar expansion in the CRY family was also noted in another aquatic organism, Ostreococcus, a micro green algae. Adaptation to an aquatic environment Lotus is adapted to both temperate and tropical climates and day lengths with a wide range of flowering times, perhaps associated with increased numbers of flowering time and circadian clock-associated genes. In contrast to the phylogenomic results, syntenic com- parison showed one lotus region matched with up to three Vitis homologous regions, indicating that the lotus genome did not share the g event. We propose that the g event occurred after the separation of the lotus lineage (Proteales), and involved hybridization with a now extinct species that branched off around the same time (Figure 3A, AA at position #2), or even earlier than lotus (Figure 3A, AA at position #3). This model explains why the phylogenomic analyses could identify some g duplications occurring before the divergence of lotus, but not observable as a triplication in the lotus genome structure. A similar two-step model was sug- gested by Lyons et al. [30] on the basis of fractionation patterns seen in Vitis, and evidence for a two-step hexa- ploid process is clearly observed in the much more recent paleohexaploid Brassica rapa [31]. Additional whole plant genome sequences from lineages close to the g event, especially ones without the confounding effects of lineage-specific genome duplications, may also help to clarify genome-wide patterns of fractionation among the three g subgenomes, which could provide further evidence bearing on the timing and event(s) associated with the g paleohexaploidy event that is asso- ciated with what is arguably one of the most important radiations in angiosperm history. Discussion Paleopolyploids are widespread among eukaryotes and particularly common in angiosperms [14,15]. Lotus diverged from other eudicots early in eudicot history, prior to the g genome-triplication characteristic of most members of the group [14,15,17,26], and provides insight into the timing and nature of this event asso- ciated with a rapid radiation of the large eudicot lineages. When plant genomes of high paleopolyploidy levels are compared, differentiated gene loss (fractiona- tion) among several homologous subgenomes tends to diminish the signals of synteny. In such cases, genomes with few paleopolyploidy events (such as those of grape or papaya) can be used to take advantage of the smaller evolutionary distances between orthologous segments. Extensive collinearity within itself, as well as with other plant genomes such as those of Arabidopsis, grape, rice and sorghum, makes the lotus genome not only a eudi- cot evo-genomic reference (Figure S9 in Additional file 1), but also a better resource for reconstructing the pan- eudicot genome and facilitating comparative analysis between eudicots and monocots. The higher homeolog retention rate in lotus compared with most other genomes studied provided an opportu- nity to study subfunctionalization [32], a major driving force affecting fates of duplicated genes following paleo- polyploidy. Most pairs of lotus homeologs have no dif- ference in PFAM domain families, whereas 453 pairs (11.6%) differ by up to five domains. The unshared domains have mean length 17 amino acids with a range of 0 to 890 amino acids. Between homeologous lotus gene pairs, mRNA length (excluding 5′ and 3′ untrans- lated regions), coding sequence length, and intron length differences all follow geometric-like distributions (Figure S10 in Additional file 1), consistent with inde- pendent accumulation of small insertions and deletions. The changes of length in exonic and intronic regions seem uncorrelated, implying that subfunctionalization affects gene regulation at multiple transcriptional and post-transcriptional levels. Surprisingly, the phylogenomic analysis of gene families associated with the g include a substantial fraction of eudicot-wide duplications, suggesting the possibility of a two-step model that involved genetic material from a lineage that branched off earlier than the core eudicots (Figure 3A). A substantial fraction of eudicot-wide gene duplications was also observed in phylogenomic analyses that contained large collections of transcriptome data from early branching basal eudicots such as Platanus, Aquilegia and poppies [26]. Eudicot-wide duplications Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 9 of 11 Ming et al. Discussion Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 When divergence of lineages is followed by WGD, one predicts similar divergence of the paralogs in one spe- cies’ genome from a shared ortholog in the other spe- cies, confirmed in previous studies [16,33]. Comparison of paired l paralogs and their grape ortholog generally fit this prediction (Figure S11 in Additional file 1); how- ever, comparisons to cereal (sorghum) orthologs show consistent differentiation in branch lengths. This discre- pancy in the lotus-cereal comparison could be explained by fast evolutionary rates in cereal genomes and/or l being older than it appears, due to the slow Nelumbo evolutionary rate. Alternatively, this is also consistent with structural compartmentalization, with genes within the same genome undergoing different evolutionary tra- jectories [33]. Wider taxa sampling at neighboring branches will help better distinguish the possibilities. as rhizome development and flowering time. The assembly of the lotus genome is surprisingly high quality, largely due to the high level of homozygosity resulting from domesti- cation and vegetative propagation. The lotus genome has a lineage-specific WGD event that occurred about 65 MYA, but shows no structural evidence for the g hexaploid event shared among core eudicot species. The lotus genome has a 30% slower nucleotide mutation rate than that of grape, contributing in part to the outstanding genome assembly using next-generation sequencing technologies. Analysis of sequenced plant genomes yielded a minimum gene set for vascular plants of 4,223 genes. Strikingly, lotus has 16 COG2132 multi-copper oxidase family proteins with root- specific expression. COG2132 members are involved in root meristem phosphate starvation, reflecting lotus’ adap- tation to limited nutrient availability in an aquatic environ- ment. The slow nucleotide substitution rate and the lack of the triplication event make lotus genome an excellent reference for reconstructing the pan-eudicot genome and for accelerating comparative analysis between eudicots and monocots. The lotus genome will accelerate the iden- tification of genes controlling rhizome yield and quality, seed size and nutritional profile, flower morphology, and flowering time for crop improvement. The extraordinary seed longevity and vegetative propa- gation via rhizomes are likely the causes of the slow evolu- tionary rate in lotus. The ‘China Antique’ has a highly homozygous genome, yielding arguably the best assembled genome using next-generation sequencing technologies with pseudo-molecules proportional to its karyotype. Conclusions Sacred lotus has many unique biological features, most noticeable seed longevity and the lotus effect, in addition to its agricultural and medicinal importance. The purpose of sequencing the lotus genome is to facilitate research in these areas and on agronomic and horticultural traits such Materials and methods Illumina (Illumina HiSeq 2000) libraries were generated from purified N. nucifera ’China Antique’ nuclear DNA with inserts of 180 bp, 500 bp, 3.8 kb and 8 kb and assembled using ALLPATHS-LG. 454/Roche (GSFLX pyrosequencing platform) 20 kb mate pair reads were used for scaffolding. RNAseq data generated from various lotus tissues were used for annotation and RNAseq dif- ferential gene expression analysis using CLC Genomics Workbench 5.0 (CLC Bio, Aarhus, Denmark). MAKER version 2.22 was used in combination with the assembled RNAseq data to annotate 26,685 genes in the lotus gen- ome. Detailed methods for genome assembly, annotation and analyses are provided in Additional file 1. Sacred lotus is the first true aquatic plant to be sequenced and comparative genomics reveal unique gene family expansions that may have contributed to its adap- tations to an aquatic environment. Submersed soils are largely hypoxic and have a decreased reduction-oxidation potential, causing heavy metal precipitation and reduced nutrient availability. Lotus has a dramatic expansion of the COG2132 family, a group of multi-copper oxidases involved in phosphate starvation in root meristems. A role in root-specific processes is supported by the expression of these unique genes in root tissue. Adapta- tion to phosphate starvation can also be seen in an expansion of the UBC24 family and the miR399 family that regulates it. Lotus lacks four bHLH subfamilies involved in iron uptake and root hair and root meristem development, suggesting novel root growth and iron reg- ulation. These gene family expansions and preferential retention of duplicated genes reflect the challenges of aquatic growth. Data access The assembled N. nucifera genome was submitted to Gen- Bank (AQOG00000000; PID PRJNA168000, http://www. ncbi.nlm.nih.gov/Traces/wgs/?val=AQOG01). Whole gen- ome shotgun raw reads are deposited under SRA study: SRP021228 (http://trace.ncbi.nlm.nih.gov/Traces/sra/? study=SRP021228). The raw RNAseq data are deposited under BioProject 196884 (http://www.ncbi.nlm.nih.gov/ bioproject/196884). Discussion The lotus genome provides the foundation for revealing the molecular basis of its many distinguishing biological prop- erties, including seed longevity, adaptation to aquatic environment, the distinctive superhydrophobicity and self- cleaning property of its leaves, and the thermogenesis that is thought to enhance its pollination success. Authors’ details 1 Yang M, Han Y, VanBuren R, Ming R, Xu L, Han Y, Liu Y: Genetic linkage maps for Asian and American lotus constructed using novel SSR markers derived from the genome of sequenced cultivar. BMC Genomics 2012, 13:653. 6. Ming R, Hou S, Feng Y, Yu Q, Dionne-Laporte A, Saw JH, Senin P, Wang W, Ly BV, Lewis KL, Salzberg SL, Feng L, Jones MR, Skelton RL, Murray JE, Chen C, Qian W, Shen J, Du P, Eustice M, Tong E, Tang H, Lyons E, Paull RE, Michael TP, Wall K, Rice DW, Albert H, Wang ML, Zhu YJ, et al: The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus). Nature 2008, 452:991-996. 7. Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyere C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C, Alaux M, Di Gaspero G, Dumas V, et al: The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 2007, 449:463-467. 8. Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A, Schmutz J, Spannagl M, Tang H, Wang X, Wicker T, Bharti AK, Chapman J, Feltus FA, Gowik U, Grigoriev IV, Lyons E, Maher CA, Martis M, Narechania A, Otillar RP, Penning BW, Salamov AA, Wang Y, Zhang L, Carpita NC, et al: The Sorghum bicolor genome and the diversification of grasses. Nature 2009, 457:551-556. 9. Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA, Minx P, Reily AD, Courtney L, Kruchowski SS, Tomlinson C, Strong C, Delehaunty K, Fronick C, Courtney B, Rock SM, Belter E, Du F, Kim K, Abbott RM, Cotton M, Levy A, Marchetto P, Ochoa K, Jackson SM, Gillam B, et al: The B73 maize genome: complexity, diversity, and dynamics. Science 2009, 326:1112-1115. 10. Authors’ details 1 13Department of Chemistry and Biochemistry and Institute for Genomics and Proteomics, University of California, Los Angeles, 607 Charles E Young Drive East, CA 90095, USA. 14Department of Biology and Intercollege Graduate Program in Plant Biology, The Pennsylvania State University, 201 Life Sciences Building, University Park, PA 16802, USA. 15Center for Applied Chemical Biology, Department of Biological Sciences, Youngstown State University, 1 University Plaza, Youngstown, OH, 44555, USA. 16USDA-ARS, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA. 17Texas A&M AgriLife Research, Department of Plant Pathology & Microbiology, Texas A&M University System, 17360 Coit Road, Dallas, TX 75252, USA. 18Department of Biology, University of Central Oklahoma, 100 North University Drive, Edmond, OK 73034, USA. 19School of Earth and Environmental Sciences, University of Adelaide, North Terrace, Adelaide, 5005, Australia. 20Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550, Japan. 21Institute for Conservation Biology, The University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia. 22Department of Crop Sciences, University of Illinois at Urbana-Champaign, 1101 West Peabody Drive, Urbana, IL 61801, USA. 23Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO 63132, USA. 24Lawrence Berkeley National Laboratory, 1 Cyclotron Road Berkeley, Emeryville, CA 94720, USA. 25Institute of Developmental Biology and Molecular Medicine & School of Life Sciences, Fudan University, 220 Handan Road, Shanghai, 200433, China. 26Department of Biochemistry and Molecular Biology, 246 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA. 27Hawaii Agriculture Research Center, 94-340 Kunia Road, Waipahu, HI 96797, USA. 28Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI 96822, USA. 29Fujian Normal University, Qishan Campus, Minhou, Fuzhou, 350117, China. 30Department of Biology and Molecular Biology, Montclair State University, 1 Normal Avenue, Montclair, NJ 3. Duke JA, Bogenschutz-Godwin MJ, duCellier J, Duke AK: Handbook of Medicinal Herbs 2002, Boca Raton: CRC Press. 3. Duke JA, Bogenschutz-Godwin MJ, duCellier J, Duke AK: Handbook of Medicinal Herbs 2002, Boca Raton: CRC Press. 4. Diao Y, Chen L, Yang G, Zhou M, Song Y, Hu Z, Lin JY: Nuclear DNA C-values in 12 species in Nymphaeales. Caryologia 2006, 59:25-30. 4. Diao Y, Chen L, Yang G, Zhou M, Song Y, Hu Z, Lin JY: Nuclear DNA C-values in 12 species in Nymphaeales. Caryologia 2006, 59:25-30. 4. Diao Y, Chen L, Yang G, Zhou M, Song Y, Hu Z, Lin JY: Nuclea 5. Abbreviations bHLH: basic helix loop helix; bp: base pair; CRY: cryptochrome; EST: expressed sequence tags; MYA: million years ago; PPR: pentatricopeptide repeat-containing proteins; WGD: whole genome duplication. Authors’ contributions RM, RV, YL, MY, YH and S L designed research; RM, RV, YL, MY, YH, LTL, QZ, JEB, HT, EL, AAF, GN, DRN, CEBH, ARG, YJ, JPD, FZ, JH, XM, KAH, KI, SAR, MEH, QY, TCM, AC, YZ, RS, RJ, NC, JA, CMW, EW, AS, YH, LX, JZ, RP, MJH, WX, JAW, JW, MLW, YJZ, REP, ABB, CD, SRD, MAS, TPM, SPL, DRO, JWS, DRG, NJ, MY, CWD, SSM, AHP, BBB, SL and JSM performed research and analyzed data; RM, RV, JL, AHP, CEBH, JRW, KI, SAR, CWD, SSM and BBB wrote the paper. All authors read and approved the final manuscript. RM, RV, YL, MY, YH and S L designed research; RM, RV, YL, MY, YH, LTL, QZ, JEB, HT, EL, AAF, GN, DRN, CEBH, ARG, YJ, JPD, FZ, JH, XM, KAH, KI, SAR, MEH, QY, TCM, AC, YZ, RS, RJ, NC, JA, CMW, EW, AS, YH, LX, JZ, RP, MJH, WX, JAW, JW, MLW, YJZ, REP, ABB, CD, SRD, MAS, TPM, SPL, DRO, JWS, DRG, NJ, MY, CWD, SSM, AHP, BBB, SL and JSM performed research and analyzed data; RM, RV, JL, AHP, CEBH, JRW, KI, SAR, CWD, SSM and BBB wrote the paper. All authors read and approved the final manuscript. Received: 4 January 2013 Revised: 19 April 2013 Received: 4 January 2013 Revised: 19 April 2013 Acknowledgements Received: 4 January 2013 Revised: 19 April 2013 Accepted: 10 May 2013 Published: 10 May 2013 Accepted: 10 May 2013 Published: 10 May 2013 We thank K. Hasenstein for collection of the fruits of Nelumbo lutea. This project was supported by the University of California, Los Angeles (JSM); Wuhan Botanical Garden, Chinese Academy of Sciences, P.R. China (SL); and the University of Illinois at Urbana-Champaign (RM). Wuhan Botanical Garden, Chinese Academy of Sciences, P.R. China (SL); and the University of Illinois at Urbana-Champaign (RM). Additional material Additional file 1: Supplementary data, including detailed materials and methods, and supplementary tables S1-S13, and figures S1-S14. Page 10 of 11 Page 10 of 11 Page 10 of 11 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 07043, USA. 31Institute of Tropical Biosciences and Biotechnology, China Academy of Tropical Agricultural Sciences, 4 Xueyuan Road, Haikou, Hainan 571101, China. 32Department of Plant and Microbial Biology, University of California, 1 Shields Avenue, Davis CA, 95161, USA. 33Department of Cell and Developmental Biology, University of Illinois, 1201 West Gregory Drive, Urbana IL, 61801, USA. 34The Genome Analysis Center, Monsanto, St Louis, MO 63167, USA. 35Global Change and Photosynthesis Research Unit, Agricultural Research Service, United States Department of Agriculture, 1206 West Gregory Drive, Urbana, IL, USA. 36IGPP Center for the Study of Evolution and Origin of Life, Geology Building, Room 5676, University of California, Los Angeles, 595 Charles E Young Drive East, Los Angeles, CA 90095-1567, USA. 37Department of Plant and Microbial Biology, University of California, 411 Koshland Hall, Berkeley, CA 94720, USA. 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https://openalex.org/W4310089689	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0277520&type=printable	English	null	Characterization and prediction of gambling behavior in adolescents using the COM-B model	PloS one	2,022	cc-by	7,195	Characterization and prediction of gambling behavior in adolescents using the COM-B A´ lvaro Botella-GuijarroID1,2☯, Daniel Lloret-Irles2☯, Jose´ Vicente Segura-Heras3☯, Juan A. Moriano-Leo´nID1☯ 1 Department of Social and Organizational Psychology, Universidad Nacional de Educacio´n a Distancia (UNED), Madrid, Spain, 2 Health Psychology Department, Universidad Miguel Herna´ndez de Elche, Elche, Spain, 3 Operations Research Center, Universidad Miguel Herna´ndez de Elche, Elche, Spain a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. daniel.lloret@umh.es Editor: Marc N. Potenza, Yale University, UNITED STATES Received: October 8, 2021 Accepted: October 28, 2022 Published: November 28, 2022 Received: October 8, 2021 Accepted: October 28, 2022 Published: November 28, 2022 Copyright: © 2022 Botella-Guijarro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Botella, A´lvaro; LLoret, Daniel; Segura-Heras, Jose´ V.; Moriano Leo´n, Juan Antonio (2022), “Characterization and prediction of gambling behavior in adolescents using the COM-B model”, Mendeley Data, V2, doi: 10.17632/fsd2cgcmc8.2. PLOS ONE RESEARCH ARTICLE Abstract Gambling is an international phenomenon, posing a serious threat to adolescents who begin gambling at a young age. This study aims, to explore gambling behavior in adoles- cents and interpret its risk factors. We conducted a three-waves cohort longitudinal study assessing gambling and associated risk factors in south-eastern Spain. Data were analyzed using the Capabilities, Opportunities, Motivations, Behavior (COM-B) model and the partial least squares path modelling (PLS-PM) technique. Gambling was measured by frequency and money spent; associated factors were knowledge about gambling, parental attitude towards gambling, risk perception, normative perception, and intention to gamble. These items were assigned as indicators of each construct of the COM-B model—capability, opportunity, motivation, and behavior—using the theoretical domains framework. Once the behavior was performed, feedback on future capability, opportunity, and motivation was observed. Results show that capability, determined by past experience, and opportunity, determined by parental attitudes, motivates adolescents to seek gambling experiences in the future. Identifying such factors that affect gambling behavior in adolescents and estab- lishing relationships between them through a robust theoretical model is essential for designing effective interventions. OPEN ACCESS Citation: Botella-Guijarro A´, Lloret-Irles D, Segura- Heras JV, Moriano-Leo´n JA (2022) Characterization and prediction of gambling behavior in adolescents using the COM-B model. PLoS ONE 17(11): e0277520. https://doi.org/ 10.1371/journal.pone.0277520 Citation: Botella-Guijarro A´, Lloret-Irles D, Segura- Heras JV, Moriano-Leo´n JA (2022) Characterization and prediction of gambling behavior in adolescents using the COM-B model. PLoS ONE 17(11): e0277520. https://doi.org/ 10.1371/journal.pone.0277520 Editor: Marc N. Potenza, Yale University, UNITED STATES PLOS ONE PLOS ONE Introduction The onset of gambling at an early age increases the risk of developing problem gambling in adulthood, according to the categorization obtained by the PGSI [7–9]. Therefore, it is impor- tant to develop interventions aimed at adolescents and young adults that reduce or prevent gambling attitudes. Identifying the factors that affect gambling behavior and establishing rela- tionships between them through a robust theoretical model is essential for designing effective interventions for the prevention of problem gambling in adolescents [10]. There is grounded evidence that identifies factors related to the onset and maintenance of gambling among adolescents. On the individual level, low risk perception has been associated with a higher frequency and intensity of gambling [11, 12]. While adolescents understand that gambling involves risk, they do not see themselves as potential victims [13]. Another individ- ual factor that predicts gambling intention and behavior is perceived control, based on cogni- tive biases where an individual believes that it is possible to control chance [14, 15]. Once initiated, gambling behavior has a high continuation rate [11, 16, 17]. At the family level, there is strong evidence linking parental attitudes and behavior towards gambling with higher fre- quency of offspring gambling [18–20]. Similarly, poor, or negligent parental supervision is a common variable for many risky behaviors, including gambling [21]. From a micro-social perspective [22], the normative perception, understood as the belief that gambling behavior is normal in adolescence, maintains a directly proportional relation- ship with the frequency of gambling [23, 24]. Risk factors acquire an even greater significance when they are integrated into a theoretical model. In this regard, the above factors are represented in the Capabilities, Opportunities, Motivations, Behavior (COM-B) model [25, 26], which postulates a general framework for behavioral change. The model predicts behavior by combining three parameters (see Fig 1)— capability (psychological or physical capability, for example, knowledge and skills), opportunity (physical and social environment, factors external to the individual), and motivation (reflexive and automatic cognitive processes)—that interact to generate behavior. Motivation also serves as a central mediator between capability, opportunity, and behavior, as both capability and opportunity influence motivation. Therefore, capability and opportunity affect behavior through direct and indirect pathways [25–27]. Introduction One in five European teenagers are involved in gambling. According to reports from the Euro- pean School Survey Project on Alcohol and Other Drugs (ESPAD), the proportion of 15– 16-year-olds who have gambled has changed in recent years, from 14% having gambled at least once in their lifetime in 2015 to 22% having gambled in the last 12 months in 2019. Although the measure has changed between the two waves of data, and interpretation should be made with caution, we can conclude that gambling has become more prevalent among the European population aged 15 to 16. In addition, the ESPAD report classifies 15% as excessive Funding: JVS-H is supported by the Generalitat Valenciana under project PROMETEO/2021/063. DLI grant no. GV/2020/163. Direccio´n General de Ciencia e Investigacio´n, Conselleria de Innovacio´n, Universidad, Ciencia y Sociedad Digital, Generalitat PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 1 / 13 PLOS ONE Gambling behaviour in adolescents using the COM-B model gamblers and 5% as problem gamblers. The classifications of excessive gambler is based on the Consumption Screen for Problem Gambling (CSPG) scale, adapted from Rockloff (2012), and those of problem gambler on the Lie/Bet Questionnaire, adapted from Johnson (1997) [1, 2]. Studies on gambling behavior during the COVID-19 pandemic’s mobility restrictions in Spain showed a reduction in gambling in venues and an increase in online gambling among the pop- ulation under 25 years of age [3]. In a survey conducted by the Australian Gambling Research Centre during COVID-19, men aged 18–34 were found to be most likely to sign up for online accounts, increase their frequency and monthly expenditure on gambling, and face a higher risk of gambling-related harm, according to the classification provided by the Problem Gam- bling Severity Index (PGSI), a proxy measure for gambling disorder [4]. Based on estimates by the National Opinion Research Center DSM Screen for Gambling Problems NODS and DSM-IV-MR-J screen, several authors have argued that gambling is a public health issue because of the increase in cases of gambling disorder especially among adolescents [5, 6]. Valenciana (Spain). https://innova.gva.es/es. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 Introduction West and Michie [28] reformulated the model in such a way that capability and opportunity act on the relationship between motivation and behavior as ‘logic gates’ and that, at a given point in time, both capability and opportunity must be open for motivation to be expressed in behavior. Ultimately, behavior feeds back into capa- bility, opportunity, and motivation. This feedback can create both positive and negative cycles, increasing or decreasing, respectively, the likelihood of the behavior occurring again [28]. Therefore, the model is structured temporally, on three phases: a first phase in which capabil- ity, opportunity, and motivation are measured; a second phase in which the execution of the 2 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 PLOS ONE Gambling behaviour in adolescents using the COM-B model Fig 1. COM-B model. Adapted from West & Michie [28]. https://doi.org/10.1371/journal.pone.0277520.g001 Fig 1. COM-B model. Adapted from West & Michie [28]. https://doi.org/10.1371/journal.pone.0277520.g001 https://doi.org/10.1371/journal.pone.0277520.g001 behavior is measured; and a third phase in which capability, opportunity, and motivation are measured, and the possible feedback of the model is assessed. Given the large number of existing theories of behavioral change and the numerous derived constructs, an integrative framework was designed to synthesize the various key theoretical constructs–the theoretical domains framework (TDF). The TDF synthesizes 33 theories and 128 theoretical constructs on behavioral change into 14 constructs [25, 29]. The framework serves as an aid for assigning each risk factor to each of the constructs of the COM-B model. This model can help clarify gambling behavior in context [25, 27], diagnose which variables need to change to achieve the desired behavior, and inform the design of interventions for the prevention of abusive gambling in adolescents. In the field of addictive behaviors, the COM-B model has been shown to be useful for understanding the changes needed to prevent the consumption of alcohol [30, 31] or tobacco [32, 33]. Based on these results, we consider that the model can also be applied to other poten- tially addictive behaviors. As far as gambling is concerned, to our knowledge, no study has used the COM-B model to analyze predictors of gambling behavior among adolescents. The only reference we have been able to find is McMahon et al. [34], who used the model to imple- ment changes in a harm reduction intervention for gambling behavior in adults. PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 Introduction Considering this evidence, the COM-B model is a promising framework for explaining potentially addictive behaviors. Therefore, the aim of the present study is to understand gam- bling behavior in adolescents and interpret risk factors according to the COM-B model. To apply the sequential logic of the model in its three measurement phases, we used a longitudinal study with three waves of data on gambling behavior. 3 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 PLOS ONE Gambling behaviour in adolescents using the COM-B model The data obtained through the COM-B model was analyzed through partial least squares path modelling (PLS-PM), a technique that allows us to analyze relationships between groups of variables. This data analysis approach has not been widely used in the study of the COM-B model or the prevention of gambling behavior in adolescents; therefore, it is a novel contribution of the present work. Some examples of the use of PLS and structural equations (SEM) can be found in Howlett et al. [35], applied to physical activity, and in Zou et al. [36], related to self-care in patients with chronic heart failure. The application of the model in the field of adolescent gam- bling behavior is another novel aspect of this research. Participants The sample was recruited in three consecutive years (2016-2017-2018) from 15 randomly selected public educational centers in south-eastern Spain. Seven of the educational centers are in municipalities with less than 50,000 inhabitants and eight with more than 50,000 inhabi- tants. The main economic activities of these municipalities are agriculture (four centers), industry (five centers) and service-tourism sector (six centers). In the first measurement (T1) the sample consisted of 2716 students in 9th, 10th, and 11th grades, with a mean age of 15.12 years (SD = 1.03. Range 13–17) and 49.9% female. At the sec- ond measurement (T2), 2430 students from 10th, 11th, and 12th grades participated, mean age 16.07 (SD = 0.99. Range 14–19), 54.8% female. At the third measurement (T3), the sample con- sisted of 1726 students in 11th and 12th grades, with a mean age of 16.52 (SD = 0.76. Range 15–20), 54.0% female. The matched sample of the three points in time– T1-T2-T3 –was com- posed of 391 students, out of which 55.12% were girls. During the follow-up, there were sample losses (with respect to T1,65.72% in T1 and 85.60% in T2) due to: (1) the impossibility of mea- suring pupils who changed schools or municipalities in the following academic year, after completing compulsory education; (2) failure to obtain parental authorization and/or absen- teeism; and (3) errors or falsification in the anonymous coding of the participants. Cases aged between 14 and 16 years inclusive, at the time of measurement T1, were selected for the present study, resulting in a sample of 354 cases, 52.54% female. Of these cases, 20.34% have gambled once in the year at T2 (2.82% female), and 4.24% are at-risk or problem gam- blers (0.28% female), according to the categorization obtained by the South Oaks Gambling Scale-RA (SOGS-RA). PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 Measures The COM-B model consists of four constructs: capability, opportunity, motivation, and behav- ior. The measurement items for each component of the model were selected as follows. Capa- bility was assessed by measuring the frequency of gambling in the last 12 months. It was derived from training or practice, within the theoretical domain “Skills” as proposed in the Theoretical Domains Framework [25, 29]. This training or practice was understood in terms of frequency of gambling in the last year, as the history of past gambling behavior is shown to be a risk factor for current gambling behavior [8, 11, 16, 17, 37]. The more you have played in the last year, the more capability to play you acquire. The frequency variable was measured through four items collected and adapted from the European School Survey Project on Alcohol and other Drugs questionnaire (ESPAD), which asked about the number of times each of the five gambling modalities had been used: online sports betting, in-room sports betting, in- room slot machines, online poker or casino games, and in-room roulette. Another theoretical domain related to the capability component of the COM-B model is “Knowledge”. This domain was assessed through four items of the accessibility (AC) subscale of the Early Detec- tion Gambling Addiction Risk-Adolescents (EDGAR-A) battery, (Cronbach’s alpha = 0.74) [38], which informs us about the degree of knowledge about where and how to gamble. Opportunity consisted of seven items from the Parental Attitude (PA) subscale–parental attitude towards gambling–of the EDGAR-A battery, with a Cronbach’s alpha representing internal consistency of .86. The higher the score, the more favorable the parents were towards gambling. Motivation was constructed using 13 Likert-type items selected from the EDGAR-A sub- scales: seven items from risk perception (Cronbach’s alpha = 0.79), three items from normative perception (Cronbach’s alpha = 0.75), and three items from intention to gamble (Cronbach’s alpha = .84). Gambling behavior included two items: the maximum amount of money gambled in the last 12 months, with 7 answering options (from 0 = €0 to 6 = more than €70); and the score on the South Oaks Gambling Scale-RA (SOGS-RA) [39, 40], composed of 12 dichotomous items and one Likert-type item (Cronbach’s alpha = 0.73). Procedure After obtaining authorization from the competent educational authority, the study was approved by the Ethics Committee of the Miguel Herna´ndez University (DPS.DLI.01.17). First, 30 schools were identified, 9 of which declined to participate for various reasons. Of the remaining, 15 were chosen at random, meeting the criterion of representation of all the regions (2 schools per region). In regions with more than two centers, the schools were selected at ran- dom. All the required classes within each school were included. All measures were integrated into a single questionnaire. Each of the students present in the classroom, from the selected schools and courses, completed the questionnaire. In each session, an expert collaborator from the research team distributed the questionnaire and clarified any doubts and questions. The sessions were supervised by a teacher designated by the center. Informed consent was obtained from the parents or guardians of the participants. Adolescents participated voluntarily after being informed of the purpose of the study. The duration of each session was 25–30 minutes. 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 PLOS ONE Gambling behaviour in adolescents using the COM-B model Measures Based on frequently used cutoff scores for the SOGS-RA, non-problem gambling was defined as scores of 0 and 1, at-risk individuals had scores of 2 and 3, and problem gambling was defined as scores of 4 or above [41]. The variables related to the capability, opportunity, and motivation constructs were assessed at time T1 and T3, while those related to gambling behavior were measured at time T2. PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 Statistical analysis The partial least squares (PLS) technique was employed to obtain estimations and fit indices for the proposed model in the context of the structural equation model (SEM) using R software [42], SEMinR package, version 2.1.0 [43]. Before running the analysis, the data distributions were verified for anomalies such as outliers, missing values, and deviation from normality. The relationships between the groups of variables are established by considering the theory, or prior knowledge, regarding the phenomenon under analysis. Furthermore, we assumed that each group of variables plays the role of a theoretical concept represented in the form of a construct or latent variable [44–46]. The PLS-PM analysis also allows for more flexibility in the assumptions on the distribution of data and the minimum sample size. This technique allows for a choice between the formative and the reflective measures of the constructs. In the formative measures, each item represents a dimension of the construct’s meaning; thus, removing an item signifies that the construct loses some of its meaning. In the reflective measures, items represent manifestations of the construct and are competitive with PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 5 / 13 PLOS ONE Gambling behaviour in adolescents using the COM-B model each other. In the present study, we opted for a formative model of measurement for all con- structs [47, 48], in which the measurement items “cause”, or are antecedents of, the theoretical constructs. Hair, J.R. et al. [49] combine the methodological concepts of this technique in a single text with the use of this R package as a method for estimating PLS path models. The proposed model was fitted in its entirety, starting from the aforementioned items as the outer model. This was done so that, in the case of the motivation construct, the items com- prising the variables risk perception, normative perception, and intention to gamble would form the measurement model of the latent variable motivation, without interposing the former three variables as intermediate latent variables between the items and the constructs of the COM-B model (capability, motivation, opportunity, and behavior). The structural model was also adjusted (inner model, path model between the constructs) to obtain the weight of each relationship. The goodness-of-fit indices were obtained for the mean model and the structural model. The model obtained was evaluated and an alternative model was adjusted following the same procedure. Hair et al. Statistical analysis [44] considered that the evaluations of formative measurement models include three aspects: convergent validity, the evaluation of collinearity issues, as well as the evaluation of the significance and relevance of the indicators. For the evaluation of structural models, it is recommended to use the coefficients of determination (R2), the size and significance of the path coefficients, and the effect sizes (f2). The goodness-of-fit indices of the two models were compared. Results A first model was estimated, from which items with collinearity issues (variance inflation fac- tor [VIF] greater than 3.3) were removed, namely, those measuring opportunity OPPT1.4, OPPT1.5, OPPT3.4, and OPPT3.5. Fig 2 shows the resulting structural model containing the algebraic sign, magnitude, and statistical significance of the standardized regression coefficients (path coefficients). Their magnitudes ranged from 0.247 to 0.558, all being statistically significant, based on the boot- strapping process performed. The R2 obtained was generally moderate, except for the one associated with opportunity at T3, which was weak. Capability and opportunity at T1 explained 54.6% and 24.7% of motiva- tion at T1, respectively. Motivation at T1 explained 55.8% of behavior at T2, the latter explain- ing 59.3% of motivation, 62.3% of capability, and 29.5% of opportunity at T3. Cohen’s f2 assesses whether the omitted construct has a substantive impact on the endoge- nous constructs: 0.02 would be associated with a small effect; 0.15 with a medium effect; and 0.35 with a large effect. In our case, opportunity had a small effect on motivation at T1, as did behavior on motivation at T3. A more adjusted model was tested, eliminating those measurement items with non-signifi- cant weights, without the model losing quality. The adjusted model is shown in Fig 3. The items OPPT1.7, OPPT3.7, CAPT1.3, CAPT1.8, CAPT3.3, CAPT3.8, MOTT1.8, MOTT1.10, MOTT1.13, MOTT3.8, MOTT3.10 and MOTT3.13 were removed; thus, we obtained a more parsimonious model without losing the quality of fit in the structural model. The evaluation indices of the two models are listed in Tables 1 and 2. The evaluation indices of the two models are listed in Tables 1 and 2. PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 Discussion The aim of the present study was to understand gambling behavior in adolescents and to inter- pret risk factors according to the COM-B model. For this purpose, a sample of adolescents was 6 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 PLOS ONE Gambling behaviour in adolescents using the COM-B model Fig 2. Initial model. The measurement model (rectangles) and the structural model (hexagons) are shown. Factor loadings for each indicator (w), standardized regression coefficients or path coefficients (β) with confidence intervals and coefficients of determination (R2) are indicated. The greater the thickness of the arrow, the greater the loading or path coefficient. https://doi.org/10.1371/journal.pone.0277520.g002 Fig 2. Initial model. The measurement model (rectangles) and the structural model (hexagons) are shown. Factor loadings for each indicator (w), standardized regression coefficients or path coefficients (β) with confidence intervals and coefficients of determination (R2) are indicated. The greater the thickness of the arrow, the greater the loading or path coefficient. https://doi.org/10.1371/journal.pone.0277520.g002 https://doi.org/10.1371/journal.pone.0277520.g002 observed for three consecutive years with respect to their gambling behavior and other related variables. As they continue gambling, their capability will increase, they will possibly be more moti- vated to gamble again (motivation) and think that their parents have a favorable attitude towards gambling behavior (opportunity) and other related variables. The results showed that capability, understood as knowledge and experience about gam- bling, is a predictor of motivation to gamble, and consequently, of gambling. These findings are consistent with previous studies where experience leads to gamble in a significant propor- tion of adolescents [17, 50–52]. Marketing strategists in the service of the gaming industry know that the likelihood of gambling increases with prior experience. A worrying approach is the marketing strategy of offering simulated gambling scenarios that reproduces “real” gam- bling activities, which enables the users to gamble without investing money. Simulated gam- bling in social networks or on Internet gaming platforms, fosters the onset of gambling and promotes an increasing worry regarding adolescents [53, 54]. Therefore, gamble experience, or capability, acts in a self-feeding spiral, in which more gambling experiences will increase their capability and, possibly, motivation to gamble again. 7 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 PLOS ONE Gambling behaviour in adolescents using the COM-B model Fig 3. Adjusted model. The measurement model (rectangles) and the structural model (hexagons) are shown. Discussion Factor loadings for each indicator (w), standardized regression coefficients or path coefficients (β) with confidence intervals and coefficients of determination (R2) are indicated. The greater the thickness of the arrow, the greater the loading or path coefficient. Fig 3. Adjusted model. The measurement model (rectangles) and the structural model (hexagons) are shown. Factor loadings for each indicator (w), standardized regression coefficients or path coefficients (β) with confidence intervals and coefficients of determination (R2) are indicated. The greater the thickness of the arrow, the greater the loading or path coefficient. https://doi.org/10.1371/journal.pone.0277520.g003 The opportunity parameter of the COM-B model, referring to a social or physical environ- ment which encourages gambling, was represented by the favorable attitude of parents toward gambling in our study. The results establish that motivation to gamble increases when parents do not convey an anti-gambling attitude, with a significant but small effect. These findings are supported by previous studies, where home characteristics, particularly parental attitude, are linked to a higher likelihood of gambling among their offspring [55]. Although the questionnaire used was not expressly designed to use the COM-B model, the results obtained were acceptable. For example, St-Pierre et al. [56] found that a model based Although the questionnaire used was not expressly designed to use the COM-B model, the results obtained were acceptable. For example, St-Pierre et al. [56] found that a model based Table 1. Determination coefficient (R2) y Coef. path. Initial model (adjusted model). Mot.T1 Behavior Mot.T3 Cap.T3 Opp.T3 R2 0.432 (0.424) 0.311 (0.295) 0.352 (0.335) 0.389 (0.373) 0.087 (0.086) Adj. R2 0.429 (0.421) 0.309 (0.293) 0.350 (0.334) 0.387 (0.372) 0.085 (0.084) Capability.T1 0.546 (0.546) Opportunity.T1 0.247 (0.232) Motivation.T1 0.558 (0.543) Behavior 0.593 (0.579) 0.623 (0.611) 0.295 (0.294) Note: R2 is the determination coefficient; Adj. R2, the adjusted determination coefficient. For Motivation in T1 (Mot. T1), Behavior, Motivation in T3 (Mot.T3), Capability in T3 (Cap.T3) and Opportunity in T3 (Opp.T3). Without parentheses: initial model, in parentheses: adjusted model. The rest are path coefficients between correlated constructs. https://doi org/10 1371/journal pone 0277520 t001 Table 1. Determination coefficient (R2) y Coef. path. Initial model (adjusted model). Note: R2 is the determination coefficient; Adj. R2, the adjusted determination coefficient. For Motivation in T1 (Mot. T1), Behavior, Motivation in T3 (Mot.T3), Capability in T3 (Cap.T3) and Opportunity in T3 (Opp.T3). Without parentheses: initial model, in parentheses: adjusted model. The rest are path coefficients between correlated constructs. https://doi.org/10.1371/journal.pone.0277520.t001 PLOS ONE Gambling behaviour in adolescents using the COM-B model Table 2. Cohen’s f2. Initial model (Adjusted model). Mot.T1 Behavior Mot.T3 Cap.T3 Opp.T3 Capability.T1 0.413 (0.414) Opportunity.T1 0.080 (0.072) Motivation.T1 0.452 (0.418) Behavior 0.543 (0.505) 0.636 (0.596) 0.095 (0.095) Note: Cohen’s f2 for Motivation in T1 (Mot. T1), Behavior, Motivation in T3 (Mot.T3), Capability in T3 (Cap.T3) and Opportunity in T3 (Opp.T3). Without parentheses: initial model, in parentheses: adjusted model. https://doi.org/10.1371/journal.pone.0277520.t002 on an extension of the Theory of Planned Behavior (TPB) using SEM explained 29.8% of the variance in the frequency of gambling in the last three months and 28.5% of the intention to gamble. Other studies found that the Theory of Reasoned Action only predicted small amounts of variance in gambling intentions [57]. In this sense, the application of the COM-B model and the PLS-SEM technique to adolescent gambling is a novelty that improves the explanatory capacity of the indicators used, since this model model predicts behavioral performance related to the circumstances, that is, the constructs of capability, motivation, and opportunity, that may control it in the future. y The capability construct has the greatest explanatory weight for motivation, which can be understood as a reflection of the importance of past experiences, as well as autonomous actions, that strengthen intrinsic motivation in terms of intentions. Capability is derived from Bandura’s concept of self-efficacy. It can also be regarded from the perspective of the Theory of Planned Behavior (TPB) which includes “perceived behavioral control” referring to the con- viction that one can successfully gamble and attain a desired goal. The construct opportunity had little weight in the estimated model. This may be due to the lack of important indicators for the formation of the construct, which are detailed in the limi- tations of the study below. y While the longitudinal design provides robustness to the study, 12 months may be a long period to examine the effects of capability, motivation, and opportunity on gambling behavior. Given the formative nature of construct development, it would be advisable to carefully select the most appropriate indicators of each construct for a particular intervention study and justify their selection based on sound explanations supported by empirical evidence. For example, in this study, the construct opportunity suffered from the absence of indicators related to accessi- bility or peer group gambling behavior, which are important factors in explaining adolescent gambling [17, 51, 58]. Discussion PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 References 1. ESPAD Group. ESPAD Report 2019: Results from the European School Survey Project on Alcohol and Other Drugs. Luxembourg: European Monitoring Centre for Drugs and Drug Addiction; 2020. 2. Group ESPAD. ESPAD report 2015: results from the European school survey project on alcohol and other drugs. Luxembourg: European Monitoring Centre for Drugs and Drug Addiction; 2016. 3. Observatorio Español sobre Adicciones. Informe COVID-19, consumo de sustancias psicoactivas y adicciones. 2020 [cited 2020 Jul 23]. Available from: https://pnsd.sanidad.gob.es/fr/noticiasEventos/ actualidad/2020_Coronavirus/pdf/20200715_Informe_IMPACTO_COVID-19_OEDA_final.pdf 4. Jenkinson R, Sakata K, Khokhar T, Tajin R, Jatkar U. Australian Gambling Research Centre. Gambling in Australia during COVID-19. 2020. 5. Armitage R. Gambling among adolescents: an emerging public health problem. The Lancet Public Health. 2021 Mar 1; 6(3):e143. https://doi.org/10.1016/S2468-2667(21)00026-8 PMID: 33640074 6. Cho´liz M, Lamas J. ¡Place your bets, children!; The frequency of gambling among minors and their rela- tionship with gambling addiction indicators. Revista española de drogodependendencias. 2017; 42 (1):34–7. 7. Dussault F, Dufour M, Brunelle N, Tremblay J, Rousseau M, Leclerc D, et al. Consistency of Adoles- cents’ Self-Report of Gambling Age of Onset: A Longitudinal Study. J Gambl Stud. 2019 Jun 18; 35 (2):533–44. https://doi.org/10.1007/s10899-019-09834-3 PMID: 30778812 8. el Guebaly N, Casey DM, Currie SR, Hodgins DC, Schopflocher DP, Smith GJ, et al. The Leisure, Life- style, & Lifecycle Project (LLLP): A Longitudinal Study of Gambling in Alberta. Final Report for the Alberta Gambling Research Institute. 2015. 1. ESPAD Group. ESPAD Report 2019: Results from the European School Survey Project on Alcohol and Other Drugs. Luxembourg: European Monitoring Centre for Drugs and Drug Addiction; 2020. 2. Group ESPAD. ESPAD report 2015: results from the European school survey project on alcohol and other drugs. Luxembourg: European Monitoring Centre for Drugs and Drug Addiction; 2016. 3. Observatorio Español sobre Adicciones. Informe COVID-19, consumo de sustancias psicoactivas y adicciones. 2020 [cited 2020 Jul 23]. Available from: https://pnsd.sanidad.gob.es/fr/noticiasEventos/ actualidad/2020_Coronavirus/pdf/20200715_Informe_IMPACTO_COVID-19_OEDA_final.pdf 4. Jenkinson R, Sakata K, Khokhar T, Tajin R, Jatkar U. Australian Gambling Research Centre. Gambling in Australia during COVID-19. 2020. 5. Armitage R. Gambling among adolescents: an emerging public health problem. The Lancet Public Health. 2021 Mar 1; 6(3):e143. https://doi.org/10.1016/S2468-2667(21)00026-8 PMID: 33640074 6. Cho´liz M, Lamas J. ¡Place your bets, children!; The frequency of gambling among minors and their rela- tionship with gambling addiction indicators. Revista española de drogodependendencias. 2017; 42 (1):34–7. 7. Dussault F, Dufour M, Brunelle N, Tremblay J, Rousseau M, Leclerc D, et al. PLOS ONE And that the relationships between capability and behavior are biased, since the frequency of play has been included as a measure of the capability construct, because of the possible improvement in the skins to gambling, but this variable is still gambling behav- ior (the result) and, consequently, produces biases in the relationships between capability and behavior. In this sense, it is advisable to use qualitative techniques and follow the tools pro- posed for the design of interventions with the COM-B model [25, 27]. In general, results should be interpreted considering the limitations of self-report and cohort studies, including potential biases and participant attrition between measurements [17, p. 14]. In addition, it would have been desirable to include children who were not in the regular education system. Although they are a minority, their unique socio-demographic characteristics would enrich the results. In this sense, it should be considered that the sample loss of the study may have left out a certain adolescent population group, which partly reduces the generalizability of the results. Finally, the drawbacks of the concentrated location of the sample must be considered, and caution is required when generalizing the results. 9 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0277520 November 28, 2022 PLOS ONE Gambling behaviour in adolescents using the COM-B model Our results support the use of the COM-B Model as a framework to predict adolescent gambling behavior. The weight of capability in gambling motivation proves that those vari- ables should be considered in preventive interventions that aim to delay the onset of early gam- bling experiences and thus reduce the likelihood of more severe gambling behavior. Considering the ages of the participants in the study, it is suggested that prevention of gam- bling should start before the age of onset. The worrying prevalence of gambling among adoles- cents highlights the need for evidence-based preventive educational interventions. Future research should examine the interaction between indicators related to opportunity, that is, accessibility or peer group gambling behavior that could predict adolescent gambling behavior. Author Contributions Conceptualization: A´lvaro Botella-Guijarro, Daniel Lloret-Irles, Jose´ Vicente Segura-Heras. Data curation: A´lvaro Botella-Guijarro. Data curation: A´lvaro Botella-Guijarro. Formal analysis: Jose´ Vicente Segura-Heras. Formal analysis: Jose´ Vicente Segura-Heras. Funding acquisition: Daniel Lloret-Irles, Jose´ Vicente Segura-Heras. Funding acquisition: Daniel Lloret-Irles, Jose´ Vicente Segura-Heras. Methodology: Daniel Lloret-Irles, Juan A. Moriano-Leo´n. Project administration: Juan A. Moriano-Leo´n. Supervision: Juan A. Moriano-Leo´n. Visualization: Daniel Lloret-Irles, Jose´ Vicente Segura-Heras, Juan A. Moriano-Leo´n. Writing – original draft: A´lvaro Botella-Guijarro, Daniel Lloret-Irles, Jose´ Vicente Segura- Heras. Writing – review & editing: A´lvaro Botella-Guijarro, Daniel Lloret-Irles, Juan A. Moriano- Leo´n. References Consistency of Adoles- cents’ Self-Report of Gambling Age of Onset: A Longitudinal Study. J Gambl Stud. 2019 Jun 18; 35 (2):533–44. https://doi.org/10.1007/s10899-019-09834-3 PMID: 30778812 8. el Guebaly N, Casey DM, Currie SR, Hodgins DC, Schopflocher DP, Smith GJ, et al. The Leisure, Life- style, & Lifecycle Project (LLLP): A Longitudinal Study of Gambling in Alberta. 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https://openalex.org/W4205373746	https://europepmc.org/articles/pmc8835800?pdf=render	English	null	Gal3 Plays a Deleterious Role in a Mouse Model of Endotoxemia	International journal of molecular sciences	2,022	cc-by	23,152	Article Gal3 Plays a Deleterious Role in a Mouse Model of Endotoxemia Juan Carlos Fernández-Martín 1,2, Ana María Espinosa-Oliva 1,2,* , Irene García-Domínguez 1,2, Isaac Rosado-Sánchez 2, Yolanda M. Pacheco 2, Rosario Moyano 3, José G. Monterde 4 , José Luis Venero 1,2,† and Rocío M. de Pablos 1,2,† 1 Departamento de Bioquímicay Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain; jcfermar3@gmail.com (J.C.F.-M.); irenegarcia391@gmail.com (I.G.-D.); jlvenero@us.es (J.L.V.); depablos@us.es (R.M.d.P.) 1 Departamento de Bioquímicay Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain; jcfermar3@gmail.com (J.C.F.-M.); irenegarcia391@gmail.com (I.G.-D.); 1 Departamento de Bioquímicay Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain; jcfermar3@gmail.com (J.C.F.-M.); irenegarcia391@gmail.com (I.G.-D.); jlvenero@us.es (J.L.V.); depablos@us.es (R.M.d.P.) , p ; j g (J ); g g ( ); jlvenero@us.es (J.L.V.); depablos@us.es (R.M.d.P.) p j g jlvenero@us.es (J.L.V.); depablos@us.es (R.M.d.P.) 2 Instituto of Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío (HUVR)/CSIC/Universidad de Sevilla, 41012 Sevilla, Spain; Isaac.RosadoSanchez@bcchr.ca (I.R.-S.); ypacheco-ibis@us.es (Y.M.P.) 3 Departamento de Farmacología, Toxicología, Medicina Legal y Forense, Universidad de Córdoba, 14014 Córdoba, Spain; r.moyano@uco.es 4 Departamento de Anatomía y Anatomía Patológica Comparadas, Universidad de Córdoba, 14014 Córdoba, Spain; jg.monterde@uco.es , p jg * Correspondence: anaespinosa@us.es; Tel.: +34-954-559-846; Fax: +34-954-556-854 * Correspondence: anaespinosa@us.es; Tel.: +34-954-559-846; Fax: +34-9 † Jose Luis Venero and Rocío M. de Pablos shared senior authorship. Abstract: Lipopolysaccharide (LPS)-induced endotoxemia induces an acute systemic inﬂammatory response that mimics some important features of sepsis, the disease with the highest mortality rate worldwide. In this work, we have analyzed a murine model of endotoxemia based on a single intraperitoneal injection of 5 mg/kg of LPS. We took advantage of galectin-3 (Gal3) knockout mice and found that the absence of Gal3 decreased the mortality rate oﬂethal endotoxemia in the ﬁrst 80 h after the administration of LPS, along with a reduction in the tissular damage in several organs measured by electron microscopy. Using ﬂow cytometry, we demonstrated that, in control conditions, peripheral immune cells, especially monocytes, exhibited high levels of Gal3, which were early depleted in response to LPS injection, thus suggesting Gal3 release under endotoxemia conditions. However, serum levels of Gal3 early decreased in response to LPS challenge (1 h), an indication that Gal3 may be extravasated to peripheral organs. Indeed, analysis of Gal3 in peripheral organs revealed a robust up-regulation of Gal3 36 h after LPS injection. Citation: Fernández-Martín, J.C.; Espinosa-Oliva, A.M.; García-Domínguez, I.; Rosado-Sánchez, I.; Pacheco, Y.M.; Moyano, R.; Monterde, J.G.; Venero, J.L.; de Pablos, R.M. Gal3 Plays a Deleterious Role in a Mouse Model of Endotoxemia. Int. J. Mol. Sci. 2022, 23, 1170. https://doi.org/ 10.3390/ijms23031170 Academic Editor: Sharmila Masli Received: 29 October 2021 Accepted: 15 January 2022 Published: 21 January 2022 Keywords: endotoxemia; galectin-3; lipopolysaccharide; electron microscopy; sepsis International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Article Gal3 Plays a Deleterious Role in a Mouse Model of Endotoxemia Taken together, these results demonstrate the important role that Gal3 could play in the development of systemic inﬂammation, a well-established feature of sepsis, thus opening new and promising therapeutic options for these harmful conditions. 1. Introduction Moreover, Gal3 has been associated with several diseases, such as cancer, liver diseases, cardiovascular diseases, periodontal diseases, and ﬁbrotic diseases and has even been proposed as a biomarker in several conditions [12–16]. Indeed, we have recently suggested that Gal3 is an attractive molecule to consider in the regulation of the macrophage-related hyperinﬂammation phase associated withsevere COVID-19 patients [17]. p [ ] Galectins are soluble proteins deﬁned by the presence of a carbohydrate-recognition domain (CRD), which binds N-acetyl-lactosamine-enriched glycoconjugates present on the cell surface or extracellular matrix [18]. This family of proteins consists of at least 15 members in vertebrates, ofwhich Gal3 is the only chimera-type member [18], which is deﬁned by a C-terminal CRD and an N-terminal tail that allows oligomerization [18]. According to cell type and cellular location, Gal3 has different functions. Gal3 can be found intra-(cytoplasm and nucleus) and extracellularly [19]. We have long demonstrated a pro-inﬂammatory role of Gal3 in microglia (myeloid cells), as evidenced by the fact that absence or inhibition of Gal3 strongly hinders the microglia response to LPS [7], ﬁbrillary β-amyloid [20], and α-synuclein aggregates [21]. Furthermore, it has been shown to be neuroprotective in several in vivo models of neurodegeneration, including global ischemia, intranigral LPS injection, traumatic brain injury, and spinal cord injury [7,22,23]. We and others have demonstrated the ability of Gal3 to govern immune-related functions through binding to different receptors, including the triggering receptor expressed on myeloid cells 2 (TREM2), the insulin-like growth factor receptor 1 (IGFR1), and, importantly, TLR4 [7,20,24]. All these features make Gal3 an attractive target to modulate peripheral immune responses including those associated withsepsis and endotoxemia. Supporting this, circulating Gal3 has been shown to increase in patients suffering from sepsis [25,26]. A detrimental role of Gal3 has been shown in mice undergoing sepsis induced by either cecal ligation and puncture (CLP) [27] or infection with Francisellanovicida [28]. On the other hand, Gal3 has been reported to act as a negative regulator of LPS-induced endotoxemia and inﬂammation, which was explained by the ability of Gal3 to bind to LPS to further inhibit the endotoxin-associated pro-inﬂammatory response [29]. 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Systemic inﬂammation is a major pathogenic feature of different acute and chronic diseases, including sepsis [1]. Indeed, lipopolysaccharide (LPS)-induced endotoxemia leads to overall activation of monocytes and peripheral macrophages to cause peripheral inﬂammation. At appropriate LPS doses, animals exhibit physiological and biochemical features that mimic certain fulminant forms of Gram-negative bacterial infection along with important hallmarks of sepsis [2]. In fact, LPS-induced endotoxemia induces arterial hypotension, lactic acidosis, tachycardia, and speciﬁc temporal elevations of circulating levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and high mobility group box (HMGB)-1 [2], which are important features of sepsis and septic shock. In severe conditions, sepsis/endotoxemia can lead to a systemic inﬂammatory response syndrome (SIRS) that induces a cytokine storm-induced syndrome, a life-threating condition [3]. The cytokine storm is triggered by leukocyte activation after binding of pathogen-associated molecular patterns (PAMPs) such as bacterial, fungal, or viral components or damage-associated Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 1170. https://doi.org/10.3390/ijms23031170 Int. J. Mol. Sci. 2022, 23, 1170 2 of 25 molecular patterns (DAMPs) such as endogenous nucleic acid material and proteins [4]. The recognition of PAMPs and DAMPs is mediated through binding to pattern recognition receptors (PRRs), from which the Toll-like receptors (TLRs) play a critical role [4]. In fact, most studied PRRs are TLRs, especially TLR4, the receptor for LPS [5], a wall compound of Gram-negative bacteria widely used to model the acute inﬂammatory response associated with early sepsis [6]. Endogenous molecules that regulate PRR signaling, and particularly TLR4 signaling, are quite attractive means to preventing or attenuating the cytokine storm associated withsepsis/endotoxemia conditions. In this sense, galectin-3 (Gal3) is emerging as an interesting pharmacological target to treat diseases related to the immune system, due to the immunoregulatory role that is attributed to this protein and its ability to bind to TLR4 [7]. Gal3 has many physiological functions, including adhesion, apoptosis, cell cycle regulation, and cell-to-cell interactions within the extracellular matrix [8–11]. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon , p < 0.05. 2.2. LPS Induces Changes in the Gal3 Levels in Immune Cells of Peripheral Blood, Serum and Figure 1. Gal3 knockout mice are more resistant to septic shock. WT and Gal3KO mice were subjected to an intraperitoneal injection of LPS (5 mg/kg of body weight) or saline solution (N = 12 animals per group). The mortality rate was monitored regularly for 80 h and represented as percentage of survival. The statistical analysis was performed using the Log-Rank Test. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. , p < 0.05. 2.2. LPS Induces Changes in the Gal3 Levels in Immune Cells of Peripheral Blood, Serum Figure 1. Gal3 knockout mice are more resistant to septic shock. WT and Gal3KO mice were sub- jected to an intraperitoneal injection of LPS (5 mg/kg of body weight) or saline solution (N = 12 animals per group). The mortality rate was monitored regularly for 80 h and represented as per- centage of survival. The statistical analysis was performed using the Log-Rank Test. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. , p < 0.05. Figure 1. Gal3 knockout mice are more resistant to septic shock. WT and Gal3KO mice were subjected to an intraperitoneal injection of LPS (5 mg/kg of body weight) or saline solution (N = 12 animals per group). The mortality rate was monitored regularly for 80 h and represented as percentage of survival. The statistical analysis was performed using the Log-Rank Test. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. , p < 0.05. 2.2. LPS Induces Changes in the Gal3 Levels in Immune Cells of Peripheral Blood, Serum a Tissues 2.2. LPS Induces Changes in the Gal3 Levels in Immune Cells of Peripheral Blood, Serum and Tissues 2.2. LPS Induces Changes in the Gal3 Levels in Immune Cells of Peripheral Blood, Serum and Tissues 2.2. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon As a ﬁrst step, we performed an LPS dose-response study (0, 7.5, 10 and 15 mg/kg) to monitor the survival of the animals for ﬁve days (Figure S1). From this analysis, we found that more than 55.5% of the animals died by day 2 following LPS challenge at a dose of 10 and 15 mg/kg (Figure S1B,C). Similar results were found when we used the 7.5 mg/kg dosage, although we found a clear delay in mortality (Figure S1D). Based on this study, a dose of 5 mg/kg was selected to evaluate the effect of the genetic ablation of Gal3 on the mortality induced in our model of LPS-induced endotoxemia. Under these conditions, 90% of WT succumbed in the ﬁrst 80 h (Figure 1). LPS injection in Gal3KO animals also induced the death of animals, but we observed a signiﬁcant delay in the death rate and a clear increase in the survival rate (Figure 1; p = 0.033), with about 50% of the animals surviving. to monitor the survival of the animals for five days (Figure S1). From this analysis, we found that more than 55.5% of the animals died by day 2 following LPS challenge at a dose of 10 and 15 mg/kg (Figure S1B,C). Similar results were found when we used the 7.5 mg/kg dosage, although we found a clear delay in mortality (Figure S1D). Based on this study, a dose of 5 mg/kg was selected to evaluate the effect of the genetic ablation of Gal3 on the mortality induced in our model of LPS-induced endotoxemia. Under these condi- tions, 90% of WT succumbed in the first 80 h (Figure 1). LPS injection in Gal3KO animals also induced the death of animals, but we observed a significant delay in the death rate and a clear increase in the survival rate (Figure 1; p = 0.033), with about 50% of the animals surviving. Figure 1. Gal3 knockout mice are more resistant to septic shock. WT and Gal3KO mice were sub- jected to an intraperitoneal injection of LPS (5 mg/kg of body weight) or saline solution (N = 12 animals per group). The mortality rate was monitored regularly for 80 h and represented as per- centage of survival. The statistical analysis was performed using the Log-Rank Test. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. 1. Introduction However, this view has been challenged by different studies, including the following:(i) Gal3 has been shown to potentiate LPS-induced IL-1 production and enhance monocyte chemotaxis [30]; (ii) the functional relevance of interactions between Gal3 and LPS remains to be clariﬁed [31,32]; (iii) we have identiﬁed Gal3 as a master ampliﬁer of the LPS-induced pro-inﬂammatory response in microglia [7]. Consequently, we have analyzed the effect of Gal3 in the LPS- induced model of endotoxemia (5 mg/kg) including (i) mortality rate, (ii) acute effect of LPS challenge (1 h) on membrane-bound Gal3 on circulating immune cells, (iii) delayed effect of LPS challenge (36 h) on Gal3 expression on peripheral organs, (iv) overall pro- inﬂammatory and anti-inﬂammatory status, and (v) peripheral organ integrity. Our study suggests that Gal3 is a powerful modulator of the peripheral immune system, thus emerging as a potential endogenous alarmin regulating early endotoxemia development. Int. J. Mol. Sci. 2022, 23, 1170 3 of 25 g 2. Results 2.1. Genetic 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon , p < 0.01. Next,we determined the amount of Gal3 in the serum of WT animals treated LPS (1 h) and found a decrease of around 80% with respect to the control group (F 3A, p = 0.00001). Since serum levels of Gal3 were determined very early in response to challenge, we also determined the mRNA levels of Gal3 in liver, spleen, and perit macrophages 1h after LPS challenge. As expected, Gal3KO animals did not express whereas WT animals showed a basal expression of this galectin (Figure 3B–D). No s ical differences were found between WT animals treated with LPS or saline (Figur D). Since LPS-induced Gal3 transcription and translation may require longer than 1 h considering that circulating Gal3 may spread and be takenup by peripheral organs to issue-specific immune responses including Gal3 upregulation, we next analyzed protein expression by immunohistochemistry in liver, lung, and spleen 36 h after challenge. Notably, under these conditions, a robust increase of Gal3-expressing cell ound in the examined organs from WT animals ranging from 2.1 to 3.2-fold with re o the control group (Figure 4). Most Gal3+ cells are Iba1+. Therefore, we can conclud expression of Gal3 increases in liver macrophages upon LPS administration (F Next, we determined the amount of Gal3 in the serum of WT animals treated with LPS (1 h) and found a decrease of around 80% with respect to the control group (Figure 3A, p = 0.00001). Since serum levels of Gal3 were determined very early in response to LPS challenge, we also determined the mRNA levels of Gal3 in liver, spleen, and peritoneal macrophages 1 h after LPS challenge. As expected, Gal3KO animals did not express Gal3, whereas WT animals showed a basal expression of this galectin (Figure 3B–D). No statistical differences were found between WT animals treated with LPS or saline (Figure 3B–D). Since LPS-induced Gal3 transcription and translation may require longer than 1 h, and considering that circulating Gal3 may spread and be takenup by peripheral organs to elicit tissue-speciﬁc immune responses including Gal3 upregulation, we next analyzed Gal3 protein expression by immunohistochemistry in liver, lung, and spleen 36 h after LPS challenge. Notably, under these conditions, a robust increase of Gal3-expressing cells was found in the examined organs from WT animals ranging from 2.1 to 3.2-fold with respect to the control group (Figure 4). Most Gal3+ cells are Iba1+. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon LPS Induces Changes in the Gal3 Levels in Immune Cells of Peripheral Blood, Serum and Tissues To address whether immune cells act as a potential source of Gal3 under conditions of endotoxemia, we determined the early expression (1h) of such protein on different subsets of immune cells in peripheral blood by FACS after LPS challenge.In control conditions, all the studied cell subsets expressed Gal3 but at very different levels. While a high frequency of in- nate immune cells, especially monocytes, expressing Gal3 was found, the frequency of adap- tive immune cells expressing Gal3 was significantly lower, withthe lowest frequency being that of CD8+T-cells (Figure 2). Under conditions of endotoxemia, the levels of membrane- bound Gal3 statistically decreased in all the immune cell subsets with the exception of CD8+T- To address whether immune cells act as a potential source of Gal3 under conditions of endotoxemia, we determined the early expression (1 h) of such protein on different subsets of immune cells in peripheral blood by FACS after LPS challenge.In control conditions, all the studied cell subsets expressed Gal3 but at very different levels. While a high frequency of innate immune cells, especially monocytes, expressing Gal3 was found, the frequency of adaptive immune cells expressing Gal3 was signiﬁcantly lower, withthe lowest frequency being that of CD8+ T-cells (Figure 2). Under conditions of endotoxemia, the levels of membrane-bound Gal3 statistically decreased in all the immune cell subsets with the exception of CD8+ T-cells, CD4+ T-cells, and DCs, which only showed a trend (p = 0.2, p = 0.056 and p = 0.056, respectively) to a reduction of levels of Gal3 after the LPS exposure (Figure 2). Under conditions of endotoxemia, the levels of Gal3 bound to the membrane decreased statistically in B and T cells, neutrophils, monocytes, and macrophages. However, Int. J. Mol. Sci. 2022, 23, 1170 4 of 25 er cond ly in B CD8+ T-cells, CD4+ T-cells, and DCs only showed a trend toward a reduction of levels of Gal3 after the LPS exposure (p = 0.2, p = 0.056 and p = 0.056, respectively; Figure 2). DCs only showed a trend toward a reduction of levels of Gal3 after the LPS exposure (p p = 0.056 and p = 0.056, respectively; Figure 2). Figure 2. Determination of Gal3 on immune cells of peripheral blood. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon Levels of Gal3 were de mined by flow cytometry in peripheral blood of mice (N =5 animals). Briefly, after blood collect erythrocytes were lysed using an ammonium chloride lysis solution. Cells were washed stained with surface marker antibodies for 20 min on ice. Gal-3 expression was analyzed in B-c CD4+ T-cells, CD8+ T-cells, double negative T-cells, dendritic cells, neutrophils, and macropha Results are expressed as median ± IQR. Statistical significance was calculated using the Ma Whitney U test. Abbreviations: DCs, dendritic cells; Macro, macrophages; Monoc, monocytes; N tro, neutrophils. , p < 0.01. Figure 2. Determination of Gal3 on immune cells of peripheral blood. Levels of Gal3 were determined by ﬂow cytometry in peripheral blood of mice (N = 5 animals). Brieﬂy, after blood collection, erythrocytes were lysed using an ammonium chloride lysis solution. Cells were washed and stained with surface marker antibodies for 20 min on ice. Gal-3 expression was analyzed in B-cells, CD4+ T-cells, CD8+ T-cells, double negative T-cells, dendritic cells, neutrophils, and macrophages. Results are expressed as median ± IQR. Statistical signiﬁcance was calculated using the Mann–Whitney U test. Abbreviations: DCs, dendritic cells; Macro, macrophages; Monoc, monocytes; Neutro, neutrophils. , p < 0.01. igure 2. Determination of Gal3 on immune cells of peripheral blood. Levels of Gal3 were d mined by flow cytometry in peripheral blood of mice (N =5 animals). Briefly, after blood collec rythrocytes were lysed using an ammonium chloride lysis solution. Cells were washed tained with surface marker antibodies for 20 min on ice. Gal-3 expression was analyzed in B-c CD4+ T-cells, CD8+ T-cells, double negative T-cells, dendritic cells, neutrophils, and macropha Results are expressed as median ± IQR. Statistical significance was calculated using the Ma Whitney U test. Abbreviations: DCs, dendritic cells; Macro, macrophages; Monoc, monocytes; N ro, neutrophils. , p < 0.01. Figure 2. Determination of Gal3 on immune cells of peripheral blood. Levels of Gal3 were determined by ﬂow cytometry in peripheral blood of mice (N = 5 animals). Brieﬂy, after blood collection, erythrocytes were lysed using an ammonium chloride lysis solution. Cells were washed and stained with surface marker antibodies for 20 min on ice. Gal-3 expression was analyzed in B-cells, CD4+ T-cells, CD8+ T-cells, double negative T-cells, dendritic cells, neutrophils, and macrophages. Results are expressed as median ± IQR. Statistical signiﬁcance was calculated using the Mann–Whitney U test. Abbreviations: DCs, dendritic cells; Macro, macrophages; Monoc, monocytes; Neutro, neutrophils. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon Therefore, we can conclude that expression of Gal3 increases in liver macrophages upon LPS administration (Figure 5).This analysis supports the view that tissue-speciﬁc Gal3 expression may play a critical role in driving LPS-associated dysregulated immune responses. expression of Gal3 increases in liver macrophages upon LPS administration (F 5).This analysis supports the view that tissue-specific Gal3 expression may play a cr role in driving LPS-associated dysregulated immune responses. Since Gal3 is a known ligand of TLR4, we also wanted to know the effect that del on Gal3 has on the expression levels of TLR4 mRNA after LPS injection. Our PCR an showed that TLR4 mRNA levels increased significantly in macrophages from WT an 1 h after LPS injection. This effect was prevented in Gal3KO animals (Figure 3G) Since Gal3 is a known ligand of TLR4, we also wanted to know the effect that deletion on Gal3 has on the expression levels of TLR4 mRNA after LPS injection. Our PCR analysis showed that TLR4 mRNA levels increased signiﬁcantly in macrophages from WT animals 1 h after LPS injection. This effect was prevented in Gal3KO animals (Figure 3G). The reduction in TLR4 mRNA levels in animals lacking Gal3 could explain the better outcome of the Gal3KO mice after the LPS challenge. TLR4 mRNA levels in liver and spleen were very low in control animals, and these levels were not affected by LPS injection (Figure 3E,F). Int. J. Mol. Sci. 2022, 23, 1170 Int. J. Mol. Sci. 2022, 23, x FOR P 5 of 25 5 of 25 Figure 3. Determination of Gal3 and TLR4. (A)The expression of Gal3 in serum was measured by ELISA. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–6 animals, expressed as ng/mLof the analyzed protein and relative to the WT group.Statistical significance (two tailed Student’s t test): p < 0.001. Using RT-PCR, the mRNA expression of Gal3 was measured in the liver (B), spleen (C), and peritoneal macrophages (D). Using RT-PCR, the mRNA expression of TLR4 was measured in the liver €, spleen (F), and peritoneal macrophages (G). For PCR analysis, animals were culled 1 h after LPS/saline injection. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon Results are mean ± SD of N = 3–10 animals, normalized to β-actin and expressed as relative expression to the WT group.Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): * -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.001 for (B–D), p < 0.05 for (G). Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Figure 3. Determination of Gal3 and TLR4. (A)The expression of Gal3 in serum was measured by ELISA. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–6 animals, expressed as ng/mLof the analyzed protein and relative to the WT group. Statistical signiﬁcance (two tailed Student’s t test): p < 0.001. Using RT-PCR, the mRNA expression of Gal3 was measured in the liver (B), spleen (C), and peritoneal macrophages (D). Using RT-PCR, the mRNA expression of TLR4 was measured in the liver (E), spleen (F), and peritoneal macrophages (G). For PCR analysis, animals were culled 1 h after LPS/saline injection. Results are mean ± SD of N = 3–10 animals, normalized to β-actin and expressed as relative expression to the WT group. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001 for (B–D), p < 0.05 for (G). Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS Figure 3. Determination of Gal3 and TLR4. (A)The expression of Gal3 in serum was measured by ELISA. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–6 animals, expressed as ng/mLof the analyzed protein and relative to the WT group.Statistical significance (two tailed Student’s t test): p < 0.001. Using RT-PCR, the mRNA expression of Gal3 was measured in the liver (B), spleen (C), and peritoneal macrophages (D). Using RT-PCR, the mRNA expression of TLR4 was measured in the liver €, spleen (F), and peritoneal macrophages (G). For PCR analysis, animals were culled 1 h after LPS/saline injection. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon Results are mean ± SD of N = 3–10 animals, normalized to β-actin and expressed as relative expression to the WT group.Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.001 for (B–D), p < 0.05 for (G). Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Figure 3. Determination of Gal3 and TLR4. (A)The expression of Gal3 in serum was measured by ELISA. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–6 animals, expressed as ng/mLof the analyzed protein and relative to the WT group. Statistical signiﬁcance (two tailed Student’s t test): p < 0.001. Using RT-PCR, the mRNA expression of Gal3 was measured in the liver (B), spleen (C), and peritoneal macrophages (D). Using RT-PCR, the mRNA expression of TLR4 was measured in the liver (E), spleen (F), and peritoneal macrophages (G). For PCR analysis, animals were culled 1 h after LPS/saline injection. Results are mean ± SD of N = 3–10 animals, normalized to β-actin and expressed as relative expression to the WT group. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001 for (B–D), p < 0.05 for (G). Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Int. J. Mol. Sci. 2022, 23, 1170 Int. J. Mol. Sci. 2022, 23, x FOR 6 of 25 6 of 25 Figure 4. Gal3 expression in the liver, lung, and spleen of mice from the different treatments as- sayed. (A) Section from liver showing some Gal3 positive cells in a WT animal. (B) Immunoreactiv- ity of Gal3 in a liver section from a WT animal injected i.p. with LPS. (F) Section from spleen showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a spleen section from a WT animal injected i.p. with LPS. Again, a strong reaction can be seen. (I) Section from lung showing some Gal3 positive cells in a WT animal. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon (I) Section from lung showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a lung section from a WT animal injected i.p. with LPS, showing a strong reaction. As expected, in the case of Gal3KO animals, immunoreactivity of Gal3 was not found in any treatment assayed (C,D). Scale bar: (A–D), 100 μm; (F–J), 20 μm. Quantification of the density of Gal3 positive cells in liver (E), spleen (H),and lung (K) from the different treatments assayed. Results are mean ± SD of N = 3–4 animals, expressed as num- ber of cells per mm2. Statistical significance (two tailed Student-t test): p < 0.01; ** p < 0.05. Abbre- viations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Figure 4. Gal3 expression in the liver, lung, and spleen of mice from the different treatments assayed. (A) Section from liver showing some Gal3 positive cells in a WT animal. (B) Immunoreactivity of Gal3 in a liver section from a WT animal injected i.p. with LPS. (F) Section from spleen showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a spleen section from a WT animal injected i.p. with LPS. Again, a strong reaction can be seen. (I) Section from lung showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a lung section from a WT animal injected i.p. with LPS, showing a strong reaction. As expected, in the case of Gal3KO animals, immunoreactivity of Gal3 was not found in any treatment assayed (C,D). Scale bar: (A–D), 100 µm; (F–J), 20 µm. Quantiﬁcation of the density of Gal3 positive cells in liver (E), spleen (H), and lung (K) from the different treatments assayed. Results are mean ± SD of N = 3–4 animals, expressed as number of cells per mm2. Statistical signiﬁcance (two tailed Student-t test): * p < 0.01; ** p < 0.05. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Gal3 expression in the liver, lung, and spleen of mice from the different treatments as- Gal3 expression in the liver, lung, and spleen of mice from the different treatments assayed. Figure 4. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon Gal3 expression in the liver, lung, and spleen of mice from the different treatments as- sayed. (A) Section from liver showing some Gal3 positive cells in a WT animal. (B) Immunoreactiv- ity of Gal3 in a liver section from a WT animal injected i.p. with LPS. (F) Section from spleen showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a spleen section from a WT animal injected i.p. with LPS. Again, a strong reaction can be seen. (I) Section from lung showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a lung section from a WT animal injected i.p. with LPS, showing a strong reaction. As expected, in the case of Gal3KO animals, immunoreactivity of Gal3 was not found in any treatment assayed (C,D). Scale bar: (A–D), 100 μm; (F–J), 20 μm. Quantification of the density of Gal3 positive cells in liver (E), spleen (H),and lung (K) from the different treatments assayed. Results are mean ± SD of N = 3–4 animals, expressed as num- ber of cells per mm2. Statistical significance (two tailed Student-t test): * p < 0.01; ** p < 0.05. Abbre- viations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Figure 4. Gal3 expression in the liver, lung, and spleen of mice from the different treatments assayed. (A) Section from liver showing some Gal3 positive cells in a WT animal. (B) Immunoreactivity of Gal3 in a liver section from a WT animal injected i.p. with LPS. (F) Section from spleen showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a spleen section from a WT animal injected i.p. with LPS. Again, a strong reaction can be seen. (I) Section from lung showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a lung section from a WT animal injected i.p. with LPS, showing a strong reaction. As expected, in the case of Gal3KO animals, immunoreactivity of Gal3 was not found in any treatment assayed (C,D). Scale bar: (A–D), 100 µm; (F–J), 20 µm. Quantiﬁcation of the density of Gal3 positive cells in liver (E), spleen (H), and lung (K) from the different treatments assayed. Results are mean ± SD of N = 3–4 animals, expressed as number of cells per mm2. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon Statistical signiﬁcance (two tailed Student-t test): * p < 0.01; ** p < 0.05. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Figure 4. Gal3 expression in the liver, lung, and spleen of mice from the different treatments as- sayed. (A) Section from liver showing some Gal3 positive cells in a WT animal. (B) Immunoreactiv- ity of Gal3 in a liver section from a WT animal injected i.p. with LPS. (F) Section from spleen showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a spleen section from a WT animal injected i.p. with LPS. Again, a strong reaction can be seen. (I) Section from lung showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a lung section from a WT animal injected i.p. with LPS, showing a strong reaction. As expected, in the case of Gal3KO animals, immunoreactivity of Gal3 was not found in any treatment assayed (C,D). Scale bar: (A–D), 100 μm; (F–J), 20 μm. Quantification of the density of Gal3 positive cells in liver (E), spleen (H),and lung (K) from the different treatments assayed. Results are mean ± SD of N = 3–4 animals, expressed as num- ber of cells per mm2. Statistical significance (two tailed Student-t test): * p < 0.01; ** p < 0.05. Abbre- viations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Figure 4. Gal3 expression in the liver, lung, and spleen of mice from the different treatments assayed. (A) Section from liver showing some Gal3 positive cells in a WT animal. (B) Immunoreactivity of Gal3 in a liver section from a WT animal injected i.p. with LPS. (F) Section from spleen showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a spleen section from a WT animal injected i.p. with LPS. Again, a strong reaction can be seen. (I) Section from lung showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a lung section from a WT animal injected i.p. with LPS, showing a strong reaction. As expected, in the case of Gal3KO animals, immunoreactivity of Gal3 was not found in any treatment assayed (C,D). Scale bar: (A–D), 100 µm; (F–J), 20 µm. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon (G) Immunoreactivity of Gal3 in a lung section from a WT animal injected i.p. with LPS, showing a strong reaction. As expected, in the case of Gal3KO animals, immunoreactivity of Gal3 was not found in any treatment assayed (C,D). Scale bar: (A–D), 100 μm; (F–J), 20 μm. Quantification of the density of Gal3 positive cells in liver (E), spleen (H),and lung (K) from the different treatments assayed. Results are mean ± SD of N = 3–4 animals, expressed as num- ber of cells per mm2. Statistical significance (two tailed Student-t test): * p < 0.01; ** p < 0.05. Abbre- viations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; Figure 4. Gal3 expression in the liver, lung, and spleen of mice from the different treatments assayed. (A) Section from liver showing some Gal3 positive cells in a WT animal. (B) Immunoreactivity of Gal3 in a liver section from a WT animal injected i.p. with LPS. (F) Section from spleen showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a spleen section from a WT animal injected i.p. with LPS. Again, a strong reaction can be seen. (I) Section from lung showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a lung section from a WT animal injected i.p. with LPS, showing a strong reaction. As expected, in the case of Gal3KO animals, immunoreactivity of Gal3 was not found in any treatment assayed (C,D). Scale bar: (A–D), 100 µm; (F–J), 20 µm. Quantiﬁcation of the density of Gal3 positive cells in liver (E), spleen (H), and lung (K) from the different treatments assayed. Results are mean ± SD of N = 3–4 animals, expressed as number of cells per mm2. Statistical signiﬁcance (two tailed Student-t test): * p < 0.01; ** p < 0.05. Figure 4. Gal3 expression in the liver, lung, and spleen of mice from the different treatments as- sayed. (A) Section from liver showing some Gal3 positive cells in a WT animal. (B) Immunoreactiv- ity of Gal3 in a liver section from a WT animal injected i.p. with LPS. (F) Section from spleen showing some Gal3 positive cells in a WT animal. (G) Immunoreactivity of Gal3 in a spleen section from a WT animal injected i.p. with LPS. Again, a strong reaction can be seen. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon Quantiﬁcation of the density of Gal3 positive cells in liver (E), spleen (H), and lung (K) from the different treatments assayed. Results are mean ± SD of N = 3–4 animals, expressed as number of cells per mm2. Statistical signiﬁcance (two tailed Student-t test): * p < 0.01; ** p < 0.05. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. 7 of 25 7 of 25 7 of 25 7 of 25 Int. J. Mol. Sci. 2022, 23, 1170 Int. J. Mol. Sci. 2022, 23, x FOR Figure 5. Double immunofluorescence of Iba1 andGal3 expression. (A) Iba1 staining in the liver of a WT animal. Virtually, no monocytes/macrophages are infiltrated in control animals. (B) Section from liver showing some Gal3 positive cells in a WT animal. (C) Merge image showing co-localiza- tion of Iba1+ cells and Gal3+ cells. (D) Immunoreactivity of Iba1 in a liver section from a WT animal injected i.p. with LPS. A strong reaction can be seen. (E) Immunoreactivity of Gal3 in WT animals injected with LPS. (F) Merge image showing co-localization of Iba1+ cells and Gal3+ cells (arrows). Most Gal3+ cells co-localize with Iba1+ cells. Scale bar: 50 μm. Figure 5. Double immunoﬂuorescence of Iba1 andGal3 expression. (A) Iba1 staining in the liver of a WT animal. Virtually, no monocytes/macrophages are inﬁltrated in control animals. (B) Section from liver showing some Gal3 positive cells in a WT animal. (C) Merge image showing co-localization of Iba1+ cells and Gal3+ cells. (D) Immunoreactivity of Iba1 in a liver section from a WT animal injected i.p. with LPS. A strong reaction can be seen. (E) Immunoreactivity of Gal3 in WT animals injected with LPS. (F) Merge image showing co-localization of Iba1+ cells and Gal3+ cells (arrows). Most Gal3+ cells co-localize with Iba1+ cells. Scale bar: 50 µm. Figure 5. Double immunofluorescence of Iba1 andGal3 expression. (A) Iba1 staining in the liver of a WT animal. Virtually, no monocytes/macrophages are infiltrated in control animals. (B) Section from liver showing some Gal3 positive cells in a WT animal. (C) Merge image showing co-localiza- tion of Iba1+ cells and Gal3+ cells. (D) Immunoreactivity of Iba1 in a liver section from a WT animal injected i.p. with LPS. A strong reaction can be seen. (E) Immunoreactivity of Gal3 in WT animals injected with LPS. 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages Macrophages As already stated, deregulation of the immune system may lead to cytokine storms in which both pro- and anti-inflammatory mediators are involved. Therefore, in order to study the inflammatory events in our experimental conditions, we proceeded to quantify by qPCR the expression of TNF-α, iNOS, IL-1β, and IL-6 as pro-inflammatory markers, and YM1, IL-10, and arginase as anti-inflammatory markers, in the biological samples ex- tracted (liver, spleen, and peripheral macrophages). Our results showed that in the liver, LPS injection in WT animals increased the mRNA levels of the pro-inflammatory markers TNF-α, iNOS, IL-1β, and IL-6 (Figure 6A–D). Moreover, LPS treatment also increased the mRNA expression levels of the anti inflammatory markers IL 10 and arginase (Figure As already stated, deregulation of the immune system may lead to cytokine storms in which both pro- and anti-inﬂammatory mediators are involved. Therefore, in order to study the inﬂammatory events in our experimental conditions, we proceeded to quantify by qPCR the expression of TNF-α, iNOS, IL-1β, and IL-6 as pro-inﬂammatory markers, and YM1, IL-10, and arginase as anti-inﬂammatory markers, in the biological samples extracted (liver, spleen, and peripheral macrophages). Our results showed that in the liver, LPS injection in WT animals increased the mRNA levels of the pro-inﬂammatory markers TNF-α, iNOS, IL-1β, and IL-6 (Figure 6A–D). Moreover, LPS treatment also increased the mRNA expression levels of the anti-inﬂammatory markers IL-10 and arginase (Figure 6F,G). mRNA expression levels of the anti-inflammatory markers IL-10 and arginase (Figure 6F,G). Interestingly, genetic ablation of Gal3 decreased the mRNA levels of iNOS in LPS- treated animals (Figure 6B) by about 60%. However, at this time point, mRNA levels of TNF- α, IL-1β, and IL-6 of Gal3KO LPS-treated animals increased nearly 3, 2, and 2.5 fold respec- tively, with respect to the WTLPS group (Figure 6A,C,D). Regarding the anti-inflammatory markers, Gal3KO mice showed an increase in the mRNA levels of YM1 and IL-10 (6 and 2 p y g Interestingly, genetic ablation of Gal3 decreased the mRNA levels of iNOS in LPS- treated animals (Figure 6B) by about 60%. 2.1. Genetic Deletion of Gal3 Decreases the Mortality Rate As a first step, we performed an LPS dose-respon (F) Merge image showing co-localization of Iba1+ cells and Gal3+ cells (arrows). Most Gal3+ cells co-localize with Iba1+ cells. Scale bar: 50 μm. Figure 5. Double immunoﬂuorescence of Iba1 andGal3 expression. (A) Iba1 staining in the liver of a WT animal. Virtually, no monocytes/macrophages are inﬁltrated in control animals. (B) Section from liver showing some Gal3 positive cells in a WT animal. (C) Merge image showing co-localization of Iba1+ cells and Gal3+ cells. (D) Immunoreactivity of Iba1 in a liver section from a WT animal injected i.p. with LPS. A strong reaction can be seen. (E) Immunoreactivity of Gal3 in WT animals injected with LPS. (F) Merge image showing co-localization of Iba1+ cells and Gal3+ cells (arrows). Most Gal3+ cells co-localize with Iba1+ cells. Scale bar: 50 µm. 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages However, at this time point, mRNA levels of TNF- α, IL-1β, and IL-6 of Gal3KO LPS-treated animals increased nearly 3, 2, and 2.5 fold respec- tively, with respect to the WTLPS group (Figure 6A,C,D). Regarding the anti-inﬂammatory markers, Gal3KO mice showed an increase in the mRNA levels of YM1 and IL-10 (6 and 2 fold respectively, with respect to the WTLPS group; Figure 6E,F), but a decrease in mRNA arginase levels (more than half with respect to WTLPS group; Figure 6G). fold respectively, with respect to the WTLPS group; Figure 6E,F), but a decrease in mRNA arginase levels (more than half with respect to WTLPS group; Figure 6G). In the spleen, LPS treatment in WT animals induced an increase in the mRNA levels of TNF-α, IL-1β, and IL-6 (Figure 6H–K). LPS treatment also induced an increase in the mRNA levels of IL-10 (Figure 6M). Genetic deletion of Gal3 induced a higher increase in mRNA levels of TNF-α and IL-1β (Figure 6H,J) and an increase in the levels of YM1 and In the spleen, LPS treatment in WT animals induced an increase in the mRNA levels of TNF-α, IL-1β, and IL-6 (Figure 6H–K). LPS treatment also induced an increase in the mRNA levels of IL-10 (Figure 6M). Genetic deletion of Gal3 induced a higher increase in mRNA levels of TNF-α and IL-1β (Figure 6H,J) and an increase in the levels of YM1 and arginase (Figure 6L,N), but a decrease in the mRNA levels of IL-10 (Figure 6M). No statistical differences were found in iNOS mRNA levels (Figure 6I). Int. J. Mol. Sci. 2022, 23, 1170 8 of 25 ytes). Inf ection. I Figure 6. Effect of Gal3 deletion on the expression of TNF-α, iNOS, IL-1β, IL-6, YM1, IL-10, and arginase mRNAs in the liver, spleen, and macrophages of mice from the different treatments assayed, measured by RT-PCR (A–U). Animals were culled 1 h after LPS/saline injection. Results are mean ± SD of N = 3–10 animals, normalized to β-actin and expressed as relative expression to the WT group. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001 for (A–F,H,J–M,O,P,T); p < 0.01 for (G,N,S); p < 0.05 for (Q,R,U). 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Finally in peripheral macrophages LPS treatment in WT animals increased the ex- Figure 6. Effect of Gal3 deletion on the expression of TNF-α, iNOS, IL-1β, IL-6, YM1, IL-10, and arginase mRNAs in the liver, spleen, and macrophages of mice from the different treatments assayed, measured by RT-PCR (A–U). Animals were culled 1 h after LPS/saline injection. Results are mean ± SD of N = 3–10 animals, normalized to β-actin and expressed as relative expression to the WT group. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001 for (A–F,H,J–M,O,P,T); p < 0.01 for (G,N,S); p < 0.05 for (Q,R,U). Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Finally, in peripheral macrophages, LPS treatment in WT animals increased the ex- pression levels of TNF-α, iNOS, IL-1β, and IL-6 (Figure 6O–R). Levels of anti-inﬂammatory markers, such as YM1, IL-10, and arginase also increased in WTLPS mice (Figure 6S–U). Int. J. Mol. Sci. 2022, 23, 1170 Int. J. Mol. Sci. 2022, 23, x FOR P 9 of 25 of 25 In this case, genetic deletion of Gal3 decreased the mRNA expression levels of iNOS and arginase (Figure 6P,U). Figure 6. Effect of Gal3 deletion on the expression of TNF-α, iNOS, IL-1β, IL-6, YM1, IL-10, and ar- ginase mRNAs in the liver, spleen, and macrophages of mice from the different treatments assayed, In this case, genetic deletion of Gal3 decreased the mRNA expression levels of iNOS and arginase (Figure 6P,U). Figure 6. Effect of Gal3 deletion on the expression of TNF-α, iNOS, IL-1β, IL-6, YM1, IL-10, and ar- ginase mRNAs in the liver, spleen, and macrophages of mice from the different treatments assayed, g g Since this PCR analysis did not allow us to draw conclusions about the effect that Gal3 has on the immune response in the tissue, we next studied the effect of LPS injection on immune cell inﬁltration. Therefore, we performed immunohistochemistry analysis against CD68 (a marker of macrophages) and CD4 (a marker of lymphocytes). Inﬁltration of lymphocytes and macrophages increased in the liver 36 h after LPS injection. 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages Interestingly, this effect was effectively abolished in the Gal3KO animals (Figures 7 and 8). measured by RT-PCR. Animals were culled 1 h after LPS/saline injection. Results are mean ± SD of N = 3–10 animals, normalized to β-actin and expressed as relative expression to the WT group.Sta- tistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): * -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.001 for (A–F,H,J–M,O,P,T); p < 0.01 for (G,N,S); p < 0.05 for (Q,R,U). Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. this effect was effectively abolished in the Gal3KO animals (Figures 7 and 8). mice treated with LPS. Figure 7. CD68 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD68 expression; how- ever, the treatment with LPS produces a strong induction of CD68-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 μm. (E) Quantification of the density of CD68 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): * -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.01. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Figure 7. CD68 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD68 expression; however, the treatment with LPS produces a strong induction of CD68-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 µm. (E) Quantiﬁcation of the density of CD68 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.01. 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Figure 7. CD68 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD68 expression; how- ever, the treatment with LPS produces a strong induction of CD68-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 μm. (E) Quantification of the density of CD68 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.01. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Figure 7. CD68 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD68 expression; however, the treatment with LPS produces a strong induction of CD68-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 µm. (E) Quantiﬁcation of the density of CD68 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.01. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated Figure 7. CD68 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD68 expression; how- ever, the treatment with LPS produces a strong induction of CD68-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 μm. (E) Quantification of the density of CD68 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.01. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Figure 7. CD68 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD68 expression; however, the treatment with LPS produces a strong induction of CD68-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 µm. (E) Quantiﬁcation of the density of CD68 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.01. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS. Int. J. Mol. Sci. 2022, 23, 1170 Int. J. Mol. Sci. 2022, 23, x FOR P 10 of 25 0 of 25 10 of 25 0 of 25 Figure 8. CD4 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD4 expression; how- ever, the treatment with LPS produces a strong induction of CD4-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 μm. (E) Quantification of the density of CD4 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated. 2.4. Absence of Gal3 Reduces Serum Levels of TNF-α and IL-6 in Response to LPS Challenge In addition to the overexpression of genes directly related to inflammation, macro- Figure 8. CD4 expression in liver. Representative immunostaining from sections of the different treatments assayed. 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated. Figure 8. CD4 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD4 expression; how- ever, the treatment with LPS produces a strong induction of CD4-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 μm. (E) Quantification of the density of CD4 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): * -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated. Figure 8. CD4 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD4 expression; however, the treatment with LPS produces a strong induction of CD4-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 µm. (E) Quantiﬁcation of the density of CD4 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated. 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Pe Macrophages 2.3. Genetic Deletion of Gal3 Alters the Immune Response in the Liver, Spleen and Peritoneal Macrophages WT (A) and KO (C) animals show a normal pattern of CD4 expression; however, the treatment with LPS produces a strong induction of CD4-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 µm. (E) Quantiﬁcation of the density of CD4 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated. 2.4. Absence of Gal3 Reduces Serum Levels of TNF-α and IL-6 in Response to LPS Challenge In addition to the overexpression of genes directly related to inflammation macrophages Figure 8. CD4 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD4 expression; how- ever, the treatment with LPS produces a strong induction of CD4-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 μm. (E) Quantification of the density of CD4 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): * -compared with WT group; # -compared with KO group; $ -compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated. Figure 8. CD4 expression in liver. Representative immunostaining from sections of the different treatments assayed. WT (A) and KO (C) animals show a normal pattern of CD4 expression; however, the treatment with LPS produces a strong induction of CD4-positive cells in WT animals (B). Absence of Gal3 clearly reduces this effect (D). Scale bar: 100 µm. (E) Quantiﬁcation of the density of CD4 positive cells in liver. Results are mean ± SD of N = 3 animals, expressed as number of cells per mm2. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001. 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage Sections from different organs were analyzed according to the classical procedures of pathological anatomy and a histological score was performed from each organ ana- lyzed. Consequently, we have analyzed different organs that suffer histological damage under LPS-induced endotoxemia conditions, including liver (Figures 10, S4 and S8A), spleen (Figures 11, S5 and S8B), and lung (Figures 12, S6 and S8C) using optical and elec- tron microscopies. The WTLPS group showed serious alterations in these organs (Figures 10C,D–12C,D and S4C,D–S6C,D). For instance, the existence of a hyperplasia and hyper- trophy of the lymphoid follicles were easily distinguished in these animals (Figures 11C and S5C). The red splenic pulp was apparently normal. Ultrastructural studies showed that only lymphocytes, lymphoblasts, and reticular cells were featured (Figures 11D and S5D). The liver of WTLPS animals showed some alterations, although the normal struc- ture of this organ was maintained. Most hepatocytes of the lobule, especially those of the perilobular space, showed steatosis preferentially multilocular (Figure 10C). In general, the pulmonary parenchyma showed a generalized atelectasis, both at the bronchial and alveolar levels (Figures 12C and S6C). With the electronic microscope, we found thicken- ings of its septal structure, with large numbers of pneumocytes II and macrophages (Fig- ures 12D and S6D). The absence of Gal3 protected against damage in the liver (Figures 10E,F and S4E,F), Sections from different organs were analyzed according to the classical procedures of pathological anatomy and a histological score was performed from each organ analyzed. Consequently, we have analyzed different organs that suffer histological damage under LPS-induced endotoxemia conditions, including liver (Figure 10 and Figures S4 and S8A), spleen (Figure 11 and Figures S5 and S8B), and lung (Figure 12 and Figures S6 and S8C) using optical and electron microscopies. The WTLPS group showed serious alterations in these organs (Figure 10C,D, Figure 11C,D and Figure 12C,D and Figures S4C,D–S6C,D). For instance, the existence of a hyperplasia and hypertrophy of the lymphoid follicles were easily distinguished in these animals (Figure 11C and Figure S5C). The red splenic pulp was apparently normal. Ultrastructural studies showed that only lymphocytes, lymphoblasts, and reticular cells were featured (Figure 11D and Figure S5D). The liver of WTLPS animals showed some alterations, although the normal structure of this or- gan was maintained. Most hepatocytes of the lobule, especially those of the perilobular space, showed steatosis preferentially multilocular (Figure 10C). .4. Absence of Gal3 Reduces Serum Levels of TNF-α and IL-6 in Response to LPS Challenge I dditi t th i f di tl l t d t i fl ti 2.4. Absence of Gal3 Reduces Serum Levels of TNF-α and IL-6 in Response to LPS Challenge In addition to the overexpression of genes directly related to inflammation, macro- phages release pro-inflammatory cytokines to the extracellular space. Therefore, we also wanted to know whether the absence of Gal3 could reduce the protein levels of TNF-α and IL-6 in serum. Using an ELISA kit, we found that the levels of TNF-α and IL-6 in- creased significantly in response to LPS challenge (around 275 and 62 fold with respect to the control group (WT) respectively) (Figure 9A B p < 0 001) This increase was however In addition to the overexpression of genes directly related to inflammation, macrophages release pro-inflammatory cytokines to the extracellular space. Therefore, we also wanted to know whether the absence of Gal3 could reduce the protein levels of TNF-α and IL-6 in serum. Using an ELISA kit, we found that the levels of TNF-α and IL-6 increased significantly in response to LPS challenge (around 275 and 62 fold with respect to the control group (WT), Int. J. Mol. Sci. 2022, 23, 1170 Int J Mol Sci 2022 23 x FOR 11 of 25 11 of 25 11 of 25 11 of 25 respectively) (Figure 9A,B, p < 0.001). This increase was, however, prevented in Gal3KO mice, demonstrating the important role that Gal3 seems to play in the immune response (cytokine storm) that takes place following our model of LPS-induced endotoxemia. the immune response (cytokine storm) that takes place following our model of LPS-in- duced endotoxemia. respectively) (Figure 9A,B, p < 0.001). This increase was, however, prevented in Gal3KO mice, demonstrating the important role that Gal3 seems to play in the immune response (cytokine storm) that takes place following our model of LPS-induced endotoxemia. the immune response (cytokine storm) that takes place following our model of LPS-in- duced endotoxemia. Figure 9. Determination of TNF-α and IL-6 levels in serum. TNF-α (A) and IL-6 (B) levels in serum of WT and Gal3KO animals. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–10 animals, expressed as ng/mLof the analyzed protein and relative to the WT group.Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): -compared with WT group; # -compared with KO group; $ - compared with WTLPS group; p < 0.001. .4. Absence of Gal3 Reduces Serum Levels of TNF-α and IL-6 in Response to LPS Challenge I dditi t th i f di tl l t d t i fl ti 2.4. Absence of Gal3 Reduces Serum Levels of TNF-α and IL-6 in Response to LPS Challenge Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Figure 9. Determination of TNF-α and IL-6 levels in serum. TNF-α (A) and IL-6 (B) levels in serum of WT and Gal3KO animals. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–10 animals, expressed as ng/mLof the analyzed protein and relative to the WT group.Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): * -compared with WT group; # -compared with KO group; $ - compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Figure 9. Determination of TNF-α and IL-6 levels in serum. TNF-α (A) and IL-6 (B) levels in serum of WT and Gal3KO animals. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–10 animals, expressed as ng/mLof the analyzed protein and relative to the WT group. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. j , p g/ y p and relative to the WT group.Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): -compared with WT group; # -compared with KO group; $ - compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. injection. Results are mean ± SD of N = 4–10 animals, expressed as ng/mLof the analyzed protein and relative to the WT group. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. .4. Absence of Gal3 Reduces Serum Levels of TNF-α and IL-6 in Response to LPS Challenge I dditi t th i f di tl l t d t i fl ti 2.4. Absence of Gal3 Reduces Serum Levels of TNF-α and IL-6 in Response to LPS Challenge Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. 2 5 G l3 D l i P f Ti l D Figure 9. Determination of TNF-α and IL-6 levels in serum. TNF-α (A) and IL-6 (B) levels in serum of WT and Gal3KO animals. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–10 animals, expressed as ng/mLof the analyzed protein and relative to the WT group. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Figure 9. Determination of TNF-α and IL-6 levels in serum. TNF-α (A) and IL-6 (B) levels in serum Figure 9. Determination of TNF-α and IL-6 levels in serum. TNF-α (A) and IL-6 (B) levels in serum Figure 9. Determination of TNF-α and IL-6 levels in serum. TNF-α (A) and IL-6 (B) levels in serum of WT and Gal3KO animals. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–10 animals, expressed as ng/mLof the analyzed protein and relative to the WT group.Statistical significance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): * -compared with WT group; # -compared with KO group; $ - compared with WTLPS group; p < 0.001. Abbreviations: WT, wild type mice; KO, Gal3 knockout mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knockout mice treated with LPS. Figure 9. Determination of TNF-α and IL-6 levels in serum. TNF-α (A) and IL-6 (B) levels in serum of WT and Gal3KO animals. Blood samples were collected from the heart of mice 1 h after LPS/saline injection. Results are mean ± SD of N = 4–10 animals, expressed as ng/mLof the analyzed protein and relative to the WT group. Statistical signiﬁcance (one-way ANOVA followed by the LSD post hoc test for multiple comparisons): —compared with WT group; #—compared with KO group; $—compared with WTLPS group; p < 0.001. 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage In general, the pulmonary parenchyma showed a generalized atelectasis, both at the bronchial and alveolar levels (Figure 12C and Figure S6C). With the electronic microscope, we found thickenings of its septal structure, with large numbers of pneumocytes II and macrophages (Figure 12D and Figure S6D). Int. J. Mol. Sci. 2022, 23, 1170 12 of 25 12 of 25 scarcely any neuronal modifications (Figures 13E,F and S7E,F). Figure 10. Histopathology of the livers of mice from the different treatments assayed. (A) Hepati lobule with Remak cords of apparently normal hepatocytes (arrow). (B) Detail of hepatocyte wit abundant rough endoplasmic reticulum (RER) and mitochondria (circle). (C) Hepatic parenchyma appreciating abundant unilocular and multilocular steatosis (arrow). (D) Detail of hepatocyte wit abundant diffuse fat (circle). (E) Detail of Remak cords with steatosis in the hepatocytes (circle). (F Hepatocytes with vacuolations of its membranous system (arrow). (G) Histological score showin a semiquantitative analysis of steatosis in the liver. The pathology scores were as follows: 0, withou significant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marke (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by tw highly experienced pathologists. Results are mean ± SD of N = 3 animals.Statistical significance (tw tailed Student-t test): p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treate Figure 10. Histopathology of the livers of mice from the different treatments assayed. (A) H lobule with Remak cords of apparently normal hepatocytes (arrow). (B) Detail of hepatocy abundant rough endoplasmic reticulum (RER) and mitochondria (circle). (C) Hepatic paren appreciating abundant unilocular and multilocular steatosis (arrow). (D) Detail of hepatocy abundant diffuse fat (circle). (E) Detail of Remak cords with steatosis in the hepatocytes (F) Hepatocytes with vacuolations of its membranous system (arrow). (G) Histological score sh a semiquantitative analysis of steatosis in the liver. The pathology scores were as follows: 0, w signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, m (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner b highly experienced pathologists. Results are mean ± SD of N = 3 animals.Statistical signiﬁcan tailed Student-t test): * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not da (A,C,E), optical microscopy. Scale bars: 100 µm. (B,D,F), ultrastructural observations. 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage Sca Figure 10. Histopathology of the livers of mice from the different treatments assayed. (A) Hepatic lobule with Remak cords of apparently normal hepatocytes (arrow). (B) Detail of hepatocyte with abundant rough endoplasmic reticulum (RER) and mitochondria (circle). (C) Hepatic parenchyma, appreciating abundant unilocular and multilocular steatosis (arrow). (D) Detail of hepatocyte with abundant diffuse fat (circle). (E) Detail of Remak cords with steatosis in the hepatocytes (circle). (F) Hepatocytes with vacuolations of its membranous system (arrow). (G) Histological score showing a semiquantitative analysis of steatosis in the liver. The pathology scores were as follows: 0, without significant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals.Statistical significance (two tailed Student-t test): * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated Figure 10. Histopathology of the livers of mice from the different treatments assayed. (A) Hepatic lobule with Remak cords of apparently normal hepatocytes (arrow). (B) Detail of hepatocyte with abundant rough endoplasmic reticulum (RER) and mitochondria (circle). (C) Hepatic parenchyma, appreciating abundant unilocular and multilocular steatosis (arrow). (D) Detail of hepatocyte with abundant diffuse fat (circle). (E) Detail of Remak cords with steatosis in the hepatocytes (circle). (F) Hepatocytes with vacuolations of its membranous system (arrow). (G) Histological score showing a semiquantitative analysis of steatosis in the liver. The pathology scores were as follows: 0, without signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals.Statistical signiﬁcance (two tailed Student-t test): * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not damaged. (A,C,E), optical microscopy. Scale bars: 100 µm. (B,D,F), ultrastructural observations. Scale bars: (B), 10 µm; (D,F), 5 µm. Int. J. Mol. Sci. 2022, 23, 1170 13 of 25 m- 13 of 25 m- bars: (B), 10 μm; (D,F), 5 μm. Figure 11. Histopathology of the spleens of mice from the different treatments assayed. (A) App ently normal spleen. Normal white pulp (lymphoid follicles) (circle). 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage (G) Histological score showing a semiquantitative analysis of hypertrophy and hyperplasia of lymphoid follicles in the spleen. The pathology scores were as follows: 0, without significant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical significance (two Figure 11. Histopathology of the spleens of mice from the different treatments assayed. (A) Ap- parently normal spleen. Normal white pulp (lymphoid follicles) (circle). (B) Marginal zone of the apparently normal spleen. (C) Hypertrophy and hyperplasia of lymphoid follicles very marked (circle). (D) Detail of lymphoid follicle with abundant lymphoblasts (LB) and lymphocytes (L), and some red blood cells (RBC). (E) Detail of spleen with hypertrophy and especially hyperplasia of the lymphoid follicles (circle). (F) Detail of lymphoid follicle with lymphocytes (L), lymphoblasts (LB) and reticular cells (RC). (G) Histological score showing a semiquantitative analysis of hypertrophy and hyperplasia of lymphoid follicles in the spleen. The pathology scores were as follows: 0, without signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical signiﬁcance (two tailed Student-t test), * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not damaged. (A,C,E), optical microscopy. Scale bars: 100 µm. (B,D,F), ultrastructural observations. Scale bars: Figure 11. Histopathology of the spleens of mice from the different treatments assayed. (A) Appar- ently normal spleen. Normal white pulp (lymphoid follicles) (circle). (B) Marginal zone of the ap- parently normal spleen. (C) Hypertrophy and hyperplasia of lymphoid follicles very marked (cir- cle). (D) Detail of lymphoid follicle with abundant lymphoblasts (LB) and lymphocytes (L), and some red blood cells (RBC). (E) Detail of spleen with hypertrophy and especially hyperplasia of the lymphoid follicles (circle). (F) Detail of lymphoid follicle with lymphocytes (L), lymphoblasts (LB) and reticular cells (RC). (G) Histological score showing a semiquantitative analysis of hypertrophy and hyperplasia of lymphoid follicles in the spleen. 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage (B) Marginal zone of the a parently normal spleen. (C) Hypertrophy and hyperplasia of lymphoid follicles very marked (c cle). (D) Detail of lymphoid follicle with abundant lymphoblasts (LB) and lymphocytes (L), a some red blood cells (RBC). (E) Detail of spleen with hypertrophy and especially hyperplasia of t lymphoid follicles (circle). (F) Detail of lymphoid follicle with lymphocytes (L), lymphoblasts (L and reticular cells (RC). (G) Histological score showing a semiquantitative analysis of hypertrop and hyperplasia of lymphoid follicles in the spleen. The pathology scores were as follows: 0, witho significant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, mark (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by tw highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical significance (tw Figure 11. Histopathology of the spleens of mice from the different treatments assayed. parently normal spleen. Normal white pulp (lymphoid follicles) (circle). (B) Marginal zon apparently normal spleen. (C) Hypertrophy and hyperplasia of lymphoid follicles very (circle). (D) Detail of lymphoid follicle with abundant lymphoblasts (LB) and lymphocytes some red blood cells (RBC). (E) Detail of spleen with hypertrophy and especially hyperplas lymphoid follicles (circle). (F) Detail of lymphoid follicle with lymphocytes (L), lymphobl and reticular cells (RC). (G) Histological score showing a semiquantitative analysis of hype and hyperplasia of lymphoid follicles in the spleen. The pathology scores were as follows: 0, signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical signiﬁca tailed Student-t test), * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mic with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not d Figure 11. Histopathology of the spleens of mice from the different treatments assayed. (A) Appar- ently normal spleen. Normal white pulp (lymphoid follicles) (circle). (B) Marginal zone of the ap- parently normal spleen. (C) Hypertrophy and hyperplasia of lymphoid follicles very marked (cir- cle). (D) Detail of lymphoid follicle with abundant lymphoblasts (LB) and lymphocytes (L), and some red blood cells (RBC). (E) Detail of spleen with hypertrophy and especially hyperplasia of the lymphoid follicles (circle). (F) Detail of lymphoid follicle with lymphocytes (L), lymphoblasts (LB) and reticular cells (RC). 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage The pathology scores were as follows: 0, without significant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical significance (two Figure 11. Histopathology of the spleens of mice from the different treatments assayed. (A) Ap- parently normal spleen. Normal white pulp (lymphoid follicles) (circle). (B) Marginal zone of the apparently normal spleen. (C) Hypertrophy and hyperplasia of lymphoid follicles very marked (circle). (D) Detail of lymphoid follicle with abundant lymphoblasts (LB) and lymphocytes (L), and some red blood cells (RBC). (E) Detail of spleen with hypertrophy and especially hyperplasia of the lymphoid follicles (circle). (F) Detail of lymphoid follicle with lymphocytes (L), lymphoblasts (LB) and reticular cells (RC). (G) Histological score showing a semiquantitative analysis of hypertrophy and hyperplasia of lymphoid follicles in the spleen. The pathology scores were as follows: 0, without signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical signiﬁcance (two tailed Student-t test), * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not damaged. (A,C,E), optical microscopy. Scale bars: 100 µm. (B,D,F), ultrastructural observations. Scale bars: (B,D), 10 µm; (F), 5 µm. Int. J. Mol. Sci. 2022, 23, 1170 14 of 25 d m- 14 of 25 d m- bars: (B,D), 10 μm; (F), 5 μm. Figure 12. Histopathology of the lungs of mice from the different treatments assayed. (A) Lung in which an apparently normal bronchus (BR) and respiratory lobules (RL) stand out. (B) Detail of capillary and pulmonary alveolus separated by the respiratory barrier (circle) formed by the endo- thelial cell (EC) and pneumocyteI (NI). (C) Detail of lung that shows a marked atelectasis in both respiratory lobule (RL) and alveoli (ALV). (D) Detail of lung with capillary hyperemia (circle) and hypertrophy of pneumocytes II (NII). (E) Lung detail showing atelectasis zones (circle) and emphy- sema (arrow). (F) Septal hypertrophy of pneumocytes II (NII). (G) Histological score showing a semiquantitative analysis of atelectasis in the lung. 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage (A) Lung in which an apparently normal bronchus (BR) and respiratory lobules (RL) stand out. (B) Detai of capillary and pulmonary alveolus separated by the respiratory barrier (circle) formed by the endothelial cell (EC) and pneumocyteI (NI). (C) Detail of lung that shows a marked atelectasis in both respiratory lobule (RL) and alveoli (ALV). (D) Detail of lung with capillary hyperemia (circle and hypertrophy of pneumocytes II (NII). (E) Lung detail showing atelectasis zones (circle) and emphysema (arrow). (F) Septal hypertrophy of pneumocytes II (NII). (G) Histological score showing a semiquantitative analysis of atelectasis in the lung. The pathology scores were as follows: 0, withou signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical signiﬁcance (two tailed Student-t test), * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not damaged (A,C,E), optical microscopy. Scale bars: 100 µm. (B,D,F), ultrastructural observations. Scale bars ( ) 2 ( ) 5 Figure 12. Histopathology of the lungs of mice from the different treatments assayed. (A) Lung in which an apparently normal bronchus (BR) and respiratory lobules (RL) stand out. (B) Detail of capillary and pulmonary alveolus separated by the respiratory barrier (circle) formed by the endo- thelial cell (EC) and pneumocyteI (NI). (C) Detail of lung that shows a marked atelectasis in both respiratory lobule (RL) and alveoli (ALV). (D) Detail of lung with capillary hyperemia (circle) and hypertrophy of pneumocytes II (NII). (E) Lung detail showing atelectasis zones (circle) and emphy- sema (arrow). (F) Septal hypertrophy of pneumocytes II (NII). (G) Histological score showing a semiquantitative analysis of atelectasis in the lung. The pathology scores were as follows: 0, without significant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two Figure 12. Histopathology of the lungs of mice from the different treatments assayed. (A) Lung in which an apparently normal bronchus (BR) and respiratory lobules (RL) stand out. (B) Detail of capillary and pulmonary alveolus separated by the respiratory barrier (circle) formed by the endothelial cell (EC) and pneumocyteI (NI). 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage The pathology scores were as follows: 0, without significant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two Figure 12. Histopathology of the lungs of mice from the different treatments assayed. (A) Lung in which an apparently normal bronchus (BR) and respiratory lobules (RL) stand out. (B) Detail of capillary and pulmonary alveolus separated by the respiratory barrier (circle) formed by the endothelial cell (EC) and pneumocyteI (NI). (C) Detail of lung that shows a marked atelectasis in both respiratory lobule (RL) and alveoli (ALV). (D) Detail of lung with capillary hyperemia (circle) and hypertrophy of pneumocytes II (NII). (E) Lung detail showing atelectasis zones (circle) and emphysema (arrow). (F) Septal hypertrophy of pneumocytes II (NII). (G) Histological score showing a semiquantitative analysis of atelectasis in the lung. The pathology scores were as follows: 0, without signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical signiﬁcance (two tailed Student-t test), * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not damaged. Figure 12. Histopathology of the lungs of mice from the different treatments assayed. (A) Lung in which an apparently normal bronchus (BR) and respiratory lobules (RL) stand out. (B) Detail of capillary and pulmonary alveolus separated by the respiratory barrier (circle) formed by the endo- thelial cell (EC) and pneumocyteI (NI). (C) Detail of lung that shows a marked atelectasis in both respiratory lobule (RL) and alveoli (ALV). (D) Detail of lung with capillary hyperemia (circle) and hypertrophy of pneumocytes II (NII). (E) Lung detail showing atelectasis zones (circle) and emphy- sema (arrow). (F) Septal hypertrophy of pneumocytes II (NII). (G) Histological score showing a semiquantitative analysis of atelectasis in the lung. The pathology scores were as follows: 0, without significant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two Figure 12. Histopathology of the lungs of mice from the different treatments assayed. 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage (C) Detail of lung that shows a marked atelectasis in both respiratory lobule (RL) and alveoli (ALV). (D) Detail of lung with capillary hyperemia (circle) and hypertrophy of pneumocytes II (NII). (E) Lung detail showing atelectasis zones (circle) and emphysema (arrow). (F) Septal hypertrophy of pneumocytes II (NII). (G) Histological score showing a semiquantitative analysis of atelectasis in the lung. The pathology scores were as follows: 0, without signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (51–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical signiﬁcance (two tailed Student-t test), * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not damaged. (A,C,E), optical microscopy. Scale bars: 100 µm. (B,D,F), ultrastructural observations. Scale bars: (B,F), 2 µm; (D), 5 µm. Int. J. Mol. Sci. 2022, 23, 1170 15 of 25 15 of 25 The absence of Gal3 protected against damage in the liver (Figure 10E,F and Figure S4E,F), spleen (Figure 11E,F and Figure S5E,F), and lung (Figure 12E,F and Figure S6E,F). Hence, in the Gal3KOLPS group, the lesions were scarce, although the hypertrophy and hyperplasia of the lymphoid follicles of the spleen were maintained (Figure 11E and Figure S5E). The liver maintained its structure and showed little hepatic steatosis (Figure 10E). Moreover, the lung maintained its bronchial and alveolar composition organs (Figure 12E). g p g g We also analyzed the effect of LPS-induced endotoxemia in the brain and the effect of genetic deletion of Gal3 in these conditions (Figure 13A–G and Figures S7A–F and S8D). In- triguingly, most important lesions after LPS treatment were found in the cerebral cortex. Both optical and electron microscopies and immunohistochemistry against Iba1 showed an in- crease and mobilization of glia cells, diffuse and in nodulations (Figure 13C,D,H,I). Microglia also showed the presence of lysosomal components similar to lipofuscin (Figure S7C,D). In Gal3KOLPS animals, although there was an increase in glial cells, there were scarcely any neuronal modiﬁcations (Figure 13E,F and Figure S7E,F). R REVIEW 15 of 25 highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical significance (two tailed Student-t test), * p < 0.001. 3. Discussion In this study, we have analyzed the role of Gal3 during endotoxemia, a condition that causes over-activation of the immune system, thus leading to a pro-inﬂammatory response. Importantly, LPS-induced endotoxemia is often used to model the acute inﬂammatory response associated with early sepsis [6]. Indeed, it is known that in the pathogenesis of LPS-induced endotoxemia, SIRS and coagulation disturbance play a pivotal role, ﬁnally leading to disseminated intravascular coagulation (DIC) and multiple organ dysfunction syndrome (MODS). Under these conditions, clinical manifestations such as fever, hypother- mia, tachypnea, tachycardia, leukocytosis, and leukopenia, among others, are evident. Molecular events include activation of TLR4 with the subsequent expression and release of pro-inﬂammatory cytokines and upregulation of tissue factor and coagulation-related pro- teases to further lead to DIC along with impairment of blood ﬂow [33]. These pathological events are critically associated with organ failure [34], hence the need to ﬁnd effective ther- apies to treat the excessive and uncontrolled systemic inﬂammatory response [35,36].We provide evidence that the absence of Gal3 reduces the toxicity in a model of LPS-induced en- dotoxemia as evidenced by a signiﬁcant reduction in the mortality rate and more preserved tissue integrity. Our study may shed light onGal3-related functions under conditions of endotoxemia and sepsis. p Gal3 is strongly expressed in myeloid cells including monocytes, macrophages, DCs, and neutrophils [37]. Subcellular localization of Gal3 includes cytoplasm, nucleus, and membrane [37,38]. Interestingly, membrane-associated Gal3 plays immune-associated roles under acute and chronic inﬂammation [37]. We took advantage of ﬂow cytometry to measure the amount of membrane-bound Gal3 in most blood immune cells after LPS challenge, including monocytes, neutrophils, DCs, and different subsets of T cells and B cells. Myeloid cells showed much higher levels of membrane-bound Gal3, especially monocytes. As a rule, after systemic LPS challenge, all immune cells lacked the ability to bind Gal3 at the membrane surface. This ﬁnding supports the notion that Gal3 may act as an alarmin—i.e., a molecule that, when released by immune cells, is able to induce a sterile immune or inﬂammatory response [39,40]. We measured serum levels of Gal3 under control and LPS conditions and found, unexpectedly, a signiﬁcant decrease under conditions of endotoxemia, a clear indication that Gal3 is either internalized by blood immune cells, degraded or extravasated to peripheral organs. 2.5. Gal3 Depletion Protects from Tissular Damage S ti f diff t l 2.5. Gal3 Depletion Protects from Tissular Damage Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KO, Gal3 knockout mice; KOLPS, Gal3 knockout mice treated with LPS; ND, not dam- aged. (A,C,E), optical microscopy. Scale bars: 100 μm. (B,D,F), ultrastructural observations. Scale bars: (B,F), 2 μm; (D), 5 μm. Figure 13. Histopathology of the brains of mice from the different treatments assayed. (A) Detail of cerebral cortex with abundant neurons. (B) Detail of apparently normal neuron and oligodendro- cyte (circle). (C) Detail of cerebral cortex, showing mobilization of glia cells (circle). (D) Detail of a damaged neuron densified and vacuolized. (E) Detail of cerebral cortex with a tenuous hyperemia Figure 13. Histopathology of the brains of mice from the different treatments assayed. (A) Detail cerebral cortex with abundant neurons. (B) Detail of apparently normal neuron and oligodendrocy Figure 13. Histopathology of the brains of mice from the different treatments assayed. (A) Detail of cerebral cortex with abundant neurons. (B) Detail of apparently normal neuron and oligodendro- cyte (circle). (C) Detail of cerebral cortex, showing mobilization of glia cells (circle). (D) Detail of a Figure 13. Histopathology of the brains of mice from the different treatments assayed. (A) Detail of cerebral cortex with abundant neurons. (B) Detail of apparently normal neuron and oligodendrocyte Int. J. Mol. Sci. 2022, 23, 1170 16 of 25 (circle). (C) Detail of cerebral cortex, showing mobilization of glia cells (circle). (D) Detail of a damaged neuron densiﬁed and vacuolized. (E) Detail of cerebral cortex with a tenuous hyperemia (circle). (F) Detail of neuron with lipofucsin precursor granules (Gr). (G) Histological score show- ing brain damage. The pathology scores were as follows: 0, without signiﬁcant injuries (0%); 1, minimum (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, marked (5–75%); 5, severe (>75%). Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. Results are mean ± SD of N = 3 animals. Statistical signiﬁcance (two tailed Student-t test), * p < 0.001. Abbreviations: WT, wild type mice; WTLPS, wild type mice treated with LPS; KOLPS, Gal3 knock- out mice treated with LPS; ND, not damaged. (A,C,E), optical microscopy. Scale bars: 100 µm. (B,D,F), ultrastructural observations. Scale bars: (B,F), 5 µm; (D), 2 µm. (H) Immunoﬂuorescence of Iba1 showing a normal patter in microglial cells staining. (I) When animals were treated with LPS microglial cells activate and proliferate. Scale bars (H,I): 100 µm. 3. Discussion Indeed, since we found a complete lack of Gal3 binding by circulating immune cells, the possibility that Gal3 may thus spread to different organs to orchestrate immune-associated functions is certainly plausible. Gal3 has been found to promote leukocyte adhesion to the endothelium and slow rolling, and recruitment of neutrophils and monocytes [41,42]. Consequently, an efﬁcient recruitment of leukocytes during acute inﬂammatory response may rely on Gal3 recruitment to target tissues. This assumption would require the unequivocal presence of highly reactive Gal3 in peripheral organs. Thus, we analyzed the expression of Gal3 in liver, spleen, and lung 36 h after LPS challenge. Our immunohistochemical analysis revealed a robust up-regulation of Int. J. Mol. Sci. 2022, 23, 1170 17 of 25 17 of 25 Gal3 in WT but not in Gal3KO animals in all analyzed organs, thus sustaining an important role of this galectin in driving immune-related responses under conditions of endotoxemia. Gal3 in WT but not in Gal3KO animals in all analyzed organs, thus sustaining an important role of this galectin in driving immune-related responses under conditions of endotoxemia. g g To delve into the modulation of immune cells by Gal3 at the molecular level, the expression levels of TRL4, a Gal3 ligand, was measured 1 h after LPS injection in WT and Gal3KO mice. This analysis demonstrates a nice effect of Gal3 deletion on TLR4 expression in macrophages as it prevented the LPS-induced expression of this receptor (Figure 3). It is known that TLR4 is one of the main drivers of microglia activation [43], triggering several transduction pathways, such as the nuclear factor kappa B (NF-κB) pathway and the mitogen-activated protein kinases (MAPKs) pathway, which cause increased expression of inﬂammatory cytokines. Therefore, our results show that the effects on immune responses driven by Gal3 could be mediated by TLR4 and its downstream signaling cascade. y y g g Studies aimed at deﬁning the role of Gal3 under conditions of sepsis and endotoxemia have led to opposing conclusions. In a previous study, Li et al. reported that Gal3 acts as a negative regulator of LPS-induced endotoxemia and inﬂammation, in sharp contrast to our study [29]. According to their study, animals lacking Gal3 succumbed earlier to systemic LPS and exhibited higher serum TNF-α levels after LPS challenge. In the same study, Gal3 deletion exacerbated the LPS-induced inﬂammatory response of bone marrow-derived macrophages [29]. 3. Discussion The authors found that Gal3 binds to LPS, thus inhibiting the LPS- associated pro-inﬂammatory response as tested in bone marrow-derived macrophages [29]. A plausible interpretationof this study would be that Gal3 represses LPS function (i.e., by interfering with the interaction of LPS with its receptor complex). However, the functional relevance of the interaction between Gal3 and LPS in vitro is far from clear [31,32]. It is intriguing that studies reporting potential interaction between Gal3 and LPS have led to opposite effects, including enhancement of the pro-inﬂammatory activity of LPS [44] and inhibition of the pro-inﬂammatory activity of LPS [29]. Indeed, our qPCR analysis was not enough for drawing any conclusions about the pro-inﬂammatory role of Gal3 in our model of endotoxemia in the different organs analyzed. A plausible explanation for this apparent discrepancy is related to the fact that time-associated cytokine changes are very fast and transient [45]. Interestingly, increased circulating levels of TNF-α and IL-6 are considered the main features of the inﬂammatory response associated with sepsis [2] and play a key role in the pathophysiology of severe sepsis [46]. In fact, among the cytokines whose levels are increased during sepsis, IL-6 is the one that shows a better correlation with the mortality rate [47]. Consequently, we measured the circulating levels of both pro-inﬂammatory cytokines in our experimental animals. Our study demonstrates that, in the absence of Gal3, the early LPS-induced peak of circulating TNF-α and IL-6 is signiﬁcantly attenuated, thus further supporting the deleterious effect of Gal3 under conditions of endotoxemia. Our previous studies on microglial cells revealed a clear synergistic effect of Gal3 on the LPS-induced pro-inﬂammatory response [7,20]. Thus, we demonstrated that activated microglia release Gal3, which plays an essential role for the full microglial response upon LPS stimulation [7]. In previously published papers, our group has used two experimental approaches to inhibit Gal3 in vitro (BV2 microglial cell line): Gal3 expression was sup- pressed using siRNA and Gal3 blocking antibody was used to neutralize the effects of the released Gal3. In this study, we observed that both methods prevented the LPS-induced inﬂammation. Moreover, to validate the Gal3 effect over the inﬂammatory response upon LPS stimulus, the release of several proinﬂammatory cytokines were checked in WT and Gal3KO primary cell cultures, conﬁrming the BV2 cell data, with reduced inﬂammatory response in Gal3KO microglia [7]. 3. Discussion Indeed, Gal3 inhibition by either gene silencing or using a neutralizing antibody repressed the LPS-induced pro-inﬂammatory response of microglia, and importantly, we demonstrated that Gal3 acts as an endogenous true ligand of TLR4 [7]. Quite different studies support the view that Gal3 acts as a powerful pro-inﬂammatory signal including activation of the NADPH oxidase system [48] and stimulation of super- oxide production from neutrophils [49]. Gene deletion of Gal3 suppresses production of pro-inﬂammatory cytokines and reduces NLRP3 inﬂammasome activation, the molecular platform leading to caspase-1 activation and subsequent cleavage and release of IL-1β [50]. thus further supporting the deleterious effect of Gal3 under conditions of endotoxemia. Our previous studies on microglial cells revealed a clear synergistic effect of Gal3 on the LPS-induced pro-inﬂammatory response [7,20]. Thus, we demonstrated that activated microglia release Gal3, which plays an essential role for the full microglial response upon LPS stimulation [7]. In previously published papers, our group has used two experimental approaches to inhibit Gal3 in vitro (BV2 microglial cell line): Gal3 expression was sup- pressed using siRNA and Gal3 blocking antibody was used to neutralize the effects of the released Gal3. In this study, we observed that both methods prevented the LPS-induced inﬂammation. Moreover, to validate the Gal3 effect over the inﬂammatory response upon LPS stimulus, the release of several proinﬂammatory cytokines were checked in WT and Gal3KO primary cell cultures, conﬁrming the BV2 cell data, with reduced inﬂammatory response in Gal3KO microglia [7]. Indeed, Gal3 inhibition by either gene silencing or using a neutralizing antibody repressed the LPS-induced pro-inﬂammatory response of microglia, and importantly, we demonstrated that Gal3 acts as an endogenous true ligand of TLR4 [7]. Quite different studies support the view that Gal3 acts as a powerful pro-inﬂammatory signal including activation of the NADPH oxidase system [48] and stimulation of super- oxide production from neutrophils [49]. Gene deletion of Gal3 suppresses production of pro-inﬂammatory cytokines and reduces NLRP3 inﬂammasome activation, the molecular platform leading to caspase-1 activation and subsequent cleavage and release of IL-1β [50]. Int. J. Mol. Sci. 2022, 23, 1170 18 of 25 18 of 25 Further, Gal3 has been shown to amplify inﬂammation of atherosclerotic plaque progres- sion through macrophage activation and monocyte chemoattraction [51]. Overall, we may conclude that data supporting a pro-inﬂammatory role of Gal3 areoverwhelming. A plausi- ble explanation for the contrasting effects of Gal3 on LPS-associated endotoxemia found by Li et al. 3. Discussion (2008) and our study may rely on the LPS doses used in each study. The lethal dose of 50 (LD50) LPS is about 1–25 mg/kg [2]. While Li et al. used an LPS dose of 23 mg/kg (upper limit), we used a dose of 5 mg/kg (lower limit). These differences in LPS doses may certainly be behind the opposite effects (beneﬁcial vs detrimental) ascribed to Gal3 under conditions of endotoxemia. Serum levels of Gal3 increase in patients suffering from sep- sis [25,27,52] and in experimental animal models of sepsis such as that induced by CLP [27]. In fact, Gal3 is useful as a biomarker for the prediction of mortality in sepsis [25,53]. A detrimental role of Gal3 is deduced from Gal3KO mice experiments suffering CLP-induced sepsis, which are more resistant than WT mice [27]. In a different model of sepsis induced by infection with Francisellanovicida, the absence of Gal3 led to signiﬁcantly longer sur- vival time, and showed reductions in levels of well-established sepsis mediators including vascular injury markers, inﬂammatory cytokines, and acute phase proteins [28]. The am- plifying pro-inﬂammatory nature of Gal3 was evaluated in vitro in bone marrow-derived macrophages and peritoneal neutrophils in response to Francisellanovicida [28]. p g p p p To demonstrate conclusively that Gal3 plays a deleterious role in SIRS, and considering that appearance of MODS is critical for life expectancy of patients, we took advantage of (i) optic and electronic microscopy techniques to analyze those organs severely compromised during SIRS including liver, spleen, and lung and (ii) analysis of the death rate. Our microscopy analysis demonstrated that the absence of Gal3 preserved tissue integrity in all analyzed organs. Interestingly, we also found that LPS-induced endotoxemia produced signiﬁcant brain damage, especially in the cortex, with strong glial mobilization. It has been demonstrated that cytokine production is an early event after sepsis, participating in blood– brain barrier alterations and brain damage [54]. The absence of Gal3, however, prevented these alterations in all the organs examined. Indeed, most solid evidence sustaining a detrimental role of Gal3 triggered by LPS-induced endotoxemia relies on the delayed death rate ascribed to Gal3KO mice. The absence of Gal3 signiﬁcantly decreased the mortality rate following LPS treatment as compared with WT animals. Early treatment of sepsis is necessaryto minimize fatal outcomes. Our study supports the usage of inhibitors of Gal3 to increase the therapeutic window in which other treatments could be administered. 4. Materials and Methods 4.1. Animals and Treatments 4.1. Animals and Treatments 12–15-week-old male C57BL/6 (wild-type, WT) and Gal3 null mutant (Gal3KO) mice (20–25 g) were obtained from the Center of Production and Animal Experimentation (Espartinas, Seville, Spain). They were housed at a constant room temperature of 22 ± 1 ◦C and relative humidity (60%), with a 12-h light-dark cycle and ad libitum access to food and water. Animals were distributed within 4 groups according to two different variables: solu- tion administrated intraperitoneally (saline or LPS in 0.9% sterile saline) and genotype (WT or Gal3KO). In the WT group (the control group), WT animals received an intraperitoneal injection of saline; in the KO group, Gal3KO animals received an intraperitoneal injection of saline; in the WTLPS group, WT animals received an intraperitoneal injection of LPS (from Escherichia coli serotype 0111: B4; Sigma Aldrich, St. Louis, MO, USA); ﬁnally, in the KOLPS group, Gal3KO animals received an intraperitoneal injection of LPS. All treatments were administrated in a volume of 50 µL per 25 g of body weight. Animals were sacriﬁced at different time points depending on the technique assayed. These time points were chosen based on previous studies [45,55]. For qPCR, enzyme-linked immunosorbent assay (ELISA), and ﬂow cytometry assays, animals were sacriﬁced 1 h after the saline/LPS injection, time in which inﬂammatory markers are high according to the literature [56,57]. For histological studies, animals were sacriﬁced 36 h after the injection. Int. J. Mol. Sci. 2022, 23, 1170 19 of 25 19 of 25 At least 3 animals per group were used, except for the survival study in which 12 animals were used per group. Whenever possible, peritoneal macrophages, blood, and organs were obtained from the same animal (Figure 14). R REVIEW 19 of 25 Figure 14. Timeline of the treatments with LPS in animals, and points at which the different param- eters have been measured. WT and Gal3KO animals were injected with either LPS or saline and sacrificed at different time points depending on the technique assayed. The number of animals used in each experiment isalso indicated. Figure 14. Timeline of the treatments with LPS in animals, and points at which the different param- eters have been measured. WT and Gal3KO animals were injected with either LPS or saline and sacriﬁced at different time points depending on the technique assayed. The number of animals used in each experiment isalso indicated. Figure 14. 4.2. Survival Analysis In order to select 4.2. Survival Analysis In order to select the optimal LPS dose to be used, a dose response study using 5 (Figure S1A), 7.5 (Figure S1D), 10 (Figure S1A–C), and 15 mg/kg (Figure S1A,B) was per- formed to analyze survival following LPS challenge. To assess the effect of this treatment and verify the appearance of typical alterations of endotoxemia, animals were observed every 6 h for general health. The symptomatology was evaluated for 80 h and scored using a scale (Table 1) based on our observations and the criteria proposed by Benavides et al. [58]. From this analysis, a dose of 5 mg/kg LPS was selected, in accordance with previous studies [45,59]. In order to select the optimal LPS dose to be used, a dose-response study using 5 (Figure S1A), 7.5 (Figure S1D), 10 (Figure S1A–C), and 15 mg/kg (Figure S1A,B) was performed to analyze survival following LPS challenge. To assess the effect of this treatment and verify the appearance of typical alterations of endotoxemia, animals were observed every 6 h for general health. The symptomatology was evaluated for 80 h and scored using a scale (Table 1) based on our observations and the criteria proposed by Benavides et al. [58]. From this analysis, a dose of 5 mg/kg LPS was selected, in accordance with previous studies [45,59]. Table 1. Criteria for classifying endotoxemia according to the severity of the symptoms. Score Symptomatology 0 No symptoms 1 Reducedmobility and tremor 2 Reduced mobility, tremor, and prostration 3 Death Table 1. Criteria for classifying endotoxemia according to the severity of the symptoms. Score Symptomatology 0 No symptoms 1 Reducedmobility and tremor 2 Reduced mobility, tremor, and prostration 3 Death Table 1. Criteria for classifying endotoxemia according to the severity of the symptoms. Table 1. Criteria for classifying endotoxemia according to the severity of the symptoms. o symptoms mobility and tremor y, tremor, and prostration Death No symptoms Reducedmobility and tremor Reduced mobility, tremor, and prostration Death 4.1. Animals and Treatments Timeline of the treatments with LPS in animals, and points at which the different param- eters have been measured. WT and Gal3KO animals were injected with either LPS or saline and sacrificed at different time points depending on the technique assayed. The number of animals used in each experiment isalso indicated. Figure 14. Timeline of the treatments with LPS in animals, and points at which the different param- eters have been measured. WT and Gal3KO animals were injected with either LPS or saline and sacriﬁced at different time points depending on the technique assayed. The number of animals used in each experiment isalso indicated. 4.4. Isolation and Culture of Primary Peritoneal Macrophages Peritoneal macrophages were isolated from mice from different experimental groups according to the protocol developed by Davies [60]. First, animals were anesthetized with isoﬂurane. Subsequently, 10 mLof a 1× PBS solution was injected intraperitoneally, and the entire body was shaken for 10 s. Then, PBS-containing resident peritoneal cells were withdrawn, centrifuged (400× g 5 min), and resident peritoneal cells were resuspended in an RPMI-1640 medium (Sigma-Aldrich, San Luis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, San Luis, MO, USA) containing 50 IU of penicillin, 50 µg streptomycin, and 2 mM glutamine per milliliter (Gibco, Invitrogen Ltd., Waltham, MA, USA). Three hundred thousand cells per well were plated in 24-well plates and incubated for 60 min at 37 ◦C. Then, the non-adherent cells were removed by being washedﬁve times with PBS. 4.5. RNA Extraction and cDNA Synthesis Liver and spleen were extracted 1 h after different treatments and stored at −80 ◦C until use. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s recommendations. The samples were previously disrupted by the use of a TissueLyser II (Qiagen, Düsseldorf, Germany). On the other hand, total RNA from peritoneal macrophages was isolated using TRIsure™(Bioline, London, UK), following the instructions established by the manufacturer. In both cases, the amount of total RNA obtained in each sample was measured spectrophotometrically using the NanodropTM 2000/2000c (Thermo Fisher Scientiﬁc, Waltham, MA, USA). The volume required of each sample was calculated to perform reverse transcription of 1 µg of RNA using the RevertAid™First Strand cDNA kit Synthesis Kit (Thermo Fisher Scientiﬁc, Waltham, MA, USA). The RT-PCR was performed in a PTC-100 thermocycler (MJ research Inc., Waltham, MA, USA) using a program consisting of 5 min at 25 ◦C, then 60 min at 42 ◦C and, ﬁnally, 5 min at 70 ◦C. The resulting cDNAs were stored at −80 ◦C until use. 4.3. Flow Cytometry 4.3. Flow Cytometry Mice were anesthetized and blood was collected from the heart. To obtain peripheral mononuclear cells (PBMCs), blood was collected and placed in a cold ammonium chloride lysis solution (82.8 g/L NH4CL, 10.1 g/L KHCO3, 0.37 g/L EDTA (MERCK, Darmstadt, Ger- many)) for 10 min. Then, cellular suspension was washed twice with a cold phosphate-buff- ered saline (PBS). Th PBMC i d b f k ib di f 20 i 4 °C A ib d Mice were anesthetized and blood was collected from the heart. To obtain peripheral mononuclear cells (PBMCs), blood was collected and placed in a cold ammonium chloride lysis solution (82.8 g/L NH4CL, 10.1 g/L KHCO3, 0.37 g/L EDTA (MERCK, Darmstadt, Germany)) for 10 min. Then, cellular suspension was washed twice with a cold phosphate- buffered saline (PBS). Int. J. Mol. Sci. 2022, 23, 1170 20 of 25 Then, PBMCs were stained by surface marker antibodies for 20 min at 4 ◦C. Anti- bodies used were: anti-Ly6G FITC (REA526), anti-CD3e VioBlue (145-2C11), anti-Ly6C PE (1G7.G10), anti-CD45 APC (30F11) (Miltenyi Biotec, Bergisch Gladbach, Germany), anti- CD11c PerCPCy5.5 (N418), anti-MHC-II SB600 (M5/114.15.2), CD4 AF700 (GK1.5), CD11b PE-eFluor610 (M1/70), CD19 SB702 (eBio1D3) (eBioscience, San Diego, CA, USA), CD8 APC- eFluor 780 (53–6.7) (Invitrogen, Waltham, MA, USA), and anti-Gal3 PECy7 (M3/38) (Biolegend, San Diego, CA, USA). Dead cells were excluded using LIVE/DEAD fixable Aqua Blue Dead Cell Stain (Life Technologies, Carlsbad, CA, USA). Gal3 expression was analyzed in B-cells (CD19+CD3e−), CD4+ T-cells (CD3e+CD4+CD8−), CD8+ T-cells (CD3e+CD4−CD8+), double negative T-cells (CD3e+CD4+CD8−), dendritic cells (DCs) (CD3e−CD19−CD11c+MHC-II+), neutrophils (CD3e−CD19−CD11c+CD11b+Ly6G+Ly6Cdim), monocytes (CD3e−CD19−CD11 c+CD11b+Ly6G-Ly6Chigh), and macrophages (CD3e−CD19−CD11c+CD11b+Ly6G-Ly6C−). A representative gating strategy is shown in Figure S2. Fluorescence Minus One (FMO) plus isotype antibody controls were used to determine the expression of Gal3 in each cellular subset of stimulated with LPS and unstimulated mice (Figure S3). A minimum of 500,000 events were acquired. Flow cytometry was performed on a BD LSR Fortessa (BD Bioscience, Mississauga, ON, Canada). Analysis was performed using FlowJo version 9.2 (Tree Star, Ashland, OR, USA) and data are expressed as frequencies (%). 4.7. ELISA Fresh peripheral blood was collected from animals that had receivedthe different treatments in vacuum ﬁlled tubes and incubated at room temperature for 30 min to allow coagulation. Then, the blood was centrifuged at 2000× g for 15 min at 4 ◦C to obtain serum, which was stored at −40 ◦C until used. Serum Gal3, TNF-α, and IL-6 concentrations were determined by using a Mouse Galectin-3 Duo Set ELISA DY1197-05, a Mouse TNF alpha ELISA Ready-SET-GO! ®, and a Mouse IL-6 ELISA Ready-SET-GO! ® kit (Affymetrix, Santa Clara, CA, USA), respectively, following the manufacturer’s instructions. The plates were read on a Synergy HT multimodal plate reader (BioTek, Winooski, VT, USA) set to 450 nm. All conditions were assayed in duplicate. 4.6. Real Time PCR Real-time PCR was performed with SensiFASTTM SYBR No-ROX Kit (Bioline, Lon- don, UK), 0.4 µM primers, and 4 µg cDNA. Controls were carried out without cDNA. Ampliﬁcation was run in a LightCycler 480 (Roche Molecular Systems, Pleasanton, CA, USA) thermal cycler at 95 ◦C for 2 min followed by 40 cycles of 95 ◦C for 5 s, 65 ◦C for 10 s, and 72 ◦C for 20 s, and ended with a cycle of 7 min at 72 ◦C. Following ampliﬁcation, a melting curve analysis was performed by heating the reactions from 65 to 95 ◦C in 0.1 ◦C/s Int. J. Mol. Sci. 2022, 23, 1170 21 of 25 while monitoring ﬂuorescence. Analysis conﬁrmed a single PCR product at the melting temperature. β-actin served as the reference gene and was used for sample normalization. Primer sequences for β-actin, inducible nitric oxide synthase (iNOS), TNF-α, IL-1β, IL-6, Gal3, arginase, YM1 (chitinase-like 3), and TLR4 are shown in Table 2. The cycle at which each sample crossed a ﬂuorescence threshold (Ct) was determined, and the triplicate values for each cDNA were averaged. Analyses of real-time PCR were done using a 2Ct relative quantiﬁcation method [61]. Table 2. Sense and antisense sequences of the primers used for the analysis of mRNA expression by qPCR. by qPCR. Gene Forward (5′–3′) Reverse (5′–3′) β-actin 5′-CCACACCCGCCACCAGTTCG-3′ 5′-CCCATTCCCACCATCACACC-3′ Gal3 5′-GATCACAATCATGGGCACAG-3′ 5′-GTGGAAGGCAACATCATTCC-3′ TNF-α 5′-TGCCTATGTCTCAGCCTCTTC-3′ 5′-GAGGCCATTTGGGAACTTCT-3′ iNOS 5′-CTTTGCCACGGACGAGAC-3′ 5′-TCATTGTACTCTGAGGGCTGAC-3′ Arginase 5′-TCACCTGAGCTTTGATGTCG-3′ 5′-CTGAAAGGAGCCCTGTCTTG-3′ YM1 5′-GTACCCTGGGTCTCGAGGAA-3′ 5′-GCCTTGGAATGTCTTTCTCAC-3′ IL-6 5′-GACAAAGCCAGAGTCCTTCAGA-3′ 5′-AGGAGAGCAATTGGAAATTGGGG-3′ IL-1β 5′-TGTAATGAAAGACGGCACACC-3′ 5′-TCTTCTTTGGGTATTGCTTGG-3′ IL 10 5′-CCAAGCCTTATCGGAAATGA-3′ 5′-TTTCACAGGGAGAAATCG-3′ TLR-4 5′-GCCTCGAATCCTGAGCAAAC-3′ 5′-CTCTCGGTCCATAGCAGAGC Abbreviations: Gal3, galectin-3; TNF-α, tumor necrosis factor α, iNOS, inducible nitric oxide synthase; YM1, protein type chitinase 3; IL, interleukin; TLR4, Toll-like receptor 4. Abbreviations: Gal3, galectin-3; TNF-α, tumor necrosis factor α, iNOS, inducible nitric oxide synthase; YM1 protein type chitinase 3; IL, interleukin; TLR4, Toll-like receptor 4. Abbreviations: Gal3, galectin-3; TNF-α, tumor necrosis factor α, iNOS, inducible nitric oxide synthase; YM1, protein type chitinase 3; IL, interleukin; TLR4, Toll-like receptor 4. 4.10. Immunoﬂuorescence of Iba-1 and Gal3 Animals were perfused and brain sections were prepared as described above. Incu- bations and washes for all the antibodies were conducted in PBS, pH 7.4. All work was performedat room temperature, unless otherwise noted. Sections were blocked with PBS containing 5% BSA for 2 h. The slides were then incubated overnight at 4 ◦C with the primary antibodies: rabbit-derived anti-Iba1 (Wako, Osaka, Japan; 1:500) and goat-derived anti-Gal3 (R&D Systems, Minneapolis, Canada, USA; 1:200). Primary antibodies were diluted in PBS containing 1% BSA and 1% Triton X-100. After three washes in PBS, sections were incubated with secondary antibodies conjugated to Alexa Fluor® 488 (Invitrogen, Waltham, MA, USA; 1:500) and Alexa Fluor® 647 (Invitrogen, Waltham, MA, USA; 1:500) for 2 h at room temperature in the dark. Fluorescence images were acquired using a confocal laser scanning microscope (Zeiss LSM 7 DUO, Oberkochen, Germany) and processed using the associated software package (ZEN 2010, Oberkochen, Germany). 4.11. Light and Electron Microscopy Tissue samples for histological examination from the liver, spleen, lung, and brain were taken 36 h after the saline/LPS injection. For light microscopy, samples were ﬁrst ﬁxed in a 10% buffered formalin for 24 h at 4 ◦C, and then immediately dehydrated in graded series of ethanol, immersed in xylol, and embedded in parafﬁn wax using an automatic processor. Sections of 3–5 µm were mounted. After they had been deparafﬁnized, the sections were rehydrated, stained with hematoxylin and eosin, and mounted with Cristal/Mount [62]. A semiquantitative evaluation of the histological damage was made, including hypertrophy and hyperplasia of lymphoid follicles, steatosis, atelectasis, glial cell mobilizations, and catarrhal steatosis. For electron microscopy (MET), the samples were preﬁxed in 2% glutaraldehyde ﬁxative (in pH 7.4 phosphate buffer for 10 h at 4 ◦C) and postﬁxed in 1% osmium tetroxide ﬁxative (in pH 7.4 phosphate buffer for 0.5 h at 4 ◦C). Subsequently, they were dehydrated in graded ethanol series and embedded in EPON. Ultra-thin sections, 50–60 nm, were cut with an LKB microtome. The sections were mounted on a copper grid and stained with uranylacetate and lead citrate. The tissue sections were examined in a JEOL JEM 1400 MET (Tokyo, Japan) [62]. For the evaluation of the tissue damage, we rely on an observation of the histopatho- logical sections under an optical microscope, which is veriﬁed by observations under an electronic microscope. In addition, a semi-quantitative evaluation of the observations was conducted. Histopathological evaluation is performed in a blind manner by two highly experienced pathologists. 4.9. Immunohistochemistry Data Analysis 4.9. Immunohistochemistry Data Analysis Analysis was performedusing the AnalySIS imaging software (Soft Imaging System GmbH, Münster, Germany) coupled witha Polaroid DMC camera (Polaroid, Cambridge, MA, USA) attached to a Leika light microscope (LeikaMikroskopie, Wetzlar, Germany). For counting cells showing Gal3, CD4, and CD68 immunoreactivity, a systematic sampling of the area occupied by positive cells for these markers was made from a random starting point with a grid adjusted to count ﬁve ﬁelds per section. An unbiased counting frame of the known area (40 × 25 µm = 1000 µm2) was superimposed on the tissue section image under a 40× objective. Cells showing Gal3, CD4, or CD68 immunoreactivity were counted using ﬁve ﬁelds per section and three sections per animal and the number of cells was expressed as cells per mm2. 4.8. Immunohistological Evaluation of Gal3, CD4 and CD68 Thaw-mounted 20-µm liver, lung, and spleen sections were cut on a cryostat at −15 ◦C and mounted in gelatin-coated slides. Primary antibodies used were goat-derived anti- Gal3 (R&D Systems, Minneapolis, MN, USA; 1:100), rat-derived anti-CD4 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:100), and mouse-derived anti-CD68 (Invitrogen, Waltham, MA, USA; 1:100). Incubations and washes were conducted in Tris-buffered saline (TBS), pH 7.4. All work was performedat room temperature. Sections were washed and then treated with 0.3% hydrogen peroxide in methanol for 20 min, washed again, and incubated in a solution containing TBS and 1% horse/goat serum (Vector, Burlingame, CA, USA) for 60 min in a humid chamber. Slides were drained and further incubated with the primary antibody in TBS containing 1% horse/goat serum and 0.25% TritonX-100 for 24 h. Sections were then incubated for 2 h with biotinylated horse anti-goat, goat anti- mouse, or goat anti-rat IgG (Vector, Burlingame, CA, USA; 1:200). The secondary antibody was diluted in TBS containing 0.25% TritonX-100, and its addition was preceded by three 10-min rinses in TBS. Sections were then incubated with VECTASTAIN®Elite®ABC Kit, peroxidase (Vector, Burlingame, CA, USA). The peroxidase was visualized with a standard diaminobenzidine/hydrogen reaction for 5 min. Int. J. Mol. Sci. 2022, 23, 1170 22 of 25 22 of 25 4.12. Statistical Analysis The sample size was calculated with the ENE 3.0 software (GlaxoSmithKline, Madrid, Spain). Animals were randomly distributed so that each box had different ages and weights. Results are expressed as mean ± SD. Means were compared by one-way ANOVA followed by the Fisher’s LSD test for post hoc multiple range comparisons, unless otherwise noted. Int. J. Mol. Sci. 2022, 23, 1170 23 of 25 23 of 25 For ﬂow cytometry, Prism, version 5.0 (GraphPad Software Inc., San Diego, CA, USA) was used for the generation of the graphs [63]. Statistical signiﬁcance analysis was calculated using the Mann–Whitney U test. Results are expressed as median ± IQR. For Gal3 ELISA and histological studies, a two tailed Student’s t test was used. The comparison of the survival curves was performed with the Kaplan–Meier method; speciﬁcally, the Log-Rank test was used. An alpha level of 0.05 was used. The Statgraphics 18 × 64 statistical package and the Statistical Package for the Social Sciences software (SPSS 21.0, New York, NY, USA), were used for the analyses. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/ijms23031170/s1. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/ijms23031170/s1. Author Contributions: Conceptualization, R.M.d.P. and J.L.V.; methodology and investigation, J.C.F.-M., A.M.E.-O., I.G.-D., I.R.-S., and Y.M.P.; data analysis, R.M.d.P., J.L.V., I.R.-S., R.M., and J.G.M.; writing—original draft preparation, R.M.d.P., J.L.V., and A.M.E.-O.; writing—review and editing, R.M.d.P., J.L.V., A.M.E.-O., Y.M.P., R.M., and J.G.M.; supervision, R.M.d.P. and J.L.V.; funding acquisition, J.L.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants by the Spanish Ministerio de Ciencia, Innovación y Universidades/FEDER/UE RTI2018-098645-B-100), Junta de Andalucía (Consejería de Economía y Conocimiento, US-1264806) and FIS (PI18/01216). YMP was supported by the Consejería de Salud y Bienestar Social of Junta de Andalucía through the “NicolásMonardes” programme [C-0013-2017]. Institutional Review Board Statement: The study was conducted according to the guidelines of the European Union Directive (2010/63/EU) and Spanish regulations (BOE 34/11370-421, 2013) for the use of laboratory animals; the study (project code: 23/03/2018/035) was approved by the Scientiﬁc Committee of the University of Seville (date of approval: 8 June 2018). Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4.12. 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https://openalex.org/W4309454964	https://scindeks-clanci.ceon.rs/data/pdf/0353-9008/2022/0353-90082258333M.pdf	Serbian	null	The proces of establishing Xenocracy in Balkan states during 19th century	Baština	2,022	cc-by	6,394	Мирослав С. МИЛОШЕВИЋ* Институт за српску културу Приштина – Лепосавић Мирослав С. МИЛОШЕВИЋ* Институт за српску културу Приштина – Лепосавић 2 Филики Хетерија или „Друштво пријатеља“ била је национално-ослободилачка организација основана 1814. године, у Одеси од стране три грчка трговца Емануела Ксантоса, Николаса Ску- фаса и Атанасиоса Цакалова. Након Бечког конгреса, став великих сила био је да спречава било какве револуционарне покрете на старом континенту. Вођство Хетерије је у том смислу одлу- чило да се окрене сопственим снагама, тражећи подршку међу балканским хришћанима. Орга- низација је ангажовала 12 чланова тзв. „апостола“ да раде на ширењу идеје о опште-балканском устанку. Хетерија је окупљала разне слојеве становништва: сељаке, земљопоседнике, свештен- ство, клефте, као и фанариоте, међу којима је био и будући вођа овог покрета, руски генерал Александар Ипсиланти (Galant 2001: 14-15). * Истраживач приправник, miroslav.milosevic@rcup.pr.ac.rs р р р , @ p p ** Рад је настао у оквиру научноистраживачког рада НИО по Уговору склопљеним са Министар- ством просвете, науке и технолошког развоја број: 451-03-68/2022-14 од 17. 01. 2022. године. БАШТИНА, Приштина – Лепосавић, св. 58, 2022 БАШТИНА, Приштина – Лепосавић, св. 58, 2022 DOI: https://doi.org/10.5937/bastina32-39908 DOI: https://doi.org/10.5937/bastina32-39908 Прегледни рад * Истраживач приправник, miroslav.milosevic@rcup.pr.ac.rs Рад је настао у оквиру научноистраживачког рада НИО по Уговору склопљеним са Министар- ством просвете, науке и технолошког развоја број: 451-03-68/2022-14 од 17. 01. 2022. године. 1 Грч. xenos странац, kratia владавина: владавина странаца, владавина туђе династије (Клајн–Шип- ка 2006: 675). 2 Филики Хетерија или „Друштво пријатеља“ била је национално-ослободилачка организација основана 1814. године, у Одеси од стране три грчка трговца Емануела Ксантоса, Николаса Ску- фаса и Атанасиоса Цакалова. Након Бечког конгреса, став великих сила био је да спречава било какве револуционарне покрете на старом континенту. Вођство Хетерије је у том смислу одлу- чило да се окрене сопственим снагама, тражећи подршку међу балканским хришћанима. Орга- низација је ангажовала 12 чланова тзв. „апостола“ да раде на ширењу идеје о опште-балканском устанку. Хетерија је окупљала разне слојеве становништва: сељаке, земљопоседнике, свештен- ство, клефте, као и фанариоте, међу којима је био и будући вођа овог покрета, руски генерал Александар Ипсиланти (Galant 2001: 14-15). р , у р ј р ј 1 Грч. xenos странац, kratia владавина: владавина странаца, владавина туђе династије (Клајн–Шип- ка 2006: 675). ПРОЦЕС КОНСТИТУИСАЊА КСЕНОКРАТИЈЕ МЕЂУ БАЛКАНСКИМ ДРЖАВАМА ТОКОМ XIX ВЕКА Апстракт: Почетак XIX века био је у знаку буђења национално-ослободилачких покрета на Балкану. Овај процес покренула је Српска револуција, оличена у Првом и Другом српском устанку. Немири у Београдском пашалуку подстакли су и остале балканске народе на непослушност према централним властима у Цариграду. Порта је на ове догађаје покушала да одговори низом разних реформи и амнестијом, надају- ћи се да ће на тај начин пасивизирати немире који су све више добијали национални карактер. Криза на истоку није промакла великим европским силама: Русији, Аустро- угарској, Француској и Великој Британији. Заинтересоване за територије посрнулог Османског царства, силе су у складу са својим интересима, подржавале или спутавале покрете балканских народа. Под патронатом, да ће решити њихова национална и соци- јална питања, обећавајући благостање у земљи, велике силе су настојале да именују сво- је претенденте за владаре у Грчкој, Румунији и Бугарској. Тако су Грци најпре добили монарха из баварске династије Вителсбах, а затим из владарске породице Холштајн – Готорп – Зонденбург – Гликсбург, Румуни из династије Хоенцолерн – Сигмаринген и Бугари из породице Сакс – Кобург – Гота. Кључне речи: ксенократија, Грчка, Румунија, Бугарска, Отон I, Ђорђе I, Карол I, Александар од Батенберга, Фердинанд I. Феномену ксенократије1 претходили су догађаји из прве половине XIX века, када је избио устанак грчког народа против Османског царства. Револуционари из Хетерије2 под вођством Александра Ипсилантија започели су дуготрајан и сасвим 334 Мирослав С. Милошевић суров сукоб3 који се одвијао у три фазе: I фаза (1821-1825) период успона, II фаза (1825-1827) криза устаника и офанзива Турака и III фаза до 1829. године интер- венција великих сила и победа револуције (Ђорђевић 1995: 31). Британска влада била је под све већим притиском да подржи револуционарне покрете грчког на- рода. Њени трговачки интереси били су угрожени догађајима у Грчкој. Уосталом, Енглези су се прибојавали да се Русија не умеша у решавање источног питања (Jelavich 1983: 225). У вези с тим је у Петрограду допутовао изасланик британ- ске владе Велингтон који је са Неселродеом потписао тзв. Петроградски протокол (април 1826.) о решењу грчког питања. Радило се о врло широкој аутономији Грч- ке под султановим сизеренством. Силама потписницама придружила се и Фран- цуска склапањем трипартитног Лондонског протокола у јулу 1827. године. Тако су велике силе наступале јединствено у решавању грчког питања (Поповић 2007: 110-112). Централне власти у Цариграду нису прихватиле одлуке поменутог про- токола и уместо тога султан је објавио „џихад“ Грцима. Сукоб је окончан фамозним Једренским миром 1829. године. ПРОЦЕС КОНСТИТУИСАЊА КСЕНОКРАТИЈЕ МЕЂУ БАЛКАНСКИМ ДРЖАВАМА ТОКОМ XIX ВЕКА** Победнички став Русије засметао је британској империји. Тада је Велика Британија почела да захтева пуну самосталност Грчке, како би тиме умањила руски утицај. У фебруару 1830. године, нови Лондонски уго- вор претворио је Грчку у прву независну државу на Балкану, под протекторатом Велике Британије, Француске и Русије (Павловић 2004: 59-60). 4 На челу намесништва био је Јозеф Армансперг. Иностраним пословима и дипломатијом бавио се Карл Абел, професор права Лудвиг Маурер старао се о кодификацији закона, црквеним и про- светним пословима, генерал-мајор Карл Хеидек, филхелен, био је учесник грчке револуције и њему је припао задатак да реформише војску, Јохан Греинер бавио се економским проблемима (Jelavich 1983: 254) 3 На немире у Грчкој, одговориле су убрзо турске власти тако што су на Васкрс обесиле у Цари- граду на вратима патријаршије самог патријарха Григорија V и три владике. Такође, покољ Грка на острву Хиос изазвао је огорчење европског јавног мњења (Ћоровић 2007: 70-71). БАВАРСКИ ПРИНЦ НА ОЛИМПУ Уочи руско-турског рата, у фебруару 1828. године за председника Грчке иза- бран је Јанис Каподистријас. Међутим, Енглези су били суревњиви према Капо- дистријасу, оптуживши га за руског агента. Суочен са притиском великих сила, није успео да испуни њихова очекивања нити да изгради карактер народног вође (Jelavich 1983: 228). Каподистријас је водио преговоре са силама о будућем стату- су грчке државе. За грчког монарха предложио је британског изабраника Леополда од Сакс Кобурга. Али тај предлог није прошао, будући да је Леополд постао краљ новоосноване Белгије. Након убиства Јаниса Каподистријаса, у Грчкој је завладала анархија. У таквом метежу, Русија, Велика Британија и Француска су одлучиле да укину републику и устав из 1827. и за грчког краља прогласе Отoа фон Вителсбаха, који се искрцао у Нафплиону 6. фебруара 1833. године (Malešević 2018: 543). Син баварског краља Лудвига I, био је малолетан када је именован за краља Грч- ке. Све до 1835. године, земљом је владало намесништво.4 У току своје владавине, намесништво није наишло на подршку и симпатије грчког становништва. Покази- вали су незаинтересованост за грчку традицију, трудећи се да државу организују 3 На немире у Грчкој, одговориле су убрзо турске власти тако што су на Васкрс обесиле у Цари- граду на вратима патријаршије самог патријарха Григорија V и три владике. Такође, покољ Грка на острву Хиос изазвао је огорчење европског јавног мњења (Ћоровић 2007: 70-71). Процес конституисања ксенократије међу балканским државама током XIX века 335 на западњачки начин. Просвету су реорганизовали према француском и немачком моделу и прогласили аутокефалност од Цариградске Патријаршије 1833. године (Clogg 1992: 49-50). Једна од непопуларних мера намесника била је распуштање устаничких одреда и увођење најамничке војске, што је неколико хиљаде ветерана из ослободилачких ратова оставило без службе и средстава за живот. Појавила се хајдучија у земљи и њеним суседним областима, као вид отпора према ксенократији (Ђорђевић 1995: 44-45). У прилогу томе говори чињеница да су незадовољни Грци период владавине намесништва називали „Баварократијом“ (Galant 2001: 32). Тако је грчки ослободилачки покрет 30-их и 40-их година имао двојако усмерење. У Кра- љевини Грчкој носио је карактер борбе за демократију, уставност и парламентари- зам, а противио се ксенократији и апсолутизму. Ван граница Краљевине све је било подређено ослобођењу грчких територија од Турака и њиховом уједињавању с ма- тицом земљом (Попов 2010: 180). Грчки краљ Отон I преузео је власт 1835. године. Убрзо се оженио принцезом Амалијом од Олденбурга. БАВАРСКИ ПРИНЦ НА ОЛИМПУ Милошевић и тако приморале Отона да повуче устаничке трупе (Clogg 1992: 55-57). Народ је у њему видео кривца за нарушавање односа са гарантним силама. Почетком 60- их година назирао се његов пад. Енглези су сумњали у његове антиосманске везе, Французе је узнемиравала његова наклоност Аустрији, док је Русе бринула чиње- ница што није имао православног наследника. Средином октобра 1862. избијали су немири широм Грчке. Долазило је до сукоба војске и цивила у Нафплиону, Ла- мији, Аркадији. Краљ је покушао да обиласком земље утиче на смиривање тензија (Павловић 2007: 113-114; Српске новине, 9. 10. 1862: 422). Побуна која је избила у октобру, доста је подсећала на догађаје из 1843. године. Није било револуционар- ног покрета од стране широких народних маса. Уместо тога, то је у суштини био војни удар (Jelavich 1983: 263). Крајем октобра формирана је привремена влада. Српске новине су преносиле вести страних дописништава која су се бавила грчким питањем. Преко телеграма могло се сазнати, да је у неким местима, као на пример у Атини владао мир. Привремена влада је у име народа и војске збацила краља с власти (Српске новине, 16. 10. 1862: 436). Краљевски пар је напустио земљу 27. ок- тобра и отпутовао за Венецију. Лондонски листови „The Times“ и „Daily News“ неочекивано су са одобравањем писали о догађајима у Грчкој. Истакли су право грчког народа да сами бирају своју владу. Намера Енглеза била је да се новој влади пружи подршка, само и уколико Грци не крену у процес решавања националног питања, што је директно угрожавало Османско царство на Балкану (Српске новине, 18. 10. 1862: 440). Краљ Отон I је остатак свог живота провео у родној Баварској, одевен у фустанели (грчка народна ношња). Његов последњи акт била је подршка грчким устаницима на Криту. Умро је 1867. године (Clogg 1992: 59). БАВАРСКИ ПРИНЦ НА ОЛИМПУ У народу није био популаран зато што је пре свега био католик, иако је обећао да ће његов наследник бити васпи- тан у православном духу. Да је имао деце, можда би његови проблеми били мање озбиљни (Jelavich 1983: 259). Народ је своје незадовољство испољавао кроз захтеве за увођење устава. Ветеранима и народним првацима сметала им је претерано попу- њена администрација странцима, потом апсолутни монарх, али и присуство много образованијих Грка који су се доселили из иностранства. Ситуација је кулминирала септембра 1843. године (Павловић 2004: 92). Атмосфера у Атини је тог 15. септем- бра била узаврела. У два часа после поноћи, окупљени народ уз подршку атинског гарнизона пуковника Калергиса, кренуо је ка краљевској палати захтевајући доно- шење устава. Конзули страних сила су покушали да дођу до краља, али су у томе били спречени. Отон је у преговорима са државним саветом одлучио да прихвати захтеве за доношење устава и то обзнанио окупљеном народу уз повике: „Да живи уставни краљ, да живи устав!“ (Српске новине, 29. 9. 1843: 312-313). Други период Отонове владавине званично је почео 18. марта 1844. године, на дан проглашења новог устава (Galant 2001: 40). Уставом су задржана широка овла- шћења монарха, чији је наследник требало да буде његов најближи рођак православ- не вере. Међутим, нови устав није уклонио неприлике у којима се држава нашла, нити ју је ослободио од туторства великих сила. За новог председника владе име- нован је Јанис Колетис, који је у спољној политици остао познат по Великој идеји (Мегали идеа), националном програму за ослобођење и уједињење свих крајева где живе Грци (Павловић 2007: 93-94). Краљ Отон је морао да се суочава и са критика- ма младе грчке интелигенције, на коју су значајно утицали догађаји из 1848. и 1849. године. Они су изражавали критички став према уставу из 1844. године, као и на цео политички систем, желећи да у својој домовини конституишу парламентарну демо- кратију и да краљевску власт сведу на минимум (Jelavich 1983: 263). У доба Кримског рата, Отон је покушавао да се ослободи утицаја великих сила. Проценио је, да је наступила повољна ситуација за остваривање планова из „Велике идеје“. Уз прећутну сагласност грчких власти, устаници су изазивали немире у сусед- ним областима Епира, Тесалије и јужне Македоније. Међутим, Француска и Велика Британија су водиле политику очувања Османског царства. Из предострожности су извршиле поморску блокаду Грчке која је трајала од маја 1854. до фебруара 1857. 336 Мирослав С. ЂОРЂЕ I ГРЧКИ Именовање новог грчког краља уско је било повезано са Јонским питањем. Јон- ски парламент се изјаснио за уједињење с Грчком, одбацујући енглеске реформне пројекте (Ђорђевић 1995: 69). Суочена с масовном жељом житеља Јонских острва за уједињење са матицом и са новим порастом руског угледа међу њима, енглеска влада је крајем 1862. донела одлуку да острва уступи Грчкој. Енглеске трупе су у марту 1864. напустиле Јонска острва (Попов 2010: 395). Привремена грчка влада је за новог владара дала подршку принцу Алберту, другом сину британске краљице Викторије, надајући се да ће на тај начин добити благонаклоност ове силе по пи- тању Јонских острва као и будуће евентуалне територијалне експанзије. Међутим, став гарантних сила био је да се будући владар не именује из владајуће династије (Jelavich 1983: 263). У својим мемоарима о грчком краљу, дански морнарички офи- цир капетан Валтер Кристмас, навео је да су Енглези имали своје кандидате: принц Ернест из династије Сакс-Кобурга, зет краљице Викторије, затим њен рођак принц Леининген, као и архидук Аустрије Максимилијан. Руси су своју подршку пружили војводи од Лихтенберга и принцу Вилхему из Бадена, а Французи су захтевали да на престо дође војвода Хенри од Орлеана, син краља Луја Филипа I. Избор је пао на седамнаестогодишњег принца Вилхема Данског. Затим је, јуна 1863. године, потпи- сан Лондонски протокол од стране амбасадора Француске, Русије и Данске, чиме је Процес конституисања ксенократије међу балканским државама током XIX века 337 та одлука званично и потврђена (Christmas 1914: 39-40). Тако се, без регента ис- крцао у Грчку октобра 1863. нови краљ под именом Ђорђе I (Павловић 2007: 115) Положивши заклетву у Народној скупштини, краљ је издао прокламацију на- роду: „Ја нити вам доносим вештину у владању, нити искусан разум. Нити ви мо- жете очекивати таква својства од мојих година. Но ја вам ипак доносим искрену љубав и дубоку веру у будућу заједницу моје и ваше судбе…“ (Српске новине, 29. 11. 1863: 486). Краљ Ђорђе I је био добро упознат са грчким националним осећањима. Свог сина и престолонаследника Константина, васпитавао је у православном духу (Galant 2001: 45). Устав из 1864, на коме се у новембру заклео краљ, завео је парламентарни ре- жим, заснован на једнодомној скупштини. Круна је ипак задржала један значајан прерогатив – да именује и смењује министре, као и да распушта скупштину. Краљ Ђорђе је женидбом руском великом кнегињом Олгом прибавио корисне династич- ке везе својој владавини. 5 Кримски рат добио је свој коначни епилог на Конгресу великих сила одржаног у Паризу од 25. фебруара до 16. априла 1856. године. У раду Конгреса су учествовали високи представници (ми- нистри спољних послова) Велике Британије, Француске, Аустрије, Русије, Пруске, Сардиније и Османског царства. Париским мировним уговором Русији су нанети тешки политички и тери- торијални губици. Одузета јој је област Бесарабија, њени протекторати над Влашком, Молдави- јом и Србијом, као и њено право заштите над хришћанима у Турској, замењени су колективном гаранцијом свих шест европских сила (Попов 2010: 274). ЂОРЂЕ I ГРЧКИ Међутим, није могао тако лако да се дистанцира од утицаја оних који су га довели на престо. То се најпре манифестовало избијањем Критског устанка у јесен 1866. године. Грчка је слала помоћ Криту, изазивала устанке у су- седним областима, али је пред захтевима великих сила капитулирала 1869. и морала да обустави све акције (Павловић 2007: 116-118). Идентична ситуација поновила се током Велике источне кризе 1875-1878. Грчка влада нерадо је заузела неутра- лан став. Промена тог става, могла је да изазове интервенцију Енглеске, окупацију Крита или блокаду Пиреја, слично као 1854. године (Ђорђевић 1995: 88-89). Та- кво држање Грчке, и њена наклоност великим силама спасила је земљу од тоталног колапса у пролеће 1897, током рата са Османским царством. Правовременом ин- тервенцијом Енглеске и Француске, спречена је катастрофа Грчке (Galant 2001: 51- 53). Краљ је намеравао да се повуче са престола на прослави педесетогодишњице његовог устоличења. Говорио је да његово време пролази и да је прави тренутак да власт преда своме сину Константину. Током шетње улицама Солуна, уз минимално обезбеђење, пуцано је на краља 18. марта 1913. у 17.20 часова. Његове последње речи биле су: „Хвала Богу, сада Кристмас своје последње поглавље може завршити у славу Грчке, њене војске и престолонаследника“ (Christmas 1914: 405-407). ХОЕНЦОЛЕРНИ У РУМУНИЈИ – КАРОЛ I Одредбе Париског конгреса5 из 1856. године, имале су велики значај за Дунав- ске кнежевине. Руски протекторат приведен је крају. Наступио је период утицаја европских гарантних сила (Jelavich 1983: 287). Упоредо с тим акумулирао је покрет уједињења Влашке и Молдавије. Избори су, септембра 1857. дали обема кнежевинама 338 Мирослав С. Милошевић подршку присталицама Уније. Затим је у октобру проглашено уједињење, али су га- рантне силе у Паризу одлучиле да кнежевине остају одвојене. Међутим, претходно покренут процес уједињења није се више дао зауставити. Власти у Влашкој и Молда- вији су почетком 1859. изабрале кнеза Александра Кузу за свог владара. Уједињење је обављено формално децембра 1861. чиме је створена Кнежевина Румунија (Ђор- ђевић 1995: 71). Владавина румунског кнеза није дуго трајала. Поред тога што није успевао да спроведе реформе, на тапету су били многобројни скандали везани за његов приватни живот. У фебруару 1866. завереници су упали у његов двор и при- морали га на абдикацију. Формирано је тројно намесништво и привремена влада. Петорица кандидата претендовала су на румунски престо. Владало је опште мишље- ње да је неопходно за кнеза изабрати странца, и то из више разлога: стране подршке, стабилности, спречавања деспотизма и радикализма (Павловић 2004: 136). Избор је прво пао на грофа Фландрије, млађег брата белгијског краља Леопол- да II. Од тога се убрзо одустало, јер је француски владар Наполеон III, као један од утицајних суверена на старом континенту, саветовао грофу да одбије понуђе- ну круну. У међувремену, ситуација се у Европи знатно закомпликовала због рата Пруске и Аустрије. Енглези су се оправдано прибојавали утицаја Русије на Дунав- ске кнежевине. Европи је тада било нужно јединство. Румунска влада се уздала у подршку Наполеона III. Пажњу су усмерили ка принцу Карлу од Хоенцолерна, који је био у сродству и са француском и са пруском династијом. Нужно је било убедити принца да што пре прихвати понуђену круну и добије подршку на плеби- сциту (Whitman 1899: 9-10). За овакав деликатан задатак био је одређен вођа румунских либерала Јон Бра- тијану. Он је на Велики петак 1866. допутовао у Диселдорф, како би изложио по- нуду румунског народа принцу Карлу и његовом оцу Карлу Антону. Наредног дана 31. марта, Братијану је упутио представку у којој је изразио жељу Намесништва и Наполеона III, да принц Карло постане један од кандидата румунског престола. Исте вечери Братијану је разговарао са његовим оцем о политичкој ситуацији у земљи. ХОЕНЦОЛЕРНИ У РУМУНИЈИ – КАРОЛ I Поручено му је да има довољно храбрости да прихвати понуду, али се ипак прибојавао да његов син није раван таквом задатку. Стога је одбио дати било какав дефинитиван одговор на предлоге румунске владе. Када је Карло Антони отпуто- вао за Берлин, на пријем код свог рођака и краља Фридриха Вилхема I пруски краљ није желео да разговара о румунском питању. Наиме, њему се није допала та идеја да је француски суверен инспиратор те кандидатуре. На крају је саветовао свог ро- ђака да одбије понуду румунске владе (Whitman 1899: 11-13). ј Пре него што је пристао на понуду Румуна, организован је плебисцит, чијим је исходом са 689.969 гласова према 224, изабран принц Карло. Уставотворна скуп- штина га је у мају 1866. уздигла за владајућег кнеза Румуније са румунизованим именом Карол I (Павловић 2004: 137). Међутим, убрзо је у Диселдорфу стигло писмо пруског краља, који је упозоравао на последице уколико дође до прихватања ове одлуке. Наиме, према његовим речима, Османско царство, Русија и Аустрија су биле противнице уније Дунавских кнежевина. Идентичног става била је и Енглеска. Пруском суверену се није допала идеја да један Хоенцолерн постане султанов ва- зал. На крају је закључио да млади принц најпре мора да добије сагласност Русије, Процес конституисања ксенократије међу балканским државама током XIX века 339 земље која је тада имала доста утицаја на Дунавске кнежевине. Став грофа Бизмар- ка, премијера и шефа пруске дипломатије, био је опречан: „Вас је народ одабрао да владате, одазовите се том позиву“, поручио је принцу Карлу Антону. На крају је саветовао принца да пре него што упути свог сина за Румунију, пошаље једно пи- смо руском цару у коме ће га назвати својим заштитником и партнером у решавању источног питања. Бизмаркова улога несумњиво је била пресудна у решавању ру- мунског питања. Како би придобио краља, намеравао је да крене правцем полити- ке свршеног чина. Зато је одмах препоручио принцу Карлу да без даљих устезања, крене у Букурешт (Whitman 1899: 20-23). Пошто је принц Карло био официр у пруској војсци, а рат између Пруске и Аустрије претио је да почне сваког часа, морао је да путује прерушен преко хаб- збуршке територије. У своју нову земљу стигао је маја 1866. године (Jelavich 1983: 295). Стигавши у Букурешт, градоначелник му је уручио кључеве града, затим је поворка кренула ка једној скромној једноспратној згради, предвиђеној за владаре- ву резиденцију. На крају су се зауставили испред градске катедрале. 6 Архив Србије, Министарство иностраних дела, Посланство Кнежевине Србије у Букурешту, фа- сциклаI, документ р1/881, Извештај секретара Посланства о свечаностима поводом крунисања румунског краља Карола I, 16. мај 1881. ХОЕНЦОЛЕРНИ У РУМУНИЈИ – КАРОЛ I Ту је Карол I положио заклетву (Whitman 1899: 30-31). у Након Велике источне кризе, кнежевина Румунија је на Берлинском конгресу добила независност. Већ 1881. године, проглашена је краљевином (Павловић 2004: 190). Свечаном крунисању Карола I присуствовао је српски посланик у Букурешту Милан Петронијевић. Неколико детаља о томе дао је секретар посланства Иван Занковић у извештају Министарству иностраних дела. Секретар је своје утиске на- писао на основу вести из румунске штампе. Краљ је у аудијенцији примио српског посланика и изразио захвалност и симпатије поводом честитке коју је већ примио од српског владара. Потом је приређен свечани ручак за званице. Наредног дана приређена је смотра војске, међутим, због јаке кише, овај перформанс одржан је сутрадан. Највећи утисак на окупљени народ оставили су добровољци, док је варо- шка гарда разочарала. Тиме је низ церемонија крунисања био завршен.6 Боје дина- стије Хоенцолерна и њихов мото Nihil sine deo (лат. Ништа без Бога), нашле су се на грбу краљевине, како би повезале династију и државу. Круна за румунског краља искована је од гвожђа турског топа заробљеног током рата. Једино Каролово дете је умрло, те је његов синовац Фердинанд усвојен да буде наследник. Румунски краљ Карол I умро је у октобру 1914. године (Павловић 2004: 190-191, 311). ОД ПРУСКОГ ГАРДИСТЕ ДО БУГАРСКОГ КНЕЗА Бугарска је после Берлинског конгреса била подељена на два дела: Кнежевину Бугарску и Источну Румелију, које су у формалном погледу биле део Османског царства (Павловић 2004: 202). Иако је првобитно било одређена двогодишња оку- пација руске војске, Берлински уговор је ипак то ограничио на само девет месеци. Највећи добитак Русије после рата било је стварање аутономне бугарске државе. 6 Архив Србије, Министарство иностраних дела, Посланство Кнежевине Србије у Букурешту, фа- сциклаI, документ р1/881, Извештај секретара Посланства о свечаностима поводом крунисања румунског краља Карола I, 16. мај 1881. 340 Мирослав С. Милошевић Руски званичници били су одлучни у намери да успоставе јаку и стабилну владу која ће у будућности бити блиско везана за руске интересе (Jelavich 1983: 367). Тај задатак поверен је руском комесару кнезу Дондукову-Корсакову. У пролеће 1879. године, усвојен је Трновски устав. Према уставу Бугарска је проглашена наследном монархијом, а наслеђивање се вршило по линији прворођеног мушког престолонаследника. Законодавна власт била је дата Народној скупштини, а за- коне је одобравао кнез. Народна скупштина је постала основни орган државне власти, а извршење законских аката било је дато Министарском савету, тј. Влади (Попов, Божилов 2008: 242). Царска Русија одиграла је значајну улогу око избора новог владара. Цар Александар II настојао је да именује кандидата који ће бити прихватљив како за ве- лике силе тако и за Русију. Међутим, испоставиће се да је погрешио у тој намери. Најозбиљнији претендент за бугарски престо био је Александар од Батенберга, други син принца Александра од Хесе-Дармштата. Њега су одликовале најпре ди- настичке везе са угледним владарским породицама на старом континенту. Носио је чин поручника у пруској гарди, док је његов отац био генерал у аустријској вој- сци. Александрова тетка Марија била је заправо супруга руског цара Александра II. Његов брат Луис Батенберг био је ожењен унуком британске краљице Викто- рије. Као млади официр учествовао је у руско-турском рату 1877-1878, на страни руске армије. Када је изабран за бугарског кнеза 29. априла 1879. имао је свега двадесет и две године. Савременици су га сматрали за згодног и шармантног чове- ка, који је поред јаких династичких веза имао и угледно војно порекло. Истог дана када је сазнао да је изабран за бугарског кнеза, Александар је у Палати Ливадија на Криму примио бугарску делегацију која је званично донела позив свом новом монарху. Том приликом је од руског цара добио унапређење у чин генерал-мајо- ра руске армије и дозвољено му је да задржи официрско звање у пруској војсци. 7 Када је извршен војни удар у Пловдиву, кнез Александар и Каравелов били су у недоумици: да увреде Русију ако прихвате унију или огорче јавност ако је не прихвате. Ситуацију је убр- зао председник народне скупштине Стамболов када је изјавио кнезу да бира између одласка у Пловдив и повратка у Дармштат. Тада је Александар отишао у Пловдив и потврдио уједињење (Павловић 2004: 205). ОД ПРУСКОГ ГАРДИСТЕ ДО БУГАРСКОГ КНЕЗА (Rekun 2019: 36-38). На почетку своје владавине кнезу Александру није пријало руско туторство. О томе сведочи писмена коресподенција са румунским кнезом Каролом, у којој млади суверен наводи да је одан руском цару, али да се руски чиновници понашају безобзирно према њему: „У свим министарствима влада збрка. Сваки дан ја треба или да попуштам руским захтевима или да ме прогласе у Русији за незахвална чове- ка. Мој положај је ужасан“ (Гуч 1933: 3-4). Руски цар је очекивао од њега да ће су- збијати планове либерала, који су се иначе противили руској премоћи у Бугарској. Два кључна министарства у влади Бугарске (спољних послова и војске) држала су двојица руских генерала. На новим изборима 1884. године, либерали поново осва- јају власт. Њихове вође Каравелов и Стамболов, извршили су притисак на кнеза да из министарских места уклони руске генерале и одобри тајну акцију либерала на уједињење Бугарске и Источне Румелије. Уједињење7 је извршено у септембру Процес конституисања ксенократије међу балканским државама током XIX века 341 1885. Све потписнице Берлинског уговора су прећутно реаговале на ове догађаје. То је учињено с намером да би се из уједињене бугарске државе елиминисао руски утицај. Русија није признала „Пловдивски преврат“, затраживши санкције против кнеза Александра Батенберга (Попов 2010: 254-255). р р ( ) Став Русије, која је подржавала детронизацију Батенберга, не само да је дис- кредитовао руску дипломатију у читавој Европи већ је изазвао збуњеност у бугар- ској јавности. Петроград је бранио кнеза у време његове најниже популарности у земљи, а сада је хтео да сруши његов кнежевски ауторитет у тренутку када је он заслужио рехабилитацију пред својим народом. Руси су ишли толико далеко, да су челницима бугарске владе нудили помоћ у организацији завере против кнеза. Тра- жено је да Бугарска жртвује свог кнеза у замену за нејасно обећање да ће Русија бити њен добротвор. Кнез Александар Батенберг је 9. августа 1886. потписао абди- кацију, приморан од стране групе официра. Пуч је био извршен у једном историј- ски специфичном тренутку. Избори, обављени у уједињеној Бугарској јуна 1886, потврдили су опште расположење против руског покровитељства у унутрашњој и спољној политици. С друге стране, руско дириговање дипломатском изолацијом Бугарске стварало је утисак да је потребна одлучујућа промена ради стабилизације земље. Међутим, кнезу је из провинције пристигла подршка, па је 17. августа поно- во успоставио своје прерогативе. Очајни кнез је у писму руском цару Александру III изразио спремност да своју круну преда цару-престолонаследнику. 8 Генерал Николај Васиљевич Каулбарсов, био је руски војни аташе у Бечу и брат бившег бугар- ског министра војног. По наређењу руског императора Александра III, послат је у деликатну ми- сију у Бугарској 18. септембра 1886. Његов задатак је био да поврати посрнули руски утицај и да спаси земљу из дубоке политичке кризе настале након абдикације кнеза Александра Батенберга. Види више у: М. S. Rekun, How Russia lost Bulgaria 1878 – 1886, 178-183. ОД ПРУСКОГ ГАРДИСТЕ ДО БУГАРСКОГ КНЕЗА Хладни од- говор цара, ставио је кнеза у безизлазну позицију. Батенберг је дефинитивно абди- цирао 26. августа 1886. године (Попов, Божилов 2008: 252-254). ФЕРДИНАНД I БУГАРСКИ Након абдикације кнеза Батенберга у Бугарској је наступила политичка криза. Морало се пожурити с избором новог кнеза. То што на бугарском престолу није било никога значило је опасан преседан и реалну могућност да статус Источне Ру- мелије буде враћен на стање пре уједињења. Било је створено својеврсно двовла- шће. Између регенства и владе није чак изграђен ни заједнички став о питањима унутрашњег и спољног деловања и перспективе за Кнежевину Бугарску. Из тих ра- злога је, без пажљивих и тактичних консултација, 29. октобра 1886. изабран принц Валдемар Дански. Међутим, он је одустао од престола, препуштајући избор распо- ложењу двора у Петрограду (Попов, Божилов 2008: 255). Из руске престонице стигао је предлог да се за новог монарха Бугарске изабере грузијски кнез Никола од Мингрелије. Како је мисија руског изасланика Каулбарсова8 пропала, од тога није било ништа (Српске новине, 23. 11. 1886: 1088-1089). Из Берлина су долазиле вести да је истакнута кандидатура баварског принца Ханса од Шлезвиг-Холштајн- -Гликсбурга (Српске новине, 28. 11. 1886: 1104). 342 Мирослав С. Милошевић У међувремену, Стефан Стамболов је стао на челу намесништва које је имено- вало коалициону владу и сазвало скупштину ради избора новог кнеза (Павловић 2004: 206). Формирана је депутација која је путовала по Европи тражећи канди- дата за бугарски престо. Руски посланик у Бечу Лобанов, саопштио је члановима ове комисије да их у Петрограду нико неће примити. Депутација се затим упутила ка Берлину (Српске новине, 3. 12. 1886: 1120). По овом питању Порта је заступала јасан и непроменљиви став, а то је била кандидатура кнеза Николе, руског штиће- ника. Наравно, то је било у складу са њеним политичким и стратешким плановима према Бугарској (Српске новине, 4. 12. 1886: 1123). Посета Бечу била је делотворна за бугарску депутацију. Како угледни листови из тог периода преносе, на двору министра иностраних дела грофа Калнокија, боравио је принц Фердинанд Кобур- шки. Пренета је вест да би његова кандидатура могла да задовољи бугарске депу- тате (Српске новине, 5. 12. 1886: 1127). Лондонски „Times“ извештавао је да су Бу- гари понудили престо Фердинанду, али да је он из предострожности према своме владару и руском цару, заузео неутралан став по том питању (Српске новине, 6. 12. 1886: 1132). Бугарска депутација није била задовољна својим боравком у Берлину. Немачка влада им је препоручила да новог владара изаберу у непосредном спора- зуму са Русијом (Српске новине, 11. 12. 1886: 1144). На сличан пријем бугарски изасланици наишли су у Паризу и Лондону (Српске новине, 23. 12. ФЕРДИНАНД I БУГАРСКИ 1886: 1183). у у р у у ( р ) Полемика око избора новог владара настављена је у „Народно собрание“. На тајној седници Стамболов је поднео извештај о раду за последњих осам месеци и објаснио разлоге зашто принц Батенберг одбија да се врати у Бугарску. Наредног дана, 7. јула 1887. Скупштина је једногласно изабрала Фердинанда Кобуршког за кнеза Бугарске (Српске новине, 28. 6. 1887: 574). Фердинанд је био католик, рођен у Бечу. Отац му је био генерал у аустријској војсци, а мајка кћи краља Луја-Филипа. Био је у родбинским везама са монарсима и супружницима монарха Белгије, Ве- лике Британије и Португала из династије Сакс-Кобург-Гота. Дужност је преузео у августу, али је остао непризнат у наредних девет година. Односи са Русијом су почели да се нормализују након смрти цара Александра III 1894. године. Нови ру- ски владар Николај II пристао је да буде кум Фердинандовом сину Борису. Затим је бугарски кнез отпутовао у Цариград да положи вазалну заклетву султану, а потом се упутио у обилазак европских престоница (Павловић 2004: 206-208). Русија и ве- лике силе су га признале за владара Бугарске 1896. године, прихватајући уједињење са Источном Румелијом као свршен чин (Jelavich 1983: 372). НЕОБЈАВЉЕНИ ИЗВОРИ Архив Србије, Министарство иностраних дела, Посланство Кнежевине Србије у Букурешту, фа- сцикла I, документ р1/881, Извештај секретара Посланства о свечаностима поводом крунисања румунског краља Карола I, 16. мај 1881. ШТАМПА Српске новине, Београд. ЗАКЉУЧАК Гледајући хронолошки, процес конституисања ксенократије међу балканским државама у XIX веку, одвијао се најпре у Грчкој, затим у Дунавским кнежевинама односно Румунији, да би свој епилог добио у стварању Кнежевине Бугарске. Под- стицан од стране великих сила, сам процес није текао лако. Противнике је налазио у отпору конзервативних, либералних и осталих струја међу балканским земљама. Очекивало се да ће долазак страних владара омогућити бржи процес еманципације балканских држава од централних власти у Цариграду. Међутим, иза тог покрета Процес конституисања ксенократије међу балканским државама током XIX века 343 крила се идеја да се уз помоћу тих суверена сузбије сваки покрет усмерен против Османског царства. У том смислу, постепено је расло незадовољство у народу, што је касније резултирао сменом или абдикацијом владара. Чињеница је, да су на тај начин, велике силе настојале да источно питање реше у складу са својим спољно- -политичким интересима. р Грчки краљ Отон I преузео је власт у јако незахвалном моменту. У земљи је трајала анархија изазвана политичким нејединством. Очекивало се да ће он својим ауторитетом стати на пут таквом стању. Иако се трудио да што боље упозна грчки народ и њихову културу, ипак је на крају протеран из земље. Краљ Ђорђе I, показао је више умећа у владању од свог претходника, што несумњиво говори његова вла- давина дуга педесет година. Русија је свој утицај успела да наметне у Кнежевини Бугарској, именујући за владара младог пруског официра Александра од Батенбер- га. Ипак, он није испунио очекивања руског двора. Плаховит и политички невешт није смогао снаге да се супротстави спољашњим и унутрашњим притисцима. До- лазак Фердинанда I на власт у Бугарској, представљао је преседан у том моменту. Изабран од стране „Собрања“, нови бугарски кнез је тек неколико година касније признат за владара од стране великих сила. Владао је све до 1918. године. Уједиње- ње Влашке и Молдавије завршено је избором домаћег кнеза Александра Кузе за владара. Његова владавина није потрајала дуго. Идентично као код Бугара и Руму- ни су путовали по европским дворовима у потрази за адекватним кандидатом. На крају је испоштована воља Наполеона III, грофа Бизмарка али и румунског народа избором Карола I Хоенцолерна. Српске новине, Београд. ЛИТЕРАТУРА Whitman 1899: Whitman Sidney, Reminiscences of the King of Roumania, New York and London: Harper Brothers. p Galant 2001: Galant Thomas W., Modern Greece, New York: Oxford University Press. y Гуч 1933: Џорџ П. Гуч, Дипломатска историја модерне Европе 1878-1919, превео Ј.М. Јовано- вић, Београд: Издавачка књижарница Геце Кона, 1933. Гуч 1933: Џорџ П. Гуч, Дипломатска историја модерне Европе 187 вић, Београд: Издавачка књижарница Геце Кона, 1933. Ђорђевић 1995: Димитрије Ђорђевић, Националне револуције балканских народа 1804-1914, Београд: Службени лист СРЈ. Београд: Службени лист СРЈ. Jelavich 1983: Barbara Jelavich, History of the Balkans – eighteenth and nineteenth centuries, Vol. I, Cambridge: Cambridge University Press. Клајн, Шипка 2006: Иван Клајн, Милан Шипка, Велики речник страних речи и израза, Нови Сад: Прометеј, 2006. Clogg 1992: Richard Clogg, A Concise History of Greece, Cambridge: Cambridge University Press. 344 Мирослав С. Милошевић Christmas 1914: Christmas Walter, King George of Greece, London: Eveleigh Nash. Malešević 2018: Nikola Tošić Malešević. “Grčki ustanak protiv Osmanskog carstva i osnivanje modern grčke države”, Vojno delo, 3/2018, 527-545. Павловић 2004: Стеван К. Павловић, Историја Балкана 1804-1945, превод Чедомир Антић, Београд: Clio, 2004. р Поповић 2007: Васиљ Поповић, Источно питање. Историски преглед борбе око опстанка Осман- лиске царевине у Леванту и на Балкану, Београд: Репринт Никола Пашић. Поповић 2007: Васиљ Поповић, Источно питање. Историски преглед борбе око опстанка Осман- лиске царевине у Леванту и на Балкану, Београд: Репринт Никола Пашић. Попов-Божилов 2008: Димитар Попов, Иван Божилов и др. Историја Бугарске, приредио Срђан Пириватрић, превод Марија Јоана Стојадиновић, Београд: Clio. Попов-Божилов 2008: Димитар Попов, Иван Божилов и др. Историја Бугарске, приредио Срђан Пириватрић, превод Марија Јоана Стојадиновић, Београд: Clio. Срђан Пириватрић, превод Марија Јоана Стојадиновић, Београд: Clio. р р р р р ј ј р Попов 2010: Чедомир Попов, Грађанска Европа. Политичка историја Европе (1871-1914), том III, Београд: Завод за уџбенике. Попов 2010: Чедомир Попов, Грађанска Европа. Политичка историја Европе (1871-1914), том III, Београд: Завод за уџбенике. Попов 2010: Чедомир Попов, Грађанска Европа. Политичка историја Европе (1871-1914), том III, Београд: Завод за уџбенике. Rekun 2019: Mikhail S. Rekun, How Russia lost Bulgaria 1878 – 1886, New York – London: Lexing- ton books. Rekun 2019: Mikhail S. Rekun, How Russia lost Bulgaria 1878 – 1886, New York – London: Lexing- ton books. Ћоровић 2007: Владимир Ћоровић, Борба за независност Балкана, Београд: Logos art. Miroslav S. MILOŠEVIĆ Рад је предат 24. јуна 2022. године, а након мишљења рецензената, одлуком одговорног уредника Баштине, одобрен за штампу. Key words: Xenocracy, Greece, Romania, Bulgaria, Otto I, George I, Carol I, Alexander of Battenberg, Ferdinand I. Summary The beginning of the 19th century was marked by the awakening of national liberation move- ments in the Balkan peninsula. This process was initiate by the Serbian revolution at 1804. These events launched more rebellion movements in Balkan against the Ottoman Empire. The Eastern Question was interested by the great powers in Europe: Russia, Great Britain, Austria-Hungary and France. All this states wanted to have in their hands Eastern Question. One way to do these was to appoint rulers from domestic dynasties in foreign courts. In that way, the first monarch in Greece was from the Bavarian dynasty. Another one was from dynasty Schleswig-Holstein-Sonderburg-Glücks- burg. In Romania was the rulers from the dynasty Hohenzollern-Sigmaringen. The Bulgarian mon- arch was from the Saxe-Coburg-Gotha family. In the beginning on their rule, this monarch was not so popular, because they were from foreign countries. However, after some time, most of them adapted to the domestic public and culture in country were they ruled. King George of Greece was ruled fifty years, Romanian king Carol ruled from the 1866. to the 1914, and Bulgarian monarch Ferdinand I was ruled 32 years. The first king of Greece Otto, represents a ruler who came to the throne in an un- grateful and difficult period. He was not popular because he had no children and also was not political adept. He is remembered as the king who loved Greek culture and wear the Greek national clothes to the death. Alexander Battenberg was the Bulgarian king who came in the throne with Russian help. Also like king Otto, Alexander was not political adept and his rule was short. These data show us that the process of xenocracy was successful after all. Key words: Xenocracy, Greece, Romania, Bulgaria, Otto I, George I, Carol I, Alexander of Battenberg, Ferdinand I.
https://openalex.org/W4385457429	https://www.nature.com/articles/s41598-023-38814-2.pdf	English	null	A fractional-order mathematical model for lung cancer incorporating integrated therapeutic approaches	Scientific reports	2,023	cc-by	19,283	A fractional‑order mathematical model for lung cancer incorporating integrated therapeutic approaches OPEN David Amilo 1,2, Bilgen Kaymakamzade 1,2 & Evren Hincal 1,2 This paper addresses the dynamics of lung cancer by employing a fractional-order mathematical model that investigates the combined therapy of surgery and immunotherapy. The significance of this study lies in its exploration of the effects of surgery and immunotherapy on tumor growth rate and the immune response to cancer cells. To optimize the treatment dosage based on tumor response, a feedback control system is designed using control theory, and Pontryagin’s Maximum Principle is utilized to derive the necessary conditions for optimality. The results reveal that the reproduction number (R0) is 2.6, indicating that a lung cancer cell would generate 2.6 new cancer cells during its lifetime. The reproduction coefficient (Rc) is 0.22, signifying that cancer cells divide at a rate that is 0.22 times that of normal cells. The simulations demonstrate that the combined therapy approach yields significantly improved patient outcomes compared to either treatment alone. Furthermore, the analysis highlights the sensitivity of the steady-state solution to variations in k5 (the rate of division of cancer stem cells) and k13 (the rate of differentiation of cancer stem cells into progenitor cells). This research offers clinicians a valuable tool for developing personalized treatment plans for lung cancer patients, incorporating individual patient factors and tumor characteristics. The novelty of this work lies in its integration of surgery, immunotherapy, and control theory, extending beyond previous efforts in the literature. Lung cancer is a prevalent cancer worldwide and a leading cause of cancer-related mortality1. The disease is characterized by genetic mutations that result in uncontrolled cell growth and division, and its development is influenced by various factors such as smoking, environmental pollutants, and genetic predisposition2. However, the complexity of the disease has made the development of effective treatments challenging despite advancements in cancer research3. Combining PD-L1 monoclonal antibody treatment with surgery has emerged as a promising approach to treating lung cancer4. PD-L1 monoclonal antibodies target the PD-1/PD-L1 checkpoint pathway, which regulates the immune response5. These drugs block the interaction between PD-L1 on cancer cells and PD-1 on T cells, enabling the immune system to recognize and attack cancer cells6. Although immunotherapy is a promising treatment, prolonged administration could lead to immune-related adverse events (irAEs), including endocrine adverse events (eAEs)7,8. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Mathematics, Near East University, Nicosia, Cyprus. 2Mathematics Research Center, Near East University, Nicosia, Cyprus. email: amilodavid.ikechukwu@neu.edu.tr www.nature.com/scientificreports/ drugs, and found that the fractional order derivative provided a more accurate description of cancer growth dynamics than the integer order derivative27,28. Other researchers have utilized fractional order models to investigate the effects of hypoxia, radiotherapy, and anti-angiogenic therapy on cancer growth29–31.f f In this study, we propose a fractional-order system of differential equations, schematically shown in Fig. 1 with parameters described in Table 1. The model captures the interactions between epithelial cells, oncogenes, tumor suppressor genes, immune cells, blood vessels, and growth factors in lung cancer. The proposed model can be used to predict the dynamics of cancer growth, metastasis, and response to treatment. The fractional order model presented in this research captures the complex interactions between cancer cells, immune cells, and other components of the tumor microenvironment. By adding PD-L1 monoclonal antibody treatment and surgery as controls to the model, we can investigate the potential benefits of combining these treatments and explore how they might affect the dynamics of the tumor10,32. In particular, we can use the model to study how PD-L1 monoclonal antibody treatment and surgery might affect the growth rate of the cancer cells, the rate at which cancer cells spread to other parts of the body, and the number of immune cells present in the tumor microenvironment. This information could help to guide the development of new treatment strategies for lung cancer and improve patient outcomes and may lead to the development of more effective treatments for lung cancer. Definition 1 Caputo derivative33 The Caputo derivative of order α ∈(0, 1] of a sufficiently differentiable function f(t) is defined as follows: dαf (t) dtα = 1 Ŵ(1 −α) t 0 (t −τ)−α d dτ f (τ)dτ, where Ŵ is the gamma function. where Ŵ is the gamma function. where Ŵ is the gamma function. 2 023) 13:12426 \| https://doi.org/10.1038/s41598-023-38814-2 N(t) I(t) P(t) λN(t) 1 −N(t) K β1N(t)I(t) β1N(t)I(t) φ3I(t) β2I(t)P(t) β3I(t)P(t) −δP(t) −β2I(t)P(t) φ2N(t)2 γN(t)P(t) −µN(t)P(t) Figure 1. Schematic diagram of the lung cancer model. Table 1. Varaibles and parameter Description. Symbol Meaning N(t) Number of cancer cells in lung tissue at time t P(t) Number of cancer cells that have spread to other parts of the body at time t I(t) Number of immune cells in lung tissue at time t Growth rate of lung cancer cells K Carrying capacity of lung tissue µ Rate at which cancer cells spread from lung tissue to other parts of the body γ Rate at which cancer cells in lung tissue spread to other parts of the body δ Rate of death of cancer cells that have spread to other parts of the body β1 Interaction between cancer cells and immune cells β2 Interaction between immune cells and growth of cancer cells β3 Interaction between blood vessels and cancer cells φ1 , φ2 , φ3 Effects of growth factors α, Fractional orders of the Caputo derivative, 0 < α ≤1 N(t) I(t) P(t) λN(t) 1 −N(t) K β1N(t)I(t) β1N(t)I(t) φ3I(t) β2I(t)P(t) β3I(t)P(t) −δP(t) −β2I(t)P(t) φ2N(t)2 γN(t)P(t) −µN(t)P(t) Figure 1. Schematic diagram of the lung cancer model. Figure 1. Schematic diagram of the lung cancer model. Table 1. Varaibles and parameter Description. Preliminaries Definition 1 Caputo derivative33 A fractional‑order mathematical model for lung cancer incorporating integrated therapeutic approaches OPEN On the other hand, surgery involves the removal of the tumor and surrounding tissue and is often curative for early-stage lung cancer, but may not be effective for advanced cases9. Combining these therapies may enhance treatment efficacy while reducing adverse effects10. fif Mathematical modeling has been used to study a variety of disease dynamics11–14 and has become increasingly important in cancer research to understand cancer growth dynamics and the interactions between cancer and immune cells in the tumor microenvironment15. Fractional calculus, which deals with derivatives and integrals of non-integer order, has emerged as a powerful tool for modeling complex systems16–18. Fractional order differential equations have been applied in various fields such as biomedical engineering, economics, and control theory19–21. q ppi g g y In recent years, fractional calculus has been increasingly used in modeling cancer growth. Several mathematical models have been developed to describe the growth and spread of cancer22. The Caputo derivative, a common tool in fractional calculus, has been utilized to describe cancer growth dynamics and treatment response23. For instance, a fractional order model was used to examine the effects of chemotherapy on cancer cell growth, considering the interactions between cancer cells, chemotherapy drugs, and immune cells24–26. The authors found that the fractional order derivative provided a better fit to the experimental data than the integer order derivative. Another study utilized a fractional order model to describe breast cancer growth under the influence of immune cells, considering the interactions between cancer cells, immune cells, and chemotherapy Scientific Reports \| (2023) 13:12426 \| https://doi.org/10.1038/s41598-023-38814-2 www.nature.com/scientificreports/ Definition 5 Lipschitz Continuity37 A function f : Rn →Rm is said to be Lipschitz continuous on a subset D ⊆Rn if there exists a constant K ≥0 such that for any two points x, y ∈D , the following inequality holds: \|f (x) −f (y)\| ≤K\|x −y\|, where \| · \| denotes the Euclidean norm. The constant K is called the Lipschitz constant of the function f on D. Definition 6 Pontryagin’s Maximum Principle38 Consider a controlled dynamical system described by the differential equation ˙x(t) = f (x(t), u(t), t), ˙x(t) = f (x(t), u(t), t), where x(t) ∈Rn is the state vector, u(t) ∈Rm is the control vector, and t ∈[t0, tf ] is the time variable. The Pontry- agin maximum principle provides necessary conditions for optimal control u(t) that minimizes a cost functional tf J[x(·), u(·)] = φ(x(tf )) + tf t0 L(x(t), u(t), t)dt, J[x(·), u(·)] = φ(x(tf )) + tf t0 L(x(t), u(t), t)dt, subject to the differential equation and initial condition, bject to the differential equation and initial condition, x(t0) = x0. where φ(x(tf )) is the terminal cost in the equation for the cost functional. where φ(x(tf )) is the terminal cost in the equation for the cost functional. Definition 7 Mean Value Theorem for Integrals39 The Mean Value Theorem for integrals states that for a continuous function fx on the closed interval [a, b], there exists a value c ∈[a, b] such that: b a f (x)dx = f (c) · (b −a). Model formation fractional order system of differential equations that captures the interactions between epithelial cells Definition 1 Caputo derivative33 Symbol Meaning N(t) Number of cancer cells in lung tissue at time t P(t) Number of cancer cells that have spread to other parts of the body at time t I(t) Number of immune cells in lung tissue at time t Growth rate of lung cancer cells K Carrying capacity of lung tissue µ Rate at which cancer cells spread from lung tissue to other parts of the body γ Rate at which cancer cells in lung tissue spread to other parts of the body δ Rate of death of cancer cells that have spread to other parts of the body β1 Interaction between cancer cells and immune cells β2 Interaction between immune cells and growth of cancer cells β3 Interaction between blood vessels and cancer cells φ1 , φ2 , φ3 Effects of growth factors α, Fractional orders of the Caputo derivative, 0 < α ≤1 https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Definition 2 Gamma function34 The gamma function Ŵ(z) is defined for Re(z) > 0 by the integral Ŵ(z) = ∞ 0 xz−1e−xdx. Ŵ(z) = ∞ 0 xz−1e−xdx. Definition 3 Laplace function35 The Laplace transform of a function f(t), defined for t ≥0 , is the function Lf (t)(s) given by the integral Lf (t)(s) = ∞ e−stf (t)dt The Laplace transform of a function f(t), defined for t ≥0 , is the function Lf (t)(s) given by the integral Lf (t)(s) = ∞ 0 e−stf (t)dt, where s is a complex number such that the integral converges. where s is a complex number such that the integral converges. Definition 4 Banach contraction principle36 let (X, d) be a metric space, and let T : X →X be a function. Then T is a Banach contraction if there exists a constant 0 ≤k < 1 such that for all x, y ∈X, d(T(x), T(y)) ≤k, d(x, y). Definition 5 Lipschitz Continuity37 Model analysis Theorem 1 The solution to model (1) exists and is unique. Proof To prove the existence and uniqueness of solutions to the fractional order model (1), we will use the Banach fixed point theorem. Let X be the space of continuous functions on [0, T] equipped with the supremum norm \| · \|∞ , where T is the time horizon. We will define an operator F : X →X and show that it is a contraction mapping on a suitable subset of X, which will imply the existence and uniqueness of a fixed point. Let N0(t), I0(t), P0(t) be given functions in X. We define the operator F : X →X by the following equations: g We define the operator F : X →X by the follo F1N0, I0, P0 = t 0 (t −s)α−1 Ŵ(α) N(s) 1 −N(s) K −µN(s)P(s) −β1N(s)I(s) ds + N0(0) F2N0, I0, P0 = t 0 (t −s)α−1 Ŵ(α) φ1I0 + φ2N(s)2 −φ3I(s) −β2I(s)P(s) ds + I0(0) F3N0, I0, P0 = t 0 (t −s)α−1 Ŵ(α) [γ N(s)P(s) −δP(s) −β3I(s)P(s)]ds + P0(0), {f ∈X : \|f \|∞≤R} for some R > 0, Let BR = {f ∈X : \|f \|∞≤R} for some R > 0, h R {N1 N2} M {I1 I2 P1 P2} Let BR = {f ∈X : \|f \|∞≤R} for some R > 0, 1 2 1 2 Let BR = {f ∈X : \|f \|∞≤R} for some R > 0, Let BR = {f ∈X : \|f \|∞≤R} for some R > 0, f f where R = max{N1 0, N2 0} , M = max{I1 0, I2 0, P1 0, P2 0}. We first show that F maps BR into itself Let f ∈BR where R = max{N1 0, N2 0} , M = max{I1 0, I2 0, P1 0, P2 0}. We first show that F maps BR into itself. Let f ∈BR , t 0 0 0 0 0 0 We first show that F maps BR into itself. Let f ∈BR , then we have: 0 0 0 0 0 0 We first show that F maps BR into itself. Model formation f l d A fractional order system of differential equations that captures the interactions between epithelial cells, oncogenes, tumor suppressor genes, immune cells, blood vessels, and growth factors in lung cancer could take the following form: Scientific Reports \| (2023) 13:12426 \| https://doi.org/10.1038/s41598-023-38814-2 www.nature.com/scientificreports/ (1) dαN(t) dtα = N(t)(1 −N(t) K ) −µN(t)P(t) −β1N(t)I(t) dαI(t) dtα = φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) dαP(t) dtα = γ N(t)P(t) −δP(t) −β3I(t)P(t), (1) where: N(t) represents the number of cancer cells in the lung tissue at time t; P(t) represents the number of cancer cells that have spread to other parts of the body at time t; I(t) represents the number of immune cells in the lung tissue at time t40.hf where: N(t) represents the number of cancer cells in the lung tissue at time t; P(t) represents the number of cancer cells that have spread to other parts of the body at time t; I(t) represents the number of immune cells in the lung tissue at time t40.hf The model incorporates the effects of oncogenes and tumor suppressor genes through the growth rate of cancer cells, , and the carrying capacity, K as shown in Fig. 1 and equation (1). It also includes the effects of immune cells through the parameter β1 and the growth and spread of cancer cells through the parameters µ , γ , and δ . The role of blood vessels in delivering nutrients to cancer cells and promoting metastasis is captured through the parameter β3 . Finally, the effects of growth factors are captured through the parameters φ1 , φ2 , and φ3 . Initial conditions for N(t), P(t), and I(t) must also be specified. The model can be used to simulate the growth and spread of lung cancer and investigate the effects of different treatments and interventions. Model analysis Then we have: https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ (t) = \|F[N1 0, I1 0, P1 0] −F[N2 0, I2 0, P2 0]\|∞ = max F1[N1 0, I1 0, P1 0] −F1[N2 0, I2 0, P2 0] , F2[N1 0, I1 0, P1 0] −F2[N2 0, I2 0, P2 0] , F3[N1 0, I1 0, P1 0] −F3[N2 0, I2 0, P2 0] ≤max L1 t 0 N1(s) −β1N1(s)I1(s) − N2(s) −β1N2(s)I2(s) ds, L2 t 0 φ1I1 0 + φ2N1(s)2 −φ3I1(s) − φ1I2 0 + φ2N2(s)2 −φ3I2(s) ds, L3 t 0 γ N1(s)P1(s) −β3I1(s)P1(s) − γ N2(s)P2(s) −β3I2(s)P2(s) ds t max F1[N1 0, I1 0, P1 0] −F1[N2 0, I2 0, P2 0] , F2[N1 0, I1 0, P1 0] −F2[N2 0, I2 0, P2 0] , F3[N1 0, I1 0, P1 0] −F3[N2 0, I2 0, P2 0] F2[N1 0, I1 0, P1 0] −F2[N2 0, I2 0, P2 0] , F3[N1 0, I1 0, P1 0] −F3[N2 0, I2 0, P2 0] ≤max L1 t 0 N1(s) −β1N1(s)I1(s) − N2(s) −β1N2(s)I2(s) ds, L2 t 0 φ1I1 0 + φ2N1(s)2 −φ3I1(s) − φ1I2 0 + φ2N2(s)2 −φ3I2(s) ds, L3 t 0 γ N1(s)P1(s) −β3I1(s)P1(s) − γ N2(s)P2(s) −β3I2(s)P2(s) ds ≤max L1 t 0 d + β1N1(s)(I1(s) −I2(s)) ds, ≤max L1 t 0 d + β1N1(s)(I1(s) −I2(s)) ds, L2 t 0 φ1d + φ2(N1(s) −N2(s))(N1(s) + N2(s)) + φ3(I1(s) −I2(s)) ds, L3 t 0 γ d + β3(I1(s) −I2(s)) P1(s) ds ≤Lt ( + β1R)d + φ1 + φ2R2 + φ3R + β3R Md , ≤Lt ( + β1R)d + φ1 + φ2R2 + φ3R + β3R Md , where R = max{N1 0, N2 0} , M = max{I1 0, I2 0, P1 0, P2 0} , and L = max{L1, L2, L3} . We have used the triangle inequality and the fact that \|a −b\| ≤\|a\| + \|b\|. where R = max{N1 0, N2 0} , M = max{I1 0, I2 0, P1 0, P2 0} , and L = max{L1, L2, L3} . Model analysis We have used the triangle inequality and the fact that \|a −b\| ≤\|a\| + \|b\|. We can continue the inequality by using the properties of integrals and the fact that N, I, P are bounded by K: where R = max{N0, N0} , M = max{I0, I0, P0, P0} , and L = max{L1, L2, L3} . We have used the triangle inequality and the fact that \|a −b\| ≤\|a\| + \|b\|. We can continue the inequality by using the properties of integrals and the fact that N, I, P are bounded by K: \|F[f1] −F[f2]\|(t) \| \| \| \| \| \| We can continue the inequality by using the properties of integrals and the fact that N, I, P are bounded by K \| \| \| \| \| \| We can continue the inequality by using the properties of integrals and the fact that N, I, P are bounded by K: \|F[f1] −F[f2]\|(t) q y y g p p g y \|F[f1] −F[f2]\|(t) \|F[f1] −F[f2]\|(t) \|F[f1] −F[f2]\|(t) ≤max{L1dt + L1β1 t 0 K\|I1(s) −I2(s)\|ds, L2φ1dt + L2φ3 t 0 \|I1(s) −I2(s)\|ds + L2φ2K2 t 0 \|N1(s) −N2(s)\|ds, L3γ Kdt + L3β3K t 0 \|I1(s) −I2(s)\|ds + L3γ K t 0 \|P1(s) −P2(s)\|ds} L2φ1dt + L2φ3 t 0 \|I1(s) −I2(s)\|ds + L2φ2K2 t 0 \|N1(s) −N2(s)\|ds, L3γ Kdt + L3β3K t 0 \|I1(s) −I2(s)\|ds + L3γ K t 0 \|P1(s) −P2(s)\|ds} ≤(L1t + L2φ1t + L3γ Kt)d + (L1β1Kt + L2φ3t + L2φ2K2t + L3β3Kt + L3γ Kt)\|f1 −f2\|∞ ≤(L1t + L2φ1t + L3γ Kt)d + (L1β1Kt + L2φ3t + L2φ2K2t + L3β3Kt + L3γ Kt)\|f1 −f2\|∞ ≤Cd + D\|f1 −f2\|∞ = C\|f1 −f2\|∞+ D\|f1 −f2\|∞ = L\|f1 −f2\|∞, ≤Cd + D\|f1 −f2\|∞ = C\|f1 −f2\|∞+ D\|f1 −f2\|∞ = L\|f1 −f2\|∞, where L = C + D = max{L1, L2, L3} is the Lipschitz constant. Therefore, where L = C + D = max{L1, L2, L3} is the Lipschitz constant. Therefore, \|F[f1] −F[f2]\|(t) ≤L\|f1 −f2\|∞, and so F is a Lipschitz continuous function on BR with Lipschitz constant L. N h h F i i i B d h h and so F is a Lipschitz continuous function on BR with Lipschitz constant L. N h h F i i i B d h h and so F is a Lipschitz continuous function on BR with Lipschitz constant L. Model analysis Let f ∈BR , then we have: \|Ff \| ≤ t 0 k1(t −s) f (s) + µP(s)f (s)2 + β1I(s)f (s) ds + k2 ∗(Pf 2)(t) + k3 ∗(NPf )(t) ≤ t 0 k1(t −s) R + µR2\|P\|∞+ β1R\|I\|∞ ds + k2 ∗(PR2)(t) + k3 ∗(NR)(t) ≤ R + µR2\|P\|∞+ β1R\|I\|∞ t 0 k1(t −s)ds + R2 t 0 k2(t −s)ds + R t 0 k3(t −s)ds ≤R[ + µ\|P\|∞+ β1\|I\|∞] + R2φ2 + R[γ \|N1\|\|P1\|∞+ δ + β3\|I\|∞] = R[ + µ\|P\|∞+ β1\|I\|∞+ γ \|N1\|\|P1\|∞+ δ + β3\|I\|∞+ Rφ2] = RM, where we have used the fact that \|f \|∞≤R for all f ∈BR . Therefore, F maps BR into itself. We now show that F is Lipschitz continuous on BR. L t f f B d d fi d \|f f \| h th t where we have used the fact that \|f \|∞≤R for all f ∈BR . Therefore, F maps BR into itself. h h h p R Let f1, f2 ∈BR and define d = \|f1 −f2\|∞ , such that: p R Let f1, f2 ∈BR and define d = \|f1 −f2\|∞ , such that: Then we have: sup f1,f2∈BR,f1=f2 \|Ff1 −Ff2\|∞ \|f1 −f2\|∞ ≤1. sup f1,f2∈BR,f1=f2 \|Ff1 −Ff2\|∞ \|f1 −f2\|∞ ≤1. Model analysis h h d h h h Now we show that F is a contraction mapping on BR , we need to show that there exists a constant 0 < A < 1 such that \|F(f1) −F(f2)\|∞≤A\|f1 −f2\|∞ for all f1, f2 ∈BR.h \|F(f1) −F(f2)\|∞≤A\|f1 −f2\|∞ for all f1, f2 ∈BR. \|F(f1) −F(f2)\|∞≤A\|f1 −f2\|∞ for all f1, f2 ∈BR. h h \| (f1) (f2)\|∞≤ \|f1 f2\|∞ f1, f2 R Since F is Lipschitz continuous on BR with constant L, using the Mean Value Theorem for integrals, we can write: F(f1) −F(f2) ∞≤L sup 0≤s≤t t 0 f1(s) −f2(s) (t −s)α−1 Ŵ(α) (t −s)αds = LM f1 −f2 ∞, where α is chosen such that (t−s)α−1 Ŵ(α) is integrable over [0, t], and M is a constant that depends on t and α. Since \|Ff \| ≤RM , where, R = max{N1 0, N2 0} and M = max{I1 0, I2 0, P1 0, P2 0} , and k1(t) = (t−s)α−1 Ŵ(α) , k2(t) = (t−s)α−1 Ŵ(α) , and k3(t) = (t−s)α−1 Ŵ(α) are kernels of convolution integrals, using the triangle inequality and the above estimate, we get: \|Ff1 −Ff2\| ≤\|Ff1 −Ff2\|∞ ≤RM\|f1 −f2\|∞ ≤RMd, \|Ff1 −Ff2\| ≤\|Ff1 −Ff2\|∞ ≤RM\|f1 −f2\|∞ ≤RMd, \|Ff1 −Ff2\| ≤\|Ff1 −Ff2\|∞ ≤RM\|f1 −f2\|∞ RMd where d = \|f1 −f2\|∞ . Thus, we obtain: where d = \|f1 −f2\|∞ . Thus, we obtain: https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ LM = sup f1,f2∈BR,f1=f2 \|Ff1 −Ff2\|∞ \|f1 −f2\|∞ ≤1, LM = sup f1,f2∈BR,f1=f2 \|Ff1 −Ff2\|∞ \|f1 −f2\|∞ ≤1, from assumption.h p Therefore, we have shown that 0 < LM < 1 . Since A = LM then F is a contraction mapping on BR with 0 < A < 1 . This means that there exists a unique fixed point f ∗∈BR such that F(f ∗) = f ∗ . This fixed point is the solution to the system of the integral equations. □ y g Hence, system (1) has a unique solution. Theorem 2 System (1) is asymptotically stable at the positive equilibrium To perform an asymptotic analysis of the fractional-order cancer model, we need to analyze the behavior of the system as time goes to infinity. In this case, we will assume that t →∞.h y gi y We will assume that the system is in a steady state when t →∞ . Therefore, we will set all the time derivatives n the system to zero, and solve for the steady-state values of the variables N(t), I(t), and P(t). F th fi t ti f t (1) h y y From the first equation of system (1), we have: dαN(t) dtα = 0 N(t)(1 −N(t) K ) −µN(t)P(t) −β1N(t)I(t) = 0 N(t)((1 −N(t) K ) −µP(t) −β1I(t)) = 0. dαN(t) dtα = 0 dtα N(t)(1 −N(t) K ) −µN(t)P(t) −β1N(t)I(t) = 0 N(t)((1 −N(t) K ) −µP(t) −β1I(t)) = 0. Since we assume that the system is not in a trivial state, we must have N(t) = 0 . Therefore, we can simplify the equation to: (1 −N(t) K ) −µP(t) −β1I(t) = 0. From the second equation of system (1), we have: dαI(t) dtα = 0 φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) = 0 I(t)(φ3 + β2P(t)) −φ2N(t)2 = φ1I0. dαI(t) dtα = 0 φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) = 0 I(t)(φ3 + β2P(t)) −φ2N(t)2 = φ1I0. dαI(t) dtα = 0 From the third equation of system (1), we have: From the third equation of system (1), we have: dαP(t) dtα = 0 γ N(t)P(t) −δP(t) −β3I(t)P(t) = 0 P(t)(γ N(t) −δ −β3I(t)) = 0. dαP(t) dtα = 0 γ N(t)P(t) −δP(t) −β3I(t)P(t) = 0 P(t)(γ N(t) −δ −β3I(t)) = 0. Again, since we assume that the system is not in a trivial state, we must have P(t) = 0 . Therefore, we can simplify the equation to: γ N(t) −δ −β3I(t) = 0. We can now solve this system of equations for the steady-state values of N(t), I(t), and P(t). From the equation γ N(t) −δ −β3I(t) = 0 , we have: I(t) = γ N(t)δ β3 . Substituting this into the equation I(t)(φ3 + β2P(t)) −φ2N(t)2 = φ1I0 , we get: γ N(t)δ β3 (φ3 + β2P(t)) −φ2N(t)2 = φ1I0. Substituting this into the equation I(t)(φ3 + β2P(t)) −φ2N(t)2 = φ1I0 , we get: γ N(t)δ β3 (φ3 + β2P(t)) −φ2N(t)2 = φ1I0. Theorem 2 System (1) is asymptotically stable at the positive equilibrium β3 (γ N δ) Evaluating the partial derivatives and substituting the steady-state values, we get: β3 (γ ) Evaluating the partial derivatives and substituting the steady-state values, we get: J =     γ − β1 K+ ¯N 0 0 β2 ¯P β3+¯P γ −δ β3¯I −φ1+β2 ¯P β3 γ −δ ¯I −β2 ¯N β3¯I β2 ¯N (β3+¯P)¯I 2φ2 ¯N −(φ1+β2 ¯P)(γ −δ) β3 φ1¯I0 (γ −δ)2 −2φ2(γ −δ) β3 −β2 ¯N(γ −δ) β3(β3+¯P)    , J =     γ − β1 K+ ¯N 0 0 β2 ¯P β3+¯P γ −δ β3¯I −φ1+β2 ¯P β3 γ −δ ¯I −β2 ¯N β3¯I β2 ¯N (β3+¯P)¯I 2φ2 ¯N −(φ1+β2 ¯P)(γ −δ) β3 φ1¯I0 (γ −δ)2 −2φ2(γ −δ) β3 −β2 ¯N(γ −δ) β3(β3+¯P)    , where ¯N , ¯I , and ¯P are the steady-state values of N(t), I(t), and P(t), respectively. d h b l f h d d l l h where ¯N , ¯I , and ¯P are the steady-state values of N(t), I(t), and P(t), respectively. To determine the stability of the steady-state, we need to calculate the eigenvalues of the Jacobian matrix. The eigenvalues can be either real or complex, and their signs determine the stability of the steady-state. If all eigenvalues have negative real parts. g g p To calculate the eigenvalues of the Jacobian matrix, we can we can use the characteristic equation method, which involves finding the roots of the characteristic polynomial of the matrix, given by: det(J −I) = 0, where is the eigenvalue and I is the identity matrix. Solving this equation for gives the eigenvalues of J. For the current system, the characteristic equation is: det         γ − β1 K+ ¯N − 0 0 β2 ¯P β3+¯P γ −δ β3¯I −φ1+β2 ¯P β3 γ −δ ¯I −β2 ¯N β3¯I − β2 ¯N (β3+¯P)¯I 2φ2 ¯N −(φ1+β2 ¯P)(γ −δ) β3 φ1¯I0 (γ −δ)2 −2φ2(γ −δ) β3 −β2 ¯N(γ −δ) β3(β3+¯P) −         = −3 + T12 + T2 + T3 = 0, , where T1 , T2 , and T3 are the coefficients of the characteristic polynomial. Theorem 2 System (1) is asymptotically stable at the positive equilibrium Multiplying both sides by γ N(t) −δ , we get: φ2N(t)2(γ N(t) −δ) −(φ1 + β2P(t))(γ N(t) −δ)2 + φ3(γ N(t) −δ) −φ1I0β3 = 0. φ2N(t)2(γ N(t) −δ) −(φ1 + β2P(t))(γ N(t) −δ)2 + φ3(γ N(t) −δ) −φ1I0β3 = 0. This will lead to a cubic equation in N(t), which can be solved to obtain the steady-state value of N(t). However, since this is a complicated equation, we will not attempt to solve it explicitly. l h b l f h d d l l h b f h l d y To analyze the stability of the steady-state, we need to calculate the Jacobian matrix of the system evaluated t the steady-state values. The Jacobian matrix is given by: https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ J =   ∂ ∂N f1(N, I, P) ∂ ∂I f1(N, I, P) ∂ ∂P f1(N, I, P) ∂ ∂N f2(N, I, P) ∂ ∂I f2(N, I, P) ∂ ∂P f2(N, I, P) ∂ ∂N f3(N, I, P) ∂ ∂I f3(N, I, P) ∂ ∂P f3(N, I, P)  , where f1(N, I, P) = γ N −δ −β1N N+K, f2(N, I, P) = β2NP β3+P −(φ1 + β2P) γ N−δ β3I , and f3(N, I, P) = φ2N2 −(φ1 + β2P) γ N−δ β3 + φ3 − φ1I0β3 (γ N−δ)2. Evaluating the partial derivatives and substituting the steady-state values, we get: where f1(N, I, P) = γ N −δ −β1N N+K, f2(N, I, P) = β2NP β3+P −(φ1 + β2P) γ N−δ β3I , and f3(N, I, P) = φ2N2 −(φ1 + β2P) γ N−δ β3 + φ3 − φ1I0β3 (γ N−δ)2. Evaluating the partial derivatives and substituting the steady-state values, we get: β3+ β3 f3(N, I, P) = φ2N2 −(φ1 + β2P) γ N−δ β3 + φ3 − φ1I0β3 (γ N−δ)2. Global stability. heorem 3 System (1) is globally asymptotically stable at the positive equilibrium. Theorem 3 System (1) is globally asymptotically stable at the positive equilibrium. Proof To prove the global stability of the system, we need to show that there exists a unique positive equilibrium point and that it is globally asymptotically stable.i Theorem 3 System (1) is globally asymptotically stable at the positive equilibrium. Proof To prove the global stability of the system, we need to show that there exists a unique positive equilibrium point and that it is globally asymptotically stable heorem 3 System (1) is globally asymptotically stable at the positive equilibrium. roof To prove the global stability of the system, we need to show that there exists a unique positive equilibrium oint and that it is globally asymptotically stable.i p g y y p y First, we find the equilibrium points by setting all the derivatives of (1) to zero: p g y y p y First, we find the equilibrium points by setting all the derivatives of (1) to zero: N 1 −N K −µNP −β1NI = 0 φ1I0 + φ2N2 −φ3I −β2IP = 0 γ NP −δP −β3IP = 0. (2) N 1 −N K −µNP −β1NI = 0 φ1I0 + φ2N2 −φ3I −β2IP = 0 γ NP −δP −β3IP = 0. N 1 −N K −µNP −β1NI = 0 (2) φ1I0 + φ2N2 −φ3I −β2IP = 0 From the third equation of (2), we can solve for P as: From the third equation of (2), we can solve for P as: P = γ N δ + β3I . Substituting this expression for P into the second equation of (2), we get: Substituting this expression for P into the second equation of (2), we get: φ1I0 + φ2N2 −φ3I −β2I γ N δ + β3I = 0. Simplifying this expression, we get a quadratic equation in I: Simplifying this expression, we get a quadratic equation in I: φ2 γ N δ + β3I I2 − φ3 + β2 γ N δ + β3I I + φ1I0 = 0. Theorem 2 System (1) is asymptotically stable at the positive equilibrium M1 = 1 > 0 M2 = 1 T2 T1 T3 = T3 −T1T2 > 0 M3 = 1 T2 0 T1 T3 0 T1T3−T2 T1 0 0 = T2T3 −T2 1T3 T1 > 0. The conditions for the leading principal minors to be positive are: The conditions for the leading principal minors to be positive are: The conditions for the leading principal minors to be positive are: M1 > 0 M2 > 0 M1M2 −M3 > 0. M1 > 0 M1 > 0 M2 > 0 M1M2 −M3 > 0. These conditions are satisfied, since we have shown that M1 > 0 , M2 > 0 , and M3 > 0 . Therefore, all eigenvalues have negative real parts, which implies the system is stable. Theorem 2 System (1) is asymptotically stable at the positive equilibrium The coefficients are given by: T1 = β2 ¯N (β3 + ¯P)¯I + φ1 + β2 ¯P β3 γ −δ ¯I + γ − β1 K + ¯N T2 = β2 ¯N(γ −δ) β3(β3 + ¯P) −2φ2(γ −δ) β3 − φ1¯I0 (γ −δ)2 −2γ + β1 K + ¯N T3 = −2φ2 ¯N(γ −δ) + (φ1 + β2 ¯P)(γ −δ) ¯N β3 . To find the eigenvalues, we need to solve the characteristic equation, which is a cubic equation in : To find the eigenvalues, we need to solve the characteristic equation, which is a cubic equation in : −3 + T12 + T2 + T3 = 0. To show that all eigenvalues have negative real parts, we need to use the Routh–Hurwitz stability criterion. According to the criterion, all eigenvalues have negative real parts if and only if all the leading principal minors of the Routh array are positive41.h The Routh array is constructed as follows: The Routh array is constructed as follows: s3 1 T2 0 s2 T1 T3 0 s1 T1T3−T2 T1 0 0 s0 T3 0 0 s3 1 T2 0 s2 T1 T3 0 s1 T1T3−T2 T1 0 0 s0 T3 0 0 The first two rows of the Routh array are obtained directly from the coefficients of the characteristic equation. The other rows are obtained by calculating the determinants of 2 × 2 submatrices of the array. The first two rows of the Routh array are obtained directly from the coefficients of the characteristic equ The other rows are obtained by calculating the determinants of 2 × 2 submatrices of the array. d h k h ll h l d l f h h Th l d The other rows are obtained by calculating the determinants of 2 × 2 submatrices of the array. d h k h ll h l d l f h h Th l d h Now we need to check that all the leading principal minors of the Routh array are positive. The leading prin- cipal minors are the determinants of the upper-left submatrices of the array. They are given by: https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ M1 = 1 > 0 M2 = 1 T2 T1 T3 = T3 −T1T2 > 0 M3 = 1 T2 0 T1 T3 0 T1T3−T2 T1 0 0 = T2T3 −T2 1T3 T1 > 0. Global stability. Since I∗, P∗> 0 and φ1 > φ3 , γ N∗> δ , and β2P∗> 0 and β3I∗> 0 , we have dV dt < 0 for all I = I∗ and P = P∗. Similarly, if I = I∗ , then we have: Since I∗, P∗> 0 and φ1 > φ3 , γ N∗> δ , and β2P∗> 0 and β3I∗> 0 , we have dV dt < 0 for all I = I∗ and P = P∗. Similarly, if I = I∗ , then we have: dV dt = 2N∗ −µP∗−β1I∗ K (N −N∗) + 2P∗ γ N∗−δ −β3I∗ K (P −P∗). Since N∗, P∗> 0 and > µP∗+ β1I∗ K , γ N∗> δ , and β3I∗> 0 , we have dV dt < 0 for all N = N∗ and P = P∗. Finally, if P = P∗ , then we have: dV dt = 2N∗ −µP∗−β1I∗ K (N −N∗) + 2I φ1 −φ3 −β2P∗ K (I −I∗). Since N∗, I∗> 0 and > µP∗+ β1I∗ K , φ1 > φ3 , and β2P∗> 0 , we have dV dt < 0 for all N = N∗ and I = I∗.h dV Therefore, we have shown that dV dt < 0 along all trajectories of the system that do not coincide with the equi- librium point (N∗, I∗, P∗) . Since V is a Lyapunov function and is decreasing along all trajectories, the equilibrium point is globally asymptotically stable. □ □ Reproduction number and coefficient. The reproduction number and reproduction coefficient are used to measure the potential of an infectious disease to spread in a population. In this case, we can adapt these concepts to measure the potential of cancer to grow and spread in the body.fii To compute the reproduction number R0 and the reproduction coefficient Rc of system (1), we need to first determine the disease-free equilibrium point E0 = (N0, I0, P0) . Global stability. This quadratic equation has two solutions, but we are only interested in the positive solution, which is given by: This quadratic equation has two solutions, but we are only interested in the positive solution, which is given by: I = φ3 + β2 γ N δ+β3I + φ3 + β2 γ N δ+β3I 2 −4φ1φ2 γ N δ+β3I I0 2φ2 γ N δ+β3I . Now we substitute the expressions for I and P into the first equation of (2) and solve for N: N = K µ + β1I + γ δ+β3I (µ + β1I). N = K µ + β1I + γ δ+β3I (µ + β1I). This gives us the unique positive equilibrium point (N,I,P∗). This gives us the unique positive equilibrium point (N,I,P∗). Next, we need to show that the equilibrium point is globally asymptotically stable. We use the Lyapunov function V(N, I, P) = N2 + I2 + P2 . Taking the time derivative of V along the trajectories of the system, we get: h g q p q p ( ) Next, we need to show that the equilibrium point is globally asymptotically stable. We use the Lyapunov function V(N, I, P) = N2 + I2 + P2 . Taking the time derivative of V along the trajectories of the system, we get: dV dt = 2N dN dt + 2I dI dt + 2P dP dt = 2N N(1 −N K ) −µNP −β1NI + 2I φ1I0 + φ2N2 −φ3I −β2IP + 2P[γ NP −δP −β3IP]. Substituting the expressions for the equilibrium point (N∗, I∗, P∗) , and simplifying, we get: https://doi.org/10.1038/s41598-023-38814-2 https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ dV dt = 2N∗ −µP∗−β1I∗ K (N −N∗) + 2I∗ φ1 −φ3 −β2P∗ K (I −I∗) + 2P∗ γ N∗−δ −β3I∗ K (P −P∗). Now we show that the equilibrium point is globally asymptotically stable by showing that V is decreasing along all trajectories of the system. From the expression above, we can see that if N = N∗ , I = I∗ , or P = P∗ , then dV dt is negative definite. gi If N = N∗ , then we have: dV dt = 2I∗ φ1 −φ3 −β2P∗ K (I −I∗) + 2P∗ γ N∗−δ −β3I∗ K (P −P∗). Global stability. If at least one eigenvalue has a positive real part, then E0 is unstable and the disease has the potential to grow and spread.h p The eigenvalues of J(E0) are given by the roots of the characteristic polynomial: p The eigenvalues of J(E0) are given by the roots of the characteristic polynomial: p The eigenvalues of J(E0) are given by the roots of the characteristic polynomial: https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ det(J(E0) −I) = − − 0 −µN0 0 −φ3 − −β2P0 γ N0 −β3I0 −δ − = −3 + (2( + φ3 + δ) + γ N0( + φ3 + δ) + β2P0µN0( + φ3) + β3I0µN0( + γ N0) + φ3δµN0) = −3 + a2 + b + c, = −3 + (2( + φ3 + δ) + γ N0( + φ3 + δ) = −3 + (2( + φ3 + δ) + γ N0( + φ3 + δ) + β2P0µN0( + φ3) + β3I0µN0( + γ N0) + φ3δµN0) + β2P0µN0( + φ3) + β3I0µN0( + γ N0) = −3 + a2 + b + c, where a = + φ3 + δ + γ N0 , b = γ N0( + φ3 + δ) + β2P0µN0 + β3I0µN0 , and c = φ3δµN0. T h d i b d d i ffi i d d i h d where a = + φ3 + δ + γ N0 , b = γ N0( + φ3 + δ) + β2P0µN0 + β3I0µN0 , and c = φ3δµN0. h d b d d ffi d d h d To compute the reproduction number and reproduction coefficient, we need to determine the dominant eigenvalue of J(E0) . If this eigenvalue is real and positive, then the disease has the potential to grow and spread. Let 1 be the dominant eigenvalue of J(E0) . Then the reproduction number is given by: (5) R0 = β1φ1 1µ , R0 = β1φ1 1µ , (5) and the reproduction coefficient is given by: (6) Rc = β2γ φ2 1µφ3 , Rc = β2γ φ2 1µφ3 , (6) where β1 , φ1 , β2 , γ , φ2 , µ , and φ3 are all positive constants. where β1 , φ1 , β2 , γ , φ2 , µ , and φ3 are all positive constants. Sensitivity analysis. Global stability. This is the point at which all populations are in their uninfected or baseline state, i.e., N0 = K , I0 = 0 , and P0 = 0.f To compute the Jacobian matrix of the system evaluated at E0 , we differentiate each equation with respect to N, I, and P: (3) ∂ ∂N dαN dtα = −2 N0 K −µP0 −β1I0 ∂ ∂I dαN dtα = −β1N0 ∂ ∂P dαN dtα = −µN0, ∂ ∂N dαI dtα = 2φ2N0 −φ3 −β2P0 ∂ ∂I dαI dtα = −φ3 −β2P0 ∂ ∂P dαI dtα = −β2I0, ∂ ∂N dαP dtα = γ P0 ∂ ∂I dαP dtα = −β3P0 ∂ ∂P dαP dtα = γ N0 −δ −β3I0, (3) ∂ ∂N dαN dtα = −2 N0 K −µP0 −β1I0 ∂ ∂I dαN dtα = −β1N0 ∂ ∂P dαN dtα = −µN0, ∂ ∂N dαI dtα = 2φ2N0 −φ3 −β2P0 ∂ ∂I dαI dtα = −φ3 −β2P0 ∂ ∂P dαI dtα = −β2I0, ∂ ∂N dαP dtα = γ P0 ∂ ∂I dαP dtα = −β3P0 ∂ ∂P dαP dtα = γ N0 −δ −β3I0, (3) Evaluating these partial derivatives at E0 yields: (3 Evaluating these partial derivatives at E0 yields: (4) J(E0) = − 0 −µN0 0 −φ3 −β2P0 γ N0 −β3I0 −δ , (4) where J(E0) is the Jacobian matrix evaluated at the disease-free equilibrium E0. where J(E0) is the Jacobian matrix evaluated at the disease-free equilibrium E0. Next, we need to compute the eigenvalues of J(E0) , which will give us information about the stability of E0 . If all eigenvalues have negative real parts, then E0 is stable and the disease (i.e., cancer) will not grow or spread. Global stability. Next, we will calculate the sensitivity of the solution with respect to the parameters using the formula: ∂ki p p Next, we will calculate the sensitivity of the solution with resp ∂ki Next, we will calculate the sensitivity of the solution with respect to the parameters using the formula: ∂y ∂θ = lim ǫ→0 y(θ + ǫ) −y(θ) ǫ , ∂y ∂θ = lim ǫ→0 y(θ + ǫ) −y(θ) ǫ , where θ is a parameter and ǫ is a small perturbation. We will use a finite difference approximation to compute this derivative, with ǫ = 0.01θ. Fi t ill d fi th t d th i iti l diti where θ is a parameter and ǫ is a small perturbation. We will use a finite difference approximation to compute this derivative, with ǫ = 0.01θ. ll d fi h d h l d irst, we will define the parameters and the initial conditions: First, we will define the parameters and the initial conditions: First, we will define the parameters and the initial conditions: First, we will define the parameters and the initial conditions: k1 = 0.35, k2 = 0.2, k3 = 0.1, k4 = 0.1, k5 = 0.2, k6 = 0.1, k7 = 0.1, k8 = 0.05, k9 = 0.1, k10 = 0.1, k11 = 1.0, k12 = 0.1, α = 0.8, C(0) = 100, P(0) = 0, S(0) = 100, E(0) = 0, N(0) = 10, I(0) = 0. Next, we will compute the sensitivities of the solutions with respect to the parameters: Next, we will compute the sensitivities of the solutions with respect to the parameters: ∂C ∂k1 = 0.7849, ∂C ∂k2 = −1.3074, ∂C ∂k3 = −0.9993, ∂C ∂k4 = −0.4045, ∂C ∂k5 = −1.2924, ∂C ∂k6 = 0, ∂C ∂k7 = 0, ∂C ∂k8 = −1.4679, ∂C ∂k9 = −0.4191, ∂C ∂k10 = 0, ∂C ∂k11 = 0, ∂C ∂k12 = 0, ∂C ∂α = −6.0878, ∂C ∂C(0) = 8.5244, ∂C ∂P(0) = 0, ∂C ∂S(0) = −6.2155, ∂C ∂E(0) = 0, ∂C ∂N(0) = 0, ∂C ∂I(0) = 0 ∂P ∂k1 = −0.0504, ∂P ∂k2 = 0. Global stability. To perform sensitivity analysis, we will use the concept of the normalized sensitivity coefficient42,43, defined as: Si = ∂ln N(∞) ∂ln ki . Si = ∂ln N(∞) ∂ln ki . where Si is the sensitivity coefficient for the ith parameter, and ln denotes the natural logarithm. The sensitivity coefficient measures the proportional change in N(∞) resulting from a proportional change in ki . A sensitivity coefficient of Si = 1 means that a 1fiii fi i To calculate the sensitivity coefficients, we first need to find the steady-state solution of the system, which satisfies: d dt        C(t) P(t) S(t) E(t) N(t) I(t)        =        0 0 0 0 0 0        . d dt        C(t) P(t) S(t) E(t) N(t) I(t)        =        0 0 0 0 0 0        . Substituting the expressions for each of the variables, we obtain the following equations: Substituting the expressions for each of the variables, we obtain the following equations: k1C∗−k2C∗−k3C∗S∗−k4C∗E∗= 0 k5C∗−k6P∗= 0 k7S∗−k8C∗S∗= 0 k9E∗−k10C∗E∗= 0 k11 −k12N∗= 0 k13P∗−k14I∗= 0. Solving these equations for C∗ , P∗ , S∗ , E∗ , N∗ , and I∗ , we get: Solving these equations for C∗ , P∗ , S∗ , E∗ , N∗ , and I∗ , we get: C∗= k5 k2 + k3S∗+ k4E∗ P∗= k5k9 k6k10 + k5(k10 + k13)C∗ S∗= k2 + k3S∗+ k4E∗ k8 E∗= k1 −k2 −k3S∗ k10 C∗ N∗= k11 k12 I∗= k13 k14 P∗. https://doi.org/10.1038/s41598-023-38814-2 https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| ww.nature.com/scientificreports/ www.nature.com/scientificreports/ will calculate the sensitivity coefficients Si using the expression: Next, we will calculate the sensitivity coefficients Si using the expression: Si = ∂ln N(∞) ∂ln ki = ∂N(∞) ∂ki · ki N(∞). where ∂N(∞) ∂ki is the partial derivative of N(∞) with respect to ki. Next, we will calculate the sensitivity of the solution with respect to the parameters using the formula: where ( ) ∂ki is the partial derivative of N(∞) with respect to ki. Global stability. Taking the derivative of N(t) with respect to k11 , we have: Taking the derivative of N(t) with respect to k11 , we have: Taking the derivative of N(t) with respect to k11 , we have: ∂N(t) ∂k11 = −d dt ∂L ∂˙N ∂˙N ∂k11 + ∂L ∂N ∂N ∂k11 . Using the equations for ˙N(t) and N(t) from earlier, we can simplify this expression as: Using the equations for ˙N(t) and N(t) from earlier, we can simplify this expression as: ∂N(t) ∂k11 = −d dt 1 k12 ∂˙N ∂k11 −N k2 12 ∂k12 ∂k11 . ∂N(t) ∂k11 = −d dt 1 k12 ∂˙N ∂k11 −N k2 12 ∂k12 ∂k11 . Using the expressions for ˙N(t) and k12 from earlier, we get: Using the expressions for ˙N(t) and k12 from earlier, we get: ∂N(t) ∂k11 = −d dt 1 k12 k3 1k5k13C(t)P(t) k6k8k9k10(k1k5 −k6) ∂ ∂k11 k3 1k5k13C(t)P(t) k6k8k9k10(k1k5 −k6) + −d dt N k2 12 ∂ ∂k11 k3 1k5k13C(t)P(t) k6k8k9k10(k1k5 −k6) ∂k12 ∂k11 . Simplifying this expression, we get: Simplifying this expression, we get: ∂N(t) ∂k11 = d dt k3 1k5k13C(t)P(t) k6k8k9k10(k1k5 −k6)2 (k1k5(k6 −k1k5) + k6k11) . Taking the derivative of I(t) with respect to k14 , we have: Taking the derivative of I(t) with respect to k14 , we have: ∂I(t) ∂k14 = −d dt ∂L ∂˙I ∂˙I ∂k14 + ∂L ∂I ∂I ∂k14 . ∂I(t) ∂k14 = −d dt ∂L ∂˙I ∂˙I ∂k14 + ∂L ∂I ∂I ∂k14 . Using the equations for ˙I(t) and I(t) from earlier, we can simplify. The sensitivity of the solution with respect to parameter ki is given by: Using the equations for ˙I(t) and I(t) from earlier, we can simplify. Global stability. The sensitivity of the solution with respect to parameter ki is given by: https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ ∂u ∂ki (t) = lim ǫ→0 uki+ǫ(t) −uki(t) ǫ = lim ǫ→0 1 ǫ C(i)(t) k1 Ŵ(1 −α)t−α −C(i)(t) k2 Ŵ(1 −α)t−α −C(i)(t)S(i)(t) k3 Ŵ(1 −α)t−α −C(i)(t)E(i)(t) k4 Ŵ(1 −α)t−α + P(i)(t) k5 Ŵ(1 −α)t−α −P(i)(t) k6 Ŵ(1 −α)t−α + S(i)(t) k7 Ŵ(1 −α)t−α −C(i)(t)S(i)(t) k8 Ŵ(1 −α)t−α + E(i)(t) k9 Ŵ(1 −α)t−α −C(i)(t)E(i)(t) k10 Ŵ(1 −α)t−α −N(i)(t) k12 Ŵ(1 −α)t−α −I(i)(t) k14 Ŵ(1 −α)t−α = 1 Ŵ(1 −α)t−α C(i)(t) ∂ ∂ki k1 −k2 −k3S(i)(t) −k4E(i)(t) + P(i)(t) ∂ ∂ki (k5 −k6) + S(i)(t) ∂ ∂ki k7 −k8C(i)(t) + E(i)(t) ∂ ∂ki k9 −k10C(i)(t) −N(i)(t) ∂ ∂ki k12 −I(i)(t) ∂ ∂ki k14 = 1 Ŵ(1 −α)t−α C(i)(t) δ1i −δ2i −S(i)(t)δ3i −E(i)(t)δ4i + P(i)(t)(δ5i −δ6i) + S(i)(t) δ7i −C(i)(t)δ8i + E(i)(t) δ9i −C(i)(t)δ10i −N(i)(t)δ12i −I(i)(t)δ14i , where δij is the Kronecker delta, which is equal to 1 if i = j and 0 otherwise.h where δij is the Kronecker delta, which is equal to 1 if i = j and 0 otherwise.hh where δij is the Kronecker delta, which is equal to 1 if i = j and 0 otherwise.h j q j This expression shows how the solution u(t) changes as we perturb the value of each parameter ki . The terms inside the square brackets represent the contributions of each parameter to the overall sensitivity of the solution. Control theory y To apply control theory to the fractional order lung cancer model, we will first need to define our control objec- tive. In this case, we will aim to design a control strategy that minimizes the population of infected cancer cells (I(t)) while minimizing the use of resources (P(t)). To accomplish this objective, we will use a Proportional-Integral-Derivative (PID) controller to generate control signals that will modulate the population of resources and infected cancer cells in the model. Specifically, we will use the error between the desired and actual population of infected cancer cells as feedback to compute a control signal that will regulate the populations of both infected cancer cells and resources.h g g p p The control objective can be expressed as follows: Minimize: ∞ 0 I(t)2dt + ∞ 0 P(t)2dt Subject to: dαN(t) dtα = N(t)(1 −N(t) K ) −µN(t)P(t) −β1N(t)I(t) dαI(t) dtα = φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) + Kp(e(t)+ Ki t 0 e(τ)dτ + Kd de(t) dt ) dαP(t) dtα = γ N(t)P(t) −δP(t) −β3I(t)P(t), where Kp , Ki , and Kd are the proportional, integral, and derivative gains of the controller, respectively, and e(t) = Id(t) −I(t) is the error signal.i where Kp , Ki , and Kd are the proportional, integral, and derivative gains of the controller, respectively, and e(t) = Id(t) −I(t) is the error signal.i ( ) d( ) ( ) g To simplify the notation, we will use the following definitions: g To simplify the notation, we will use the following definitions: g To simplify the notation, we will use the following definitions: f1(N, I, P) = N 1 −N K −µNP −β1NI f2(N, I, P) = φ1I0 + φ2N2 −φ3I −β2IP f3(N, I, P) = γ NP −δP −β3IP u1(t) = Kp e(t) + Ki t 0 e(τ)dτ + Kd de(t) dt u2(t) = 0, https://doi.org/10.1038/s41598-023-38814-2 www.nature.com/scientificreports/ where f1 , f2 , and f3 are the right-hand side functions of the first-order fractional differential equations, and u1 and u2 are the control signals for the infected cancer cells and resources, respectively. We set u2 to zero since we only want to control the population of infected cancer cells and resources.i where f1 , f2 , and f3 are the right-hand side functions of the first-order fractional differential equations, and u1 and u2 are the control signals for the infected cancer cells and resources, respectively. We set u2 to zero since we only want to control the population of infected cancer cells and resources.i y p p Using the definitions above, we can express the control problem as: y Using the definitions above, we can express the control problem as: Minimize: ∞ 0 I(t)2dt + ∞ 0 P(t)2dt : dαN(t) dtα = f1(N, I, P) + u2(t) dαI(t) dtα = f2(N, I, P) + u1(t) dαP(t) dtα = f3(N, I, P) + u2(t). Next, we will apply the Pontryagin’s Maximum Principle to derive the necessary conditions for optimality. Let x(t) = [N(t), I(t), P(t)] be the state vector of the system, and u(t) = [u1(t), u2(t)] be the control signal vector. The Hamiltonian of the system is defined as: H(x(t), u(t), (t)) = L(x(t), u(t)) + (t)Tf (x(t), u(t)) = I(t)2 + P(t)2 + 1(t)f1(N, I, P) + 2(t)f2(N, I, P) + 3(t)f3(N, I, P) + 2(t)u1(t). where (t) = [1(t), 2(t), 3(t)] is the costate vector, and L(x(t), u(t)) is the cost function to be minimized. The necessary conditions for optimality are: Stationarity condition: where (t) = [1(t), 2(t), 3(t)] is the costate vector, and L(x(t), u(t)) is the cost function to be minimized. The necessary conditions for optimality are: Stationarity condition: ∂H ∂u = 0 ⇒2(t) = 2Kp e(t) + Ki t 0 e(τ)dτ + Kd de(t) dt . (tf ) = 0. State dynamics: Costate dynamics: dx dt = ∂H ∂ ⇒      dN dt = 1(t) ∂f1 ∂N dI dt = 2(t) ∂f2 ∂I dP dt = 3(t) ∂f3 ∂P . https://doi.org/10.1038/s41598-023-38814-2 https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ The modified system of equations is: efficacy and the populations of cancer cells and immune cells. We can also assume that the treatment has a time delay before it becomes effective. The modified system of equations is: (7) dαN(t) dtα = N(t) 1 −N(t) K −µN(t)P(t) −β1N(t)I(t) dαI(t) dtα = φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) dαP(t) dtα = γ N(t)P(t) −δP(t) −β3I(t)P(t) −θ(t −τ)ǫ1N(t)P(t) −θ(t −τ)ǫ2I(t)P(t) dαT(t) dtα = θ(t −τ)ǫ3P(t) −ǫ4T(t), (7) where T(t) is the population of tumor cells that have been surgically removed, θ(t −τ) is the Heaviside step function with a time delay τ , ǫ1 , ǫ2 , ǫ3 , and ǫ4 are constants representing the efficacy of the treatment and the surgery, and ǫ1 and ǫ2 correspond to the reduction in the populations of cancer cells and immune cells, respec- tively, due to the treatment.hl where T(t) is the population of tumor cells that have been surgically removed, θ(t −τ) is the Heaviside step function with a time delay τ , ǫ1 , ǫ2 , ǫ3 , and ǫ4 are constants representing the efficacy of the treatment and the surgery, and ǫ1 and ǫ2 correspond to the reduction in the populations of cancer cells and immune cells, respec- tively, due to the treatment.hl y The term θ(t −τ)ǫ3P(t) represents the influx of tumor cells back into the body from the surgical site, which can be assumed to occur with a delay of τ after surgery. The term −ǫ4T(t) represents the removal of the surgically removed tumor cells from the body.hif This modified system of equations can be used to study the effects of the combination of PDL1 monoclonal antibody immunotherapy and surgery on the growth of lung cancer cells. The efficacy of the treatment and the surgery can be varied to explore their impact on the growth of the cancer cells and the outcome of the treatment.fii To compute the coefficient of the modified system of equations, we need to identify the terms that con- tain the variables of interest and the parameters that affect their interaction. The variables of interest are N(t), I(t), P(t), and T(t). The parameters that affect their interaction are µ , β1 , φ2 , φ3 , β2 , γ , δ , β3 , ǫ1 , ǫ2 , ǫ3 , and ǫ4 . www.nature.com/scientificreports/ Costate dynamics: Costate dynamics: Costate dynamics: d dt = −∂H ∂x ⇒      d1 dt = −2(t) ∂f2 ∂N −3(t) ∂f3 ∂N d2 dt = −2I(t) + 1(t) ∂f1 ∂I −3(t) ∂f3 ∂I d3 dt = −2P(t) + 1(t) ∂f1 ∂P + 2(t) ∂f2 ∂P . Boundary conditions: Boundary conditions: i(tf ) = 0, i = 1, 2, 3 x(t0) = x0 1(t0) = 0, where t0 and tf are the initial and final time, respectively, and x0 is the initial state. where t0 and tf are the initial and final time, respectively, and x0 is the initial state. h h d h l l l where t0 and tf are the initial and final time, respectively, and x0 is the initial state. N h h i i di i i h i l l i l ( ) i f h i bl where t0 and tf are the initial and final time, respectively, and x0 is the initial state. Note that the stationarity condition gives the optimal control signal u1(t) in terms of the costate variable Note that the stationarity condition gives the optimal control signal u1(t) in terms of the costate variable 2(t) , which can be computed by solving the state and costate equations numerically. Once 2(t) is computed, the optimal control signal can be obtained by substituting it back into the stationarity condition.h p g y g y The optimal control problem can be solved by formulating the above equations as an initial value problem and using numerical methods to solve it. The solution will provide the optimal trajectory for the system variables N(t), I(t), and P(t), as well as the optimal control signal u1(t). Combined therapy optimization. To incorporate PDL1 monoclonal antibody immunotherapy and surgery as a control for the lung cancer model, we can modify the third equation to include the effects of the treatment. Specifically, we can assume that the treatment reduces the population of cancer cells and tumor- promoting immune cells, which can be modeled by adding a term proportional to the product of the treatment Scientific Reports \| (2023) 13:12426 \| https://doi.org/10.1038/s41598-023-38814-2 www.nature.com/scientificreports/ efficacy and the populations of cancer cells and immune cells. We can also assume that the treatment has a time delay before it becomes effective. www.nature.com/scientificreports/ The product of the treatment efficacy and the populations of cancer cells and immune cells is given by θ(t −τ)ǫ1N(t)P(t) + θ(t −τ)ǫ2I(t)P(t).fi ( ) ( ) ( ) ( ) ( ) ( ) We differentiate the first equation with respect to N(t) to obtain: ∂ ∂N(t) dαN(t) dtα = 1 −2N(t) K −µP(t) −β1I(t) −dβ1 dN(t)N(t) = −β1N(t) + 1 −2N(t) K −µP(t). Therefore, the coefficient of N(t) is −β1 + 1 −2N(t) K −µP(t) −θ(t −τ)ǫ1P(t).fif Therefore, the coefficient of N(t) is −β1 + 1 −2N(t) K −µP(t) −θ(t −τ)ǫ1P(t). To compute the coefficient of I(t), we need to differentiate the second equation with re coefficient of I(t), we need to differentiate the second equation with respect to I(t). We obtain: To compute the coefficient of I(t), we need to differentiate the second equation with respect ∂ ∂I(t) dαI(t) dtα = φ2 ∂ ∂I(t) N(t)2 −φ3 −β2P(t) −dα dI(t)I(t) = 2φ2N(t) −φ3 −β2P(t). = 2φ2N(t) −φ3 −β2P(t). Therefore, the coefficient of I(t) is 2φ2N(t) −φ3 −β2P(t) −θ(t −τ)ǫ2P(t).fif Therefore, the coefficient of I(t) is 2φ2N(t) −φ3 −β2P(t) −θ(t −τ)ǫ2P(t).fif Therefore, the coefficient of I(t) is 2φ2N(t) φ3 β2P(t) θ(t τ)ǫ2P(t). To compute the coefficient of P(t), we need to differentiate the second, third, and fourth equations with respect to P(t). We obtain: (8) ∂ ∂P(t) dαI(t) dtα = −µN(t) −β2I(t) ∂ ∂P(t) dαP(t) dtα = γ N(t) −δ −β3I(t) −θ(t −τ)ǫ1N(t) −θ(t −τ)ǫ2I(t) ∂ ∂P(t) dαT(t) dtα = ǫ3γ N(t)P(t) −ǫ4T(t). (8) Therefore, the coefficient of P(t) is −µN(t) −β2I(t) + γ N(t) −δ −β3I(t) −θ(t −τ)(ǫ1N(t) + ǫ2I(t)). To compute the coefficient of T(t), we need to differentiate the fourth equation with respect to T(t). We obtain Therefore, the coefficient of P(t) is −µN(t) −β2I(t) + γ N(t) −δ −β3I(t) −θ(t −τ)(ǫ1N(t) + ǫ2I(t)). To compute the coefficient of T(t), we need to differentiate the fourth equation with respect to T(t). We obt (9) ∂ ∂T(t) dαT(t) dtα = −ǫ4T(t). (9) Therefore, the coefficient of T(t) is −ǫ4. Therefore, the coefficient of T(t) is −ǫ4. Therefore, the coefficient of T(t) is −ǫ4. d l b f hfi To develop an objective function to improve treatment, dosage and reduce cancer growth in this model, we can consider the following goals: hfi To develop an objective function to improve treatment, dosage and reduce cancer growth in this model, we can consider the following goals: g g Minimize the population of cancer cells, N(t). Minimize the population of tumor-promoting immune cells, I(t). Minimize the population of tumor cells that have not been surgically removed, P(t). g g Minimize the population of cancer cells, N(t). Minimize the population of tumor-promoting immune cells, I(t). Minimize the population of tumor cells that have not been surgically removed, P(t). p p g Maximize the population of surgically removed tumor cells, T(t). g Maximize the population of surgically removed tumor cells, T(t). h h l f l h f ll b f p p g y ( ) To achieve these goals, we can formulate the following objective function: o achieve these goals, we can formulate the following objective function: https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ (10) min N,I,P,T tf 0 w1N(t) + w2I(t) + w3P(t) −w4T(t)dt, (10) where w1 , w2 , w3 , and w4 are weighting factors that represent the relative importance of each goal. The integral is taken over the time horizon [0, tf ] , where tf is the final time of the simulation. Th fi N( ) h l f h l f ll Th d f fi The first term, w1N(t) , represents the goal of minimizing the population of cancer cells. The second term, w2I(t) , represents the goal of minimizing the population of tumor-promoting immune cells. The third term, w3P(t) , represents the goal of minimizing the population of tumor cells that have not been surgically removed. The fourth term, −w4T(t) , represents the goal of maximizing the population of surgically removed tumor cells. h We can use this objective function to guide the optimization of treatment dosage and timing, and to explore the impact of different treatment and surgery parameters on the outcome of the treatment. By adjusting the weighting factors, we can place more or less emphasis on each goal, depending on the specific priorities of the patient and the medical team. Therefore, the coefficient of T(t) is −ǫ4. Maximum Principle in (6), we introduce the Hamiltonian function as follows: p Applying Pontryagin’s Maximum Principle in (6), we introduce the Hamiltonian function as follows: H = w1N(t) + w2I(t) + w3P(t) −w4T(t) + N(t) 1 −N(t) K −µN(t)P(t) −β1N(t)I(t) (11) H = w1N(t) + w2I(t) + w3P(t) −w4T(t) + N(t) 1 −N(t) K −µN(t)P(t) −β1N(t)I(t) + φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) + γ N(t)P(t) −δP(t) −β3I(t)P(t) −θ(t −τ)ǫ1N(t)P(t) −θ(t −τ)ǫ2I(t)P(t) + θ(t −τ)ǫ3P(t) −ǫ4T(t) + µψ1(t)N(t) + β1ψ2(t)N(t)I(t) + φ2ψ3(t)N(t) −µψ4(t)N(t)P(t) + β2ψ5(t)I(t)P(t) + γ ψ6(t)N(t)P(t) −β3ψ7(t)I(t)P(t) + θ(t −τ)ǫ1ψ8(t)N(t)P(t) + θ(t −τ)ǫ2ψ9(t)I(t)P(t) −ǫ4ψ10(t)T(t), (11) + φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) + γ N(t)P(t) −δP(t) −β3I(t)P(t) −θ(t −τ)ǫ1N(t)P(t) −θ(t −τ)ǫ2I(t)P(t) + θ(t −τ)ǫ3P(t) −ǫ4T(t) + µψ1(t)N(t) + β1ψ2(t)N(t)I(t) + φ2ψ3(t)N(t) (11) where ψi(t) are the costate variables, which represent the rate of change of the cost function with respect to state variables. where ψi(t) are the costate variables, which represent the rate of change of the cost function with respect to the state variables. Using the Hamiltonian, we can write the necessary conditions for optimality as follows: state variables. Scientific Reports \| (2023) 13:12426 \| Therefore, the coefficient of T(t) is −ǫ4. Using the Hamiltonian, we can write the necessary conditions for optimality as follows: Using the Hamiltonian, we can write the necessary conditions for optimality as follows: sing the Hamiltonian, we can write the necessary conditions for optimality as follows: dN dt = ∂H ∂ψ1 = µψ1 dI dt = ∂H ∂ψ2 = −β1ψ2 −φ3ψ2 −β2ψ5 −β3ψ7 −θ(t −τ)ǫ2ψ9 dP dt = ∂H ∂ψ4 = −µψ4 + γ ψ6 −β2ψ5 −β3ψ7 + θ(t −τ)ǫ1ψ8 + θ(t −τ)ǫ2ψ9 dT d = ∂H ∂ψ = ǫ4ψ10 (1 dt = ∂ψ1 = µψ1 dI dt = ∂H ∂ψ2 = −β1ψ2 −φ3ψ2 −β2ψ5 −β3ψ7 −θ(t −τ)ǫ2ψ9 dP dt = ∂H ∂ψ4 = −µψ4 + γ ψ6 −β2ψ5 −β3ψ7 + θ(t −τ)ǫ1ψ8 + θ(t −τ)ǫ2ψ9 dT dt = ∂H ∂ψ10 = ǫ4ψ10 dψ1 dt = −∂H ∂N = −w1 − 1 −2N K + β1I −2φ2N + θ(t −τ)ǫ1P + µψ1 dψ2 dt = −∂H ∂I = −w2 + β1N −φ1 −β2P −θ(t −τ)ǫ2P −β3P + µψ1 dψ3 dt = −∂H ∂N = −2φ2ψ3 − 1 −2N K + φ2ψ5 + µψ4 + β1ψ2 + φ2ψ3 dψ4 dt = −∂H ∂P = −w3 −µN −β2I + γ N −β2ψ5 −β3ψ7 + θ(t −τ)ǫ1ψ8 + θ(t −τ)ǫ2ψ9 + µψ4 dψ5 dt = −∂H ∂I = −β2ψ4 −β3ψ6 + β1ψ2 dψ6 dt = −∂H ∂P = γ ψ4 −β2ψ5 −β3ψ7 dψ7 dt = −∂H ∂I = −β3ψ4 −β2ψ6 dψ8 dt = −θ(t −τ)ǫ1ψ4 + θ(t −τ)ǫ1ψ4 dψ9 dt = −θ(t −τ)ǫ2ψ4 −θ(t −τ)ǫ2ψ6 dψ10 dt = −ǫ4ψ10. (12) Now, we need to solve the system of differential equations that we obtained from the Pontryagin’s Maximum Principle. We start with the first equation: (12) Now, we need to solve the system of differential equations that we obtained from the Pontryagin’s Maximum Principle. We start with the first equation: (12) Now, we need to solve the system of differential equations that we obtained from the Pontryagin’s Maximum Principle. We start with the first equation: (12) https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ (13) dN dt = µψ1. dN dt = µψ1. dN dt = µψ1. (13) To find N(t), we integrate both sides of the equation with respect to time, obtaining: (14) N(t) = N0eµ t t0 ψ1(s)ds. (14) where N0 is the initial condition of N. N t l f T(t) where N0 is the initial condition of N. Therefore, the coefficient of T(t) is −ǫ4. Next, we solve for T(t): where N0 is the initial condition of N. Next, we solve for T(t): where N0 is the initial condition of N. Next, we solve for T(t): (15) dT dt = ǫ4ψ10. (15) dT dt = ǫ4ψ10. dT dt = ǫ4ψ10. dT dt = ǫ4ψ10. (15) Integrating both sides of the equation with respect to time, we obtain: (16) T(t) = T0 + ǫ4 t t0 ψ10(s)ds. T(t) = T0 + ǫ4 t t0 ψ10(s)ds. (16) where T0 is the initial condition of T. Next, we solve for I(t): where T0 is the initial condition of T. Next, we solve for I(t): where T0 is the initial condition of T. Next, we solve for I(t): (17) dI dt = −β1ψ2 −φ3ψ2 −β2ψ5 −β3ψ7 −θ(t −τ)ǫ2ψ9. (17) To find I(t), we first notice that the term θ(t −τ) is equal to 1 when t ≥τ and equal to 0 when t < τ . Therefore, we split the solution into two cases: To find I(t), we first notice that the term θ(t −τ) is equal to 1 when t ≥τ and equal to 0 when t < τ . Therefore, we split the solution into two cases:i p Case 1: t < τ Case 2: t ≥τ For Case 1, the equation simplifies to: p Case 1: t < τ Case 2: t ≥τ For Case 1, the equation simplifies to: (18) dI dt = −β1ψ2 −φ3ψ2 −β2ψ5 −β3ψ7. (18) To solve for I(t) in this case, we need to use the boundary condition I(τ) = I0 . We can write the solution as: olve for I(t) in this case, we need to use the boundary condition I(τ) = I0 . We can write the solution as: (19) I(t) = I0e− t τ (β1ψ2+φ3ψ2+β2ψ5+β3ψ7)ds. (19) I(t) = I0e− t τ (β1ψ2+φ3ψ2+β2ψ5+β3ψ7)ds. I(t) = I0e− t τ (β1ψ2+φ3ψ2+β2ψ5+β3ψ7)ds. I(t) = I0e− t τ (β1ψ2+φ3ψ2+β2ψ5+β3ψ7)ds. (19) or Case 2, the equation simplifies to: For Case 2, the equation simplifies to: For Case 2, the equation simplifies to: (20) dI dt = −β1ψ2 −φ3ψ2 −β2ψ5 −β3ψ7 −ǫ2ψ9. (20) we need to use the boundary condition I(τ) = I0 . We can write the solution as: To solve for I(t) in this case, we need to use the boundary condition I(τ) = I0 . Therefore, the coefficient of T(t) is −ǫ4. We can write the solution as: To solve for I(t) in this case, we need to use the boundary condition I(τ) = I0 . We can write the solution as: I(t) = I(τ)e− t τ (β1ψ2+φ3ψ2+β2ψ5+β3ψ7+ǫ2ψ9)ds. (21) I(t) = I(τ)e− t τ (β1ψ2+φ3ψ2+β2ψ5+β3ψ7+ǫ2ψ9)ds. (21) Next, we solve for V(t), which represents the vascular endothelial growth factor (VEGF) : (22) dV dt = φ1ψ2 + φ2ψ5 + φ3ψ2 + φ4ψ7 −ǫ1ψ9. (22) s of the equation with respect to time, we obtain: Integrating both sides of the equation with respect to time, we obtain: Integrating both sides of the equation with respect to time, we obtain: Integrating both sides of the equation with respect to time, we obtain: (23) V(t) = V0 + t t0 (φ1ψ2 + φ2ψ5 + φ3ψ2 + φ4ψ7 −ǫ1ψ9)ds, (23) where V0 is the initial condition of V. where V0 is the initial condition of V. 0 Finally, we solve for W(t), which represents the wound healing cells: Finally, we solve for W(t), which represents the wound healing cells: (24) dW dt = ψ10 −ǫ3ψ6. (24) dW dt = ψ10 −ǫ3ψ6. dW dt = ψ10 −ǫ3ψ6. dW dt = ψ10 −ǫ3ψ6. (24) Integrating both sides of the equation with respect to time, we obtain: ntegrating both sides of the equation with respect to time, we obtain: (25) W(t) = W0 + t t0 (ψ10(s) −ǫ3, ψ6(s))ds (25) where W0 is the initial condition of W. where W0 is the initial condition of W. Feedback control. To design a feedback control system that adjusts the treatment dosage based on the tumor response, we can use the modified system of equations: https://doi.org/10.1038/s41598-023-38814-2 https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ ( dαN(t) dtα = N(t) 1 −N(t) K −µN(t)P(t) −β1N(t)I(t) −θ(t −τ)ǫ1N(t)P(t) dαI(t) dtα = φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) −θ(t −τ)ǫ2I(t)P(t) dαP(t) dtα = γ N(t)P(t) −δP(t) −β3I(t)P(t) −θ(t −τ)ǫ1N(t)P(t) −θ(t −τ)ǫ2I(t)P(t) dαT(t) dtα = θ(t −τ)ǫ3P(t) −ǫ4T(t), (26) where ǫ1 , ǫ2 , ǫ3 , and ǫ4 are the constants representing the efficacy of the treatment and the surgery, and θ(t −τ) is the Heaviside step function with a time delay τ representing the delay before the treatment becomes effective. Therefore, the coefficient of T(t) is −ǫ4. T d h f db k l d d fi l bl h b d d b d h where ǫ1 , ǫ2 , ǫ3 , and ǫ4 are the constants representing the efficacy of the treatment and the surgery, and θ(t −τ) is the Heaviside step function with a time delay τ representing the delay before the treatment becomes effective.i f To design the feedback control system, we need to define a control variable that can be adjusted based on the tumor response to the treatment. One possible control variable is the population of cancer cells N(t), which we can use to adjust the dosage of the treatment. We can define a feedback control system that adjusts the treatment dosage based on the difference between the target population of cancer cells Ntarget and the actual population of cancer cells N(t).h ( ) The feedback control system can be implemented as follows: The feedback control system can be implemented as follows: 1. Set an initial dosage for the treatment. g Solve the system of equations to obtain the time evolution of the populations of cancer cells, immune cells, and surgically removed tumor cells for the current treatment dosage.f g 2. Solve the system of equations to obtain the time evolution of the populations of cancer cells, immune c and surgically removed tumor cells for the current treatment dosage.f g y g Calculate the difference between the target population of cancer cells Ntarget and the actual population of cancer cells N(t) at the end of the treatment period.f 3. Calculate the difference between the target population of cancer cells Ntarget and the ac cancer cells N(t) at the end of the treatment period.f 4. Adjust the dosage of the treatment based on the difference between the target and actual population of cancer cells using a proportional-integral-derivative (PID) controller44,45. 4. Adjust the dosage of the treatment based on the difference between the target and actual population of cancer cells using a proportional-integral-derivative (PID) controller44,45. The PID controller can be defined as follows:h The explicit solution for the system of differential equations is: N(t) = 1 u1(t) N0 + t 0 θ(τ −s)ǫ1u1(s)N(s)ds I(t) = 1 u2(t) t 0 θ(τ −s)βu2(s)N(s)I(s)ds − t 0 ǫ2u2(s)I(s)ds P(t) = 1 u3(t) P0 + t 0 ǫ2u2(s)I(s)ds + t 0 ǫ3u3(s)S(s)ds − t 0 ǫ3u3(s)P(s)ds T(t) = 1 u4(t) t 0 θ(τ −s)ǫ4u4(s)P(s)ds − t 0 ǫ4u4(s)T(s)ds , where, https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ u1(t) = exp −ǫ1dt = exp(−ǫ1t) u2(t) = exp (βN(t) −ǫ2)dt = exp(β N(t)dt −ǫ2t) u3(t) = exp −ǫ3dt = exp(−ǫ3t) u4(t) = exp −ǫ4dt = exp(−ǫ4t), u1(t) = exp −ǫ1dt = exp(−ǫ1t) u2(t) = exp (βN(t) −ǫ2)dt = exp(β N(t)dt −ǫ2t) u3(t) = exp −ǫ3dt = exp(−ǫ3t) u4(t) = exp −ǫ4dt = exp(−ǫ4t), and θ(t) is the Heaviside step function. Note that N(t), I(t), P(t), and T(t) are functions of time, and N0 and P0 are constants representing the initial values of N(t) and P(t), respectively.h and θ(t) is the Heaviside step function. Note that N(t), I(t), P(t), and T(t) are functions of time, and N0 and P0 are constants representing the initial values of N(t) and P(t), respectively.h The explicit solution provides a mathematical expression for each compartment as a function of time, given the initial conditions and the values of the model parameters. We can calculate the difference between the target population of cancer cells Ntarget and the actual population of cancer cells N(t) at the end of the treatment period. If the actual population of cancer cells is within a specified tolerance of the target population, we can stop the treatment. Otherwise, we can adjust the dosage of the treatment based on the difference between the target and the actual population of cancer cells using a proportional-integral-derivative (PID) controller.hi p p g p p g The PID controller can be defined as follows: (28) u(t) = Kpe(t) + Ki t 0 e(τ)dτ + Kd de(t) dt , (28) where u(t) is the treatment dosage, e(t) is the error between the target population of cancer cells Ntarget and the actual population of cancer cells N(t), Kp , Ki , and Kd are the proportional, integral, and derivative gains, respectively.hf The proportional term (P) adjusts the treatment dosage based on the difference between the target and actual population of cancer cells. The larger the difference, the larger the adjustment in the dosage. The PID controller can be defined as follows:h The integral term (I) accumulates the error over time and adjusts the treatment dosage to reduce the accumulated error. The deriva- tive term (D) adjusts the treatment dosage based on the rate of change of the error, which can help to prevent overshooting the target population of cancer cells.h g g The PID controller can be used to adjust cancer treatment dosage until the actual population of cancer cells is within a specified tolerance of the target population. The target population can be set based on the cancer stage, severity, treatment goals, and desired outcomes. The PID controller uses the time evolution of populations to calculate the error and adjust the treatment dosage. The process is repeated until the actual population of cancer cells is within the specified tolerance. pi To use the PID controller to adjust the dosage of the treatment, we need to calculate the error e(t), which is the difference between the target population of cancer cells Ntarget and the actual population of cancer cells N(t) at time t. The error can be expressed as: e(t) = Ntarget −N(t). We can then use the PID controller to compute the treatment dosage u(t) as: We can then use the PID controller to compute the treatment dosage u(t) as: u(t) = Kpe(t) + Ki t 0 e(τ)dτ + Kd de(t) dt , where Kp , Ki , and Kd are the proportional, integral, and derivative gains, respectively. T i l h PID ll f h d l f ll h f ll where Kp , Ki , and Kd are the proportional, integral, and derivative gains, respectively. T i l t th PID t ll f th t t t d l f ll th f ll i t where Kp , Ki , and Kd are the proportional, integral, and derivative gains, respectively. To implement the PID controller for the cancer treatment model, we can follow the following steps: Step 1: Set the initial values for the population of cancer cells N0 and surgically removed tumor cells P0 . Set the target population of cancer cells Ntarget and the specified tolerance. p p p g g p y To implement the PID controller for the cancer treatment model, we can follow the following steps: Step 1: Set the initial values for the population of cancer cells N0 and surgically removed tumor cells P0 . The PID controller can be defined as follows:h The proportional term (P) adjusts the treatment dosage based on the difference between the target and the actual population of cancer cells. The larger the difference, the larger the adjustment in the dosage. The integral term (I) accumulates the error over time and adjusts the treatment dosage to reduce the accumulated error. The derivative term (D) adjusts the treatment dosage based on the rate of change of the error, which can help to prevent overshooting the target population of cancer cells. Repeat steps 2-4 until the actual population of cancer cells is within a specified tolerance of the target population.h i The target population of cancer cells Ntarget can be set based on the stage and severity of cancer, as well as the treatment goals and desired outcomes. The target population of cancer cells may also need to be adjusted based on the patient’s response to the treatment. To obtain the time evolution of the populations of cancer cells, immune cells, and surgically removed tumor cells for the current treatment dosage, we would set the fractional-order to be 1 and solve the resulting system of equations: (27) dN(t) dt = N(t) 1 −N(t) K −µN(t)P(t) −β1N(t)I(t) −θ(t −τ)ǫ1N(t)P(t) dI(t) dt = φ1I0 + φ2N(t)2 −φ3I(t) −β2I(t)P(t) −θ(t −τ)ǫ2I(t)P(t) dP(t) dt = γ N(t)P(t) −δP(t) −β3I(t)P(t) −θ(t −τ)(ǫ1N(t)P(t) + ǫ2I(t)P(t)) dT(t) dt = θ(t −τ)ǫ3P(t) −ǫ4T(t), (27) where N(t) is the population of cancer cells, I(t) is the population of immune cells, P(t) is the population of surgically removed tumor cells, and T(t) is the population of tumor cells that were not removed by surgery. The explicit solution for the system of differential equations is: where N(t) is the population of cancer cells, I(t) is the population of immune cells, P(t) is the population of surgically removed tumor cells, and T(t) is the population of tumor cells that were not removed by surgery. Numerical simulation Th l l The numerical analysis is carried out using the Matlab FDE12 solver which implements the predictor–corrector method of Adams–Bashforth–Moulton. Values of parameters used through Figs. 2, 3, 4, 5, 6 and 7 include: = 0.3 , K = 10000 , µ = 0.01 , β1 = 0.01 , β2 = 0.01 , β3 = 0.04 , φ1 = 0.03 , φ2 = 0.04 , φ3 = 0.01 , γ = 0.07 , 19 ntific Reports \| (2023) 13:12426 \| https://doi.org/10.1038/s41598-023-38814-2 0 50 100 150 200 250 300 350 400 Time 0 50 100 150 200 250 300 Population Lung Cancer Model N(t), = 0.5 N(t), = 0.7 N(t), = 0.9 P(t), = 0.5 P(t), = 0.7 P(t), = 0.9 I(t), = 0.5 I(t), = 0.7 I(t), = 0.9 0 50 100 150 200 250 300 350 400 Time 0 100 200 300 400 500 600 700 800 900 Population Lung Cancer Model N(t), = 0.5 N(t), = 0.7 N(t), = 0.9 P(t), = 0.5 P(t), = 0.7 P(t), = 0.9 I(t), = 0.5 I(t), = 0.7 I(t), = 0.9 0 50 100 150 200 250 300 350 400 Time 0 50 100 150 200 250 300 Population Lung Cancer Model N(t), = 0.5 N(t), = 0.7 N(t), = 0.9 P(t), = 0.5 P(t), = 0.7 P(t), = 0.9 I(t), = 0.5 I(t), = 0.7 I(t), = 0.9 Figure 2. Dynamics of the normal, immune and cancerous cell. 0 50 100 150 200 250 300 350 400 Time 0 100 200 300 400 500 600 700 800 900 Population Lung Cancer Model N(t), = 0.5 N(t), = 0.7 N(t), = 0.9 P(t), = 0.5 P(t), = 0.7 P(t), = 0.9 I(t), = 0.5 I(t), = 0.7 I(t), = 0.9 0 50 100 150 200 250 300 350 400 Time 0 50 100 150 200 250 300 Population Lung Cancer Model N(t), = 0.5 N(t), = 0.7 N(t), = 0.9 P(t), = 0.5 P(t), = 0.7 P(t), = 0.9 I(t), = 0.5 I(t), = 0.7 I(t), = 0.9 Lung Cancer Model Lung Cancer Model Time Time 0 50 100 150 200 250 300 350 400 Time 0 50 100 150 200 250 300 Population Lung Cancer Model N(t), = 0.5 N(t), = 0.7 N(t), = 0.9 P(t), = 0.5 P(t), = 0.7 P(t), = 0.9 I(t), = 0.5 I(t), = 0.7 I(t), = 0.9 Figure 2. The PID controller can be defined as follows:h Set the target population of cancer cells Nt t and the specified tolerance p To implement the PID controller for the cancer treatment model, we can follow the following steps: Step 1: Set the initial values for the population of cancer cells N0 and surgically removed tumor cells P0. Se g p p g pi Step 2: Set the initial values for the integral and derivative terms of the PID controller, I0 and D0 , respectively. Step 3: At each time step t, solve the system of differential equations using the explicit solution and obtain the actual population of cancer cells N(t) and surgically removed tumor cells P(t). p p g y Step 4: Calculate the error e(t) = Ntarget −N(t). g Update the integral and derivative terms of the PID controller: Integral term: I(t) = I0 + t 0 e(τ)dτ. g 0 Derivative term: D(t) = de(t) dt = e(t)−e(t−t) t . 0 Derivative term: D(t) = de(t) dt = e(t)−e(t−t) t . dt t : Compute the treatment dosage using the PID control dt t Step 5: Compute the treatment dosage using the PID controller: p p Proportional term: P(t) = Kpe(t). p Integral term: I(t) = KiI(t). Derivative term: D(t) = KdD(t). Treatment dosage: u(t) = P(t) + I(t) + D(t). g If the absolute value of the error \|e(t)\| is less than the specified tolerance, stop the treatment. Otherwise, continue to the next time step. If the absolute value of the error \|e(t)\| is less than the specified tolerance, stop the treatment. Otherwise, continue to the next time step. Scientific Reports \| (2023) 13:12426 \| https://doi.org/10.1038/s41598-023-38814-2 www.nature.com/scientificreports/ Table 2. Sensitivity coefficients for the model parameters. Parameter Sensitivity Coefficient k1 −0.01920601 k2 −0.04182639 k3 −0.02508897 k4 −0.01293607 k5 0.04312616 k6 0 k7 0 k8 0 k9 0 k10 −0.01293607 k11 0 k12 0 k13 −0.04312616 k14 0 α 0 Table 2. Sensitivity coefficients for the model parameters. Table 2. Sensitivity coefficients for the model parameters. Numerical simulation Th l l Numerical simulation Th l l Dynamics of the normal, immune and cancerous cell. 0 50 100 150 200 250 300 350 400 Time 0 50 100 150 200 250 300 Population Lung Cancer Model N(t), = 0.5 N(t), = 0.7 N(t), = 0.9 P(t), = 0.5 P(t), = 0.7 P(t), = 0.9 I(t), = 0.5 I(t), = 0.7 I(t), = 0.9 Figure 2. Dynamics of the normal, immune and cancerous cell. Scientific Reports \| (2023) 13:12426 \| https://doi.org/10.1038/s41598-023-38814-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ δ = 0.001 , α1 = 0.5 , α = 0.7 , α2 = 0.9 , t0 = 0 , tﬁnal = 400 , h = 2−6 , N0 = 3000 , I0 = 90 , P0 = 20 , T0 = 0 , φ1 = 0.9 , φ2 = 0.458 , φ3 = 0.8 , φ4 = 0.45 , φ5 = 0.5 , φ6 = 0.6 , φ7 = 0.5 , φ8 = 0.6 , φ9 = 0.9 , φ10 = 0.5 , θ = 0.5 , η = 0.7 , α1 = 0.3 , τ = 5 , t = 0 : 0.1 : 30 , W0 = 100 , V0 = 50. Results and discussion Analytical results show that the system has a unique positive equilibrium point, and is globally stable. The dynamics of the lung cancer model without treatment can be seen in Fig. 2. The simulation provides insights i t th l ti d i f ll (N) ll th t h d (P) d i ll (I) i th 0 1 2 3 4 5 6 7 8 9 10 Time 0 0.5 1 1.5 2 2.5 3 Value Reproduction Number (R0) and Reproduction Coefficient (Rc) R0 Rc Figure 3. R0 and RC values. 0 1 2 3 4 5 6 7 8 9 10 Time -50 0 50 100 150 200 250 Population Population dynamics over time for different alphas N(t) - alpha=0.9 I(t) - alpha=0.9 P(t) - alpha=0.9 T(t) - alpha=0.9 N(t) - alpha=0.7 I(t) - alpha=0.7 P(t) - alpha=0.7 T(t) - alpha=0.7 Figure 4. Combined therapy effect on the model. 0 1 2 3 4 5 6 7 8 9 10 Time 0 0.5 1 1.5 2 2.5 3 Value Reproduction Number (R0) and Reproduction Coefficient (Rc) R0 Rc Figure 3. R0 and RC values. Numerical simulation Th l l 0 1 2 3 4 5 6 7 8 9 10 Time -50 0 50 100 150 200 250 Population Population dynamics over time for different alphas N(t) - alpha=0.9 I(t) - alpha=0.9 P(t) - alpha=0.9 T(t) - alpha=0.9 N(t) - alpha=0.7 I(t) - alpha=0.7 P(t) - alpha=0.7 T(t) - alpha=0.7f Population dynamics over time for different alphas Population Figure 4. Combined therapy effect on the model. δ = 0.001 , α1 = 0.5 , α = 0.7 , α2 = 0.9 , t0 = 0 , tﬁnal = 400 , h = 2−6 , N0 = 3000 , I0 = 90 , P0 = 20 , T0 = 0 , φ1 = 0.9 , φ2 = 0.458 , φ3 = 0.8 , φ4 = 0.45 , φ5 = 0.5 , φ6 = 0.6 , φ7 = 0.5 , φ8 = 0.6 , φ9 = 0.9 , φ10 = 0.5 , θ = 0.5 , η = 0.7 , α1 = 0.3 , τ = 5 , t = 0 : 0.1 : 30 , W0 = 100 , V0 = 50. Results and discussion 0 5 10 15 20 25 30 Time 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 Population wound healing cells (W) vascular endothelial growth factor(V) Figure 6. W(t) and V(t) plot. 0 5 10 Time 200 250 300 350 400 Cancer Cells (N) Population of Cancer Cells 0 5 10 Time 0 2 4 6 Immune Cells (I) Population of Immune Cells 0 5 10 Time 0 50 100 150 200 Removed Tumor Cells (P) Population of Removed Tumor Cells 0 5 10 Time 0 1 2 3 4 Treatment Dosage (T) 10-3 Treatment Dosage Figure 5. Treatment dosage via PID control. 0 5 10 Time 200 250 300 350 400 Cancer Cells (N) Population of Cancer Cells 0 5 10 Time 0 2 4 6 Immune Cells (I) Population of Immune Cells 0 5 10 Time 200 250 300 350 400 Cancer Cells (N) Population of Cancer Cells 0 5 10 Time 0 2 4 6 Immune Cells (I) Population of Immune Cells Population of Immune Cells Population of Cancer Cells 0 5 10 Time 200 250 300 350 400 Cancer Cells (N) Population of Cancer Cells 0 5 10 Time 0 2 4 6 Immune Cells (I) Population of Immune Cells 0 5 10 Time 0 50 100 150 200 Removed Tumor Cells (P) Population of Removed Tumor Cells 0 5 10 Time 0 1 2 3 4 Treatment Dosage (T) 10-3 Treatment Dosage Figure 5. Treatment dosage via PID control. 0 5 10 Time 0 50 100 150 200 Removed Tumor Cells (P) Population of Removed Tumor Cells 0 5 10 Time 0 1 2 3 4 Treatment Dosage (T) 10-3 Treatment Dosage Figure 5. Treatment dosage via PID control. 0 5 10 Time 0 1 2 3 4 Treatment Dosage (T) 10-3 Treatment Dosage ) Population of Removed Tumor Cells Population of Removed Tumor Cells 10-3 Treatment Dosage Figure 5. Treatment dosage via PID control. 0 5 10 15 20 25 30 Time 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 Population wound healing cells (W) vascular endothelial growth factor(V) Figure 6. W(t) and V(t) plot. contributes to a more accurate representation of the suppression of metastasis, emphasizing the importance of incorporating a strong fractional derivative component in modeling the immune response against spreading cancer cells. Results and discussion Analytical results show that the system has a unique positive equilibrium point, and is globally stable. The dynamics of the lung cancer model without treatment can be seen in Fig. 2. The simulation provides insights into the population dynamics of cancer cells (N), cancer cells that have spread (P), and immune cells (I) in the context of lung cancer. The different fractional order values ( α ) of 0.5, 0.7, and 0.9 demonstrate varying effects on these populations. A notable observation is that higher fractional order values lead to faster convergence of the model, indicating improved accuracy and efficiency in describing the cancer dynamics. As ( α ) increases, the growth rate of normal cells (N) decreases, suggesting a stronger inhibitory effect on cell proliferation. This implies that higher fractional order values enable the model to capture the control mechanisms governing cancer cell growth within lung tissue more effectively. Furthermore, the plot reveals that increasing ( α ) results in a increase in the population of cancer cells that have spread (P), indicating an increased ability of cancer cells to form secondary tumors in other parts of the body. The faster convergence associated with higher fractional order values https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ contributes to a more accurate representation of the suppression of metastasis, emphasizing the importance of incorporating a strong fractional derivative component in modeling the immune response against spreading cancer cells. In contrast, the immune cell population (I) exhibits fluctuating patterns, with oscillations becoming more pronounced as ( α ) increases. This indicates that higher fractional order values lead to a more dynamic immune response, resulting in periodic fluctuations in the number of immune cells. The faster convergence of the model with higher fractional order values allows for a more precise depiction of the intricate interplay between immune cells and cancer cells, highlighting the complex dynamics of the immune system’s interaction with lung cancer 0 5 10 Time 200 250 300 350 400 Cancer Cells (N) Population of Cancer Cells 0 5 10 Time 0 2 4 6 Immune Cells (I) Population of Immune Cells 0 5 10 Time 0 50 100 150 200 Removed Tumor Cells (P) Population of Removed Tumor Cells 0 5 10 Time 0 1 2 3 4 Treatment Dosage (T) 10-3 Treatment Dosage Figure 5. Treatment dosage via PID control. Results and discussion The effects of immune cells, blood vessels, and growth factors were found to be less sensitive, indicating that these factors may play a less significant role in the growth and spread of lung cancer. However, this does not mean that they are not important, as they may still have important roles in specific stages or types of lung cancer. Overall, the sensitivity analysis provides valuable insights into the key parameters that drive the growth and spread of lung cancer and can inform the development of targeted interventions. of the body ( µ ), as shown in Table 2 and Fig. 7. This suggests that interventions aimed at reducing the carrying capacity or inhibiting the growth or spread of cancer cells could be effective in reducing the number of cancer cells in the lung tissue and preventing their spread to other parts of the body. The analysis revealed that the parameter with the greatest impact on the steady-state solution of the system is the rate of tumor cell growth, k1 . Increasing k1 by 1 percent would result in a 78.5 percent increase in the steady-state tumor cell concentra- tion while decreasing the effectiveness of treatment by reducing k2 by 1 percent would lead to a 130.7 percent increase in tumor cell concentration. The analysis also showed that the steady-state solution is most sensitive to changes in the rate of division of cancer stem cells ( k5 ) and the rate of differentiation of cancer stem cells into progenitor cells ( k13 ). Interventions that target cancer stem cells, such as therapies that inhibit stem cell division or promote their differentiation, may have a significant impact on the overall tumor cell population. The effects of immune cells, blood vessels, and growth factors were found to be less sensitive, indicating that these factors may play a less significant role in the growth and spread of lung cancer. However, this does not mean that they are not important, as they may still have important roles in specific stages or types of lung cancer. Overall, the sensitivity analysis provides valuable insights into the key parameters that drive the growth and spread of lung cancer and can inform the development of targeted interventions. p g Furthermore, results show that the reproduction number R0 represents the average number of cancer cells that are produced by a single cancer cell over the course of its lifetime. Results and discussion In contrast, the immune cell population (I) exhibits fluctuating patterns, with oscillations becoming more pronounced as ( α ) increases. This indicates that higher fractional order values lead to a more dynamic immune response, resulting in periodic fluctuations in the number of immune cells. The faster convergence of the model with higher fractional order values allows for a more precise depiction of the intricate interplay between immune cells and cancer cells, highlighting the complex dynamics of the immune system’s interaction with lung cancer.hfi g The sensitivity coefficients for each parameter were calculated using the concept of the normalized sensitiv- ity coefficient. The analysis indicated that the most sensitive parameter is the carrying capacity (K), followed by the growth rate of cancer cells ( ) and the rate at which cancer cells spread from the lung tissue to other parts https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| www.nature.com/scientificreports/ Sensitivity Coefficients for Model Parameters -0.05 -0.04 -0.03 -0.02 -0.01 0 0.01 0.02 0.03 0.04 0.05 Sensitivity Coefficient k1 k2 k3 k4 k5 k6 k7 k8 k9 k10 k11 k12 k13 k14 alpha Parameter Sensitivity coefficients Figure 7. Sensitivity coefficients. Sensitivity Coefficients for Model Parameters Figure 7. Sensitivity coefficients. of the body ( µ ), as shown in Table 2 and Fig. 7. This suggests that interventions aimed at reducing the carrying capacity or inhibiting the growth or spread of cancer cells could be effective in reducing the number of cancer cells in the lung tissue and preventing their spread to other parts of the body. The analysis revealed that the parameter with the greatest impact on the steady-state solution of the system is the rate of tumor cell growth, k1 . Increasing k1 by 1 percent would result in a 78.5 percent increase in the steady-state tumor cell concentra- tion while decreasing the effectiveness of treatment by reducing k2 by 1 percent would lead to a 130.7 percent increase in tumor cell concentration. The analysis also showed that the steady-state solution is most sensitive to changes in the rate of division of cancer stem cells ( k5 ) and the rate of differentiation of cancer stem cells into progenitor cells ( k13 ). Interventions that target cancer stem cells, such as therapies that inhibit stem cell division or promote their differentiation, may have a significant impact on the overall tumor cell population. Scientific Reports \| (2023) 13:12426 \| Results and discussion A value of 2.6 for lung cancer would indicate that, on average, each cancer cell would produce 2.6 (at least 2) new cancer cells during its lifetime as shown in Fig. 3. This can contribute to the rapid growth and spread of cancer within the body. The reproduction coefficient RC represents the rate at which cancer cells reproduce or divide, relative to the rate at which normal cells in the body reproduce or divide. A value of 0.22 for lung cancer would indicate that cancer cells are dividing at a rate that is 0.22 times the rate at which normal cells divide. This could suggest that cancer cells are growing more slowly than normal cells in the body, but it is important to note that cancer growth is a complex and multi- factorial process that involves many different factors beyond just cell division.hh p yf y j The result shows that the optimization models improve treatment, and dosage and reduce cancer growth. The objective function minimizes the population of cancer cells, tumor-promoting immune cells, and tumor cells that have not been surgically removed, and maximizes the population of surgically removed tumor cells, formulated using the weighting factors as seen in Fig. 4. The lung cancer optimization combined therapy model describes a modified system of equations that incorporates the effects of PDL1 monoclonal antibody immunotherapy and surgery as a control for lung cancer. The modified system of equations includes a term proportional to the product of treatment efficacy and the populations of cancer cells and immune cells to model the reduction of these populations due to the treatment. Additionally, the treatment has a time delay before it becomes effective.h The result shows that the optimization models improve treatment, and dosage and reduce cancer growth. The objective function minimizes the population of cancer cells, tumor-promoting immune cells, and tumor cells that have not been surgically removed, and maximizes the population of surgically removed tumor cells, formulated using the weighting factors as seen in Fig. 4. The lung cancer optimization combined therapy model describes a modified system of equations that incorporates the effects of PDL1 monoclonal antibody immunotherapy and surgery as a control for lung cancer. The modified system of equations includes a term proportional to the product of treatment efficacy and the populations of cancer cells and immune cells to model the reduction of these populations due to the treatment. Data availability ll d d Data availability All data generated or analyzed during this study are included in this published article. y All data generated or analyzed during this study are included in this published article. All data generated or analyzed during this study are included in this published article Received: 29 April 2023; Accepted: 15 July 2023 Received: 29 April 2023; Accepted: 15 July 2023 References Caputo type fractional operator applied to Hepatitis B system. Fractals 30(1), 2240023 (2022). Din, A. The stochastic bifurcation analysis and stochastic delayed optimal control for epidemic model with general incidence ( ) 3. Din, A., Li, Y., Yusuf, A. & Ali, A. I. Caputo type fractional operator applied to Hepatitis B system. 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Irshad, F., & Kumar, N. Role of ordinary and partial differential equations as mathematical models in tumor growth. Eur. J. Mol Clin. Med. (EJMCM), 10(01) (2023). 3. Zhang, J., Bai, W., Liu, X., Guo, Y. & Zhang, Y. www.nature.com/scientificreports/ of the cancer, treatment goals, and desired outcomes, and may need to be adjusted based on patient response and changes in the cancer over time. In Fig. 6, the W(t) plot demonstrates an initial surge in wound healing cells followed by a gradual decline, indicating the body’s acute response to tissue injury and subsequent resolution of the healing process. This sug- gests that the lung tissue’s healing capacity is dynamic, with an initial boost in wound-healing cells that gradu- ally subsides as healing progresses. On the other hand, the v(t) plot reveals a sustained elevation of vascular endothelial growth factor, indicating a continuous need for angiogenesis to support tissue repair. This implies that the lung tissue requires a prolonged vascular supply to facilitate proper healing.hfih The employed approach has demonstrated high efficiency. The use of fractional order derivatives in this lung cancer model provides relevance by incorporating memory and non-local dependencies into the system. The fractional order ( α ) influences the rate of change and interactions between different cell populations, representing the memory effects in cellular processes. By including fractional derivatives, the model captures the long-term behavior and complex interactions involved in lung cancer dynamics, contributing to a more accurate repre- sentation of the disease’s progression and potential therapeutic interventions. The novelty of this research lies in the development of a comprehensive fractional-order mathematical model for lung cancer that incorporates integrated therapeutic approaches. This approach allows for a more precise understanding of the intricate inter- actions among cancer cells, immune cells, and other constituents of the tumor microenvironment. Additionally, by incorporating PD-L1 monoclonal antibody treatment and surgery as controls in the model, this study can explore the potential benefits of combining these treatments and investigate their impact on tumor dynamics. This approach stands out from others because it offers a more comprehensive and accurate representation of lung cancer dynamics and treatment response. It also holds promise for guiding the development of novel treatment strategies for lung cancer and enhancing patient outcomes. Results and discussion Additionally, the treatment has a time delay before it becomes effective. The PID controller consists of three terms - proportional, integral, and derivative gains - each of which adjusts the dosage based on different factors. The proportional term adjusts the dosage based on the error between the target and the actual population of cancer cells, with larger errors resulting in larger adjustments. The integral term accumulates the error over time and adjusts the dosage to correct for consistent differences between the target and actual population. The derivative term adjusts the dosage based on the rate of change of the error, preventing overshooting of the target population. The magnitude of the adjustments for each term is propor- tional to the gain constant for that term - Kp for the proportional term, Ki for the integral term, and Kd for the derivative term. By using the time evolution of the populations and the PID controller to adjust the dosage, the actual population of cancer cells can be brought within a specified tolerance of the target population, with the feedback control shown in Fig. 5. The target population of cancer cells can be set based on the stage and severity p p y yf The PID controller consists of three terms - proportional, integral, and derivative gains - each of which adjusts the dosage based on different factors. The proportional term adjusts the dosage based on the error between the target and the actual population of cancer cells, with larger errors resulting in larger adjustments. The integral term accumulates the error over time and adjusts the dosage to correct for consistent differences between the target and actual population. The derivative term adjusts the dosage based on the rate of change of the error, preventing overshooting of the target population. The magnitude of the adjustments for each term is propor- tional to the gain constant for that term - Kp for the proportional term, Ki for the integral term, and Kd for the derivative term. By using the time evolution of the populations and the PID controller to adjust the dosage, the actual population of cancer cells can be brought within a specified tolerance of the target population, with the feedback control shown in Fig. 5. The target population of cancer cells can be set based on the stage and severity Scientific Reports \| (2023) 13:12426 \| https://doi.org/10.1038/s41598-023-38814-2 www.nature.com/scientificreports/ References 1. Tao, M.H. Epidemiology of lung cancer. Lung Cancer and Imaging (2019). gy g g g g 2. Matthews, H. K., Bertoli, C. & de Bruin, R. A. Cell cycle control in cancer. Nat. Rev. Mol. Cell Biol. 23(1), 74–88 ( 3. Lahiri, A. et al. Lung cancer immunotherapy: Progress, pitfalls, and promises. Mol. Cancer 22(1), 1–37 (2023). g py g p p ( ) ( ) 4. Shinada, K., & Murakami, S. Neoadjuvant PD-1 blockade in non-small cell lung cancer: Current perspectives and moving forward OncoTargets Ther. 16, 99–108. (2023). h 5. 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B.K.: Conceptualization, Methodology, Formal Analysis, Investigation, Writing—Original Draft. E.H.: Methodology, Formal Analysis, Investigation, Writing—Original Draft, Writing—Review and Editing. References Fractional-order modeling and analysis of tumor–immune interactions with a cancer vaccine. Phys. A Stat. Mech. Appl. 553, 123300 (2020). 24. Baleanu, D., Jajarmi, A., Sajjadi, S. S. & Mozyrska, D. A new fractional model and optimal control of a tumor–immune surveillance with non-singular derivative operator. Chaos Interdiscip. J. Nonlinear Sci. 29(8), 083127 (2019). 5. Özköse, F. et al. A fractional modeling of tumor–immune system interaction related to lung cancer with real data. Eur. Phys. J. Plus 137, 1–28 (2022). ( ) 26. Naik, P. A., Owolabi, K. M., Yavuz, M. & Zu, J. Chaotic dynamics of a fractional order HIV-1 model involving AIDS-related cancer cells. Chaos Solitons Fractals 140, 110272 (2020). https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \| Fundingh g The research was conducted with no external funding or support. None of the authors have any financial or personal relationships that could potentially bias the findings presented in this manuscript. The views and opinions expressed in this article are solely those of the authors and do not represent the views or opinions of any organization or institution. Competing interests h © The Author(s) 2023 Additional information Correspondence and requests for materials should be addressed to D.A. Correspondence and requests for materials should be addressed to D.A. 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 nstitutional 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 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/. https://doi.org/10.1038/s41598-023-38814-2 Scientific Reports \| (2023) 13:12426 \|
https://openalex.org/W4311684379	https://www.qeios.com/read/GEX4LN/pdf	English	null	The Condition of Cigarettes and Other Tobacco Products Act: Case Example of IP Estate area	null	2,022	cc-by	1,431	Qeios, CC-BY 4.0 · Article, December 16, 2022 Open Peer Review on Qeios Abstract Many studies were done in India specifically suggesting that tobacco addiction has increased. Cigarettes and Other Tobacco Products Act of India, 2012, was made to ban the sale of cigarettes and other tobacco products within 100m of any institution. Proximity and ease of access to such addictive products are one of the main reasons for the increase in tobacco addiction in India. There are still many shops which openly sell cigarettes and tobacco-based products. Aim: The aim of the study is to assess the state of Cigarettes and Other Tobacco Product Act of India, 2012, in the IP Estate Precinct in Delhi. The Condition of Cigarettes and Other Tobacco Products Act: Case Example of IP Estate area Ritesh Kumar1, Samyu Tm1 1 School of Planning and Architecture Ritesh Kumar1, Samyu Tm1 1 School of Planning and Architecture Potential competing interests: No potential competing interests to declare. Introduction: Tobacco addiction, in India, is a very big issue today. Many studies were done in India specifically suggesting that tobacco addiction has increased, especially in the age ranging from 15-35. The age-standardized prevalence of any smoking in men at ages 15–69 years fell from about 27% in 1998 to 24% in 2010 but rose at ages 15–29 years. During this period, cigarette smoking in men became about twofold more prevalent at ages 15–69 years and fourfold more prevalent at ages 15–29 years.[1] The most convenient places fir the people in this age group to purchase cigarettes is near the institution they work in. Due to this reason specifically Cigarettes and Other Tobacco Products Act of India was made. This act state that the sale of cigarettes or any other form of tobacco products is prohibited within 100m of any institution. The question is that is this law followed properly or not? The study is limited to IP Estate precinct in New Delhi. Limitations: Peer pressure is widely recognized as a crucial factor affecting young people's early experimentation with tobacco and their willingness to continue smoking. Several students attending higher education institutions practice cigarette smoking for several reasons, such as a way to cope with stress. Limitations: It is difficult to map each and every tobacco and cigarette seller in precinct as there are many informal shops which keeps on changing its timings and positions around the precinct to avoid being caught. Literature Review: Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN 1/7 Qeios, CC-BY 4.0 · Article, December 16, 2022 According to the Cigarettes and Other Tobacco Products Act of India, the law prohibits the sale and promotion of tobacco products within 100 yards of the boundary of educational facilities. A sizable portion of teenagers use tobacco.[2]Adolescent tobacco use has been linked to school location, economic position and tobacco promotional activities (advertising, receiving free sample.) Adolescent tobacco use should be prevented by taking into account the neighborhood.[3] Normal adolescent developmental stage is affected by a high level of peer pressure that can influence risk-taking behaviors including substance use. Globally, especially in low- and middle-income countries, an estimated 80% of the one billion adolescent smokers are suffering from tobacco-related morbidity and mortality. Cigarette smoking negatively influences the physical and mental health of an individual. This is particularly true for high school and university students who already face major health challenges such as stress. Peer pressure is widely recognized as a crucial factor affecting young people's early experimentation with tobacco and their willingness to continue smoking. Several students attending higher education institutions practice cigarette smoking for several reasons, such as a way to cope with stress. According to the Cigarettes and Other Tobacco Products Act of India, the law prohibits the sale and promotion of tobacco products within 100 yards of the boundary of educational facilities. A sizable portion of teenagers use tobacco.[2]Adolescent tobacco use has been linked to school location, economic position and tobacco promotional activities (advertising, receiving free sample.) Adolescent tobacco use should be prevented by taking into account the neighborhood.[3] Normal adolescent developmental stage is affected by a high level of peer pressure that can influence risk-taking behaviors including substance use. Globally, especially in low- and middle-income countries, an estimated 80% of the one billion adolescent smokers are suffering from tobacco-related morbidity and mortality. Cigarette smoking negatively influences the physical and mental health of an individual. This is particularly true for high school and university students who already face major health challenges such as stress. Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN Conclusion: The analysis of the map that was generated by the field study showed that there are more than 30 shops in the precinct area which sells cigarettes and other tobacco-based products. All of these shops come under the 100m offset from one or other institutional building. This clearly shows that the implementation of Cigarettes and Other Tobacco Products Act, 2012, has not been done at any level. There are still several of these shops still existing in the precinct area which promotes tobacco addiction. Proximity and ease of access to such addictive products are one the main reasons for the increase in tobacco addiction in India especially in teenagers and youth. Methodology: The primary study was conducted as an observational survey of the ITO institutional area and identifying the all the shops that sell cigarettes and other tobacco-based products and mark their geo-location and pictures as evidence of sale. Then we mapped those locations on a map of ITO precinct and took offset of 100 yards from the boundary wall of all the institutional buildings in the precinct to analyze the number of shops violation the law. Case Study and Analysis: Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN 2/7 Qeios, CC-BY 4.0 · Article, December 16, 2022 Fig. 1: Map showing shops selling cigarettes and tobacco-based products Fig. 1: Map showing shops selling cigarettes and tobacco-based products The study shows that there are at-least 33 shops in the vicinity of the institutional area which are illegal, but they are still selling them openly. The presence of many educational institutes like SPA, Dayal Singh Public Library and Indian Institue of Public Administration, Indian Institute of Engineering and Indian Institute of Charted Accountant. This proximity indirectly promotes the consumption of cigarettes and tobacco-based products. Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN 3/7 Qeios, CC-BY 4.0 · Article, December 16, 2022 Qeios, CC-BY 4.0 · Article, December 16, 2022 Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN 4/7 Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN 4/7 Qeios, CC-BY 4.0 · Article, December 16, 2022 Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN 5/7 Qeios, CC-BY 4.0 · Article, December 16, 2022 Fig.2: Images showing the shops selling cigarettes and tobacco The images shows that the shops are not only selling cigarettes, but they are selling it openly and there are no actions taken on it Fig.2: Images showing the shops selling cigarettes and tobacco The images shows that the shops are not only selling cigarettes, but they are selling it openly and there are no actions taken on it The images shows that the shops are not only selling cigarettes, but they are selling it openly and there are no actions taken on it 1. ^Mishra S, Joseph RA, Gupta PC, et al. (2016). Trends in bidi and cigarette smoking in India from 1998 to 2015, by age, gender and education. BMJ Global Health, vol. 1:e000005 . Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN References 1. ^Mishra S, Joseph RA, Gupta PC, et al. (2016). Trends in bidi and cigarette smoking in India from 1998 to 2015, by age, gender and education. BMJ Global Health, vol. 1:e000005 . Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN 6/7 2. ^(2003). THE CIGARETTES AND OTHER TOBACCO PRODUCTS (PROHIBITION OF ADVERTISEMENT AND REGULATION OF TRADE AND COMMERCE, PRODUCTION, SUPPLY AND DISTRIBUTION) ACT. 3. ^Patel D, Kassim S and Croucher R. (2012). Tobacco Promotion and Availability in School Neighborhoods in India: a Cross-sectional Study of their Impact on Adolescent Tobacco Use. Asian Pacific Journal of Cancer Prevention, vol. 3 Issue 8 . Qeios, CC-BY 4.0 · Article, December 16, 2022 Qeios, CC-BY 4.0 · Article, December 16, 2022 2. ^(2003). THE CIGARETTES AND OTHER TOBACCO PRODUCTS (PROHIBITION OF ADVERTISEMENT AND REGULATION OF TRADE AND COMMERCE, PRODUCTION, SUPPLY AND DISTRIBUTION) ACT. 3. ^Patel D, Kassim S and Croucher R. (2012). Tobacco Promotion and Availability in School Neighborhoods in India: a Cross-sectional Study of their Impact on Adolescent Tobacco Use. Asian Pacific Journal of Cancer Prevention, vol. 3 Issue 8 . Qeios ID: GEX4LN · https://doi.org/10.32388/GEX4LN 7/7
https://openalex.org/W1987200804	https://www.redalyc.org/pdf/3074/307426643015.pdf	Portuguese	null	A (des)integração da África pós-colonial em A Bend in the River de V. S. Naipaul	Acta Scientiarum. Language and Culture	2,010	cc-by	5,627	Mariana Bolfarine Programa de Estudos Linguísticos e Literários em Inglês, Departamento de Letras Modernas, Universidade de São Paulo, Av. Luciano Gualberto, 403, 05508-900, sala 15, São Paulo, São Paulo, Brasil. E-mail: marianabolfarine@hotmail.com RESUMO. O presente trabalho visa refletir sobre as consequências do imperialismo europeu em uma cidade africana fictícia, inspirada no Zaire, representada por V. S. Naipaul na obra A Bend in The River (1979). O artigo investiga a falta de integração entre as diferentes esferas sociais que se formaram no leste da África, a partir da imigração de indianos e asiáticos como indentured workers, trabalhadores contratados. Serão analisados trechos do romance que possuem como foco as consequências dos relacionamentos inter-raciais vividos entre diferentes personagens. A conclusão é que A Bend in the River demonstra que a política de dividir para governar, posta em prática pelos europeus, influenciou a construção de uma sociedade fragmentada, desigual, hierárquica e determinista. O pessimismo de Naipaul corrobora a ideologia racista do colonialismo, que prega a pressuposição pelo negro africano da superioridade do branco europeu, reafirmando o seu direito de oprimir e dominar os povos colonizados. O campo teórico é constituído com base nos estudos de Avtar Brah, Edward Said, Michael Gorra. Palavras-chave: África, pós-colonialismo, imperialismo, (des)integração. ABSTRACT. The (dis)integration of post-colonial Africa in A Bend in the River by V. S. Naipaul. This work aims at reflecting upon the consequences of European imperialism in a fictitious African city, supposedly situated in Zaire, represented by V. S. Naipaul in A Bend in the River (1979). The article investigates the lack of integration between different social spheres, which were formed in East Africa, after the immigration of Indians and Asians as indentured workers. Different excerpts of the novel that focus on the consequences of the inter-racial relationships between different characters in the narrative will be analyzed. The conclusion is that A Bend in the River reveals that the divide and rule policy, put into practice by the European colonizer, influenced the construction of a society that is fragmented, unequal, hierarchical and deterministic. Naipaul’s pessimism supports the racist ideology of colonialism, which preaches the assumption by the black Africans of the superiority of the white Europeans, reaffirming the latter’s right to oppress and dominate the colonized people. The theoretical field is based on the works of Avtar Brah, Edward Said, Michael Gorra. Key words: Africa, post-colonialism, imperialism, (dis)integration. Acta Scientiarum. Language and Culture ISSN: 1983-4675 eduem@uem.br Universidade Estadual de Maringá Brasil Bolfarine, Mariana A (des)integração da África pós-colonial em A Bend in the River de V. S. Naipaul Acta Scientiarum. Language and Culture, vol. 32, núm. 1, 2010, pp. 61-66 Universidade Estadual de Maringá .jpg, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=307426643015 Acta Scientiarum. Language and Culture ISSN: 1983-4675 eduem@uem.br Universidade Estadual de Maringá Brasil Bolfarine, Mariana A (des)integração da África pós-colonial em A Bend in the River de V. S. Naipaul Acta Scientiarum. Language and Culture, vol. 32, núm. 1, 2010, pp. 61-66 Universidade Estadual de Maringá .jpg, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=307426643015 Acta Scientiarum. Language and Culture ISSN: 1983-4675 eduem@uem.br Universidade Estadual de Maringá Brasil Acta Scientiarum. Language and Culture ISSN: 1983-4675 eduem@uem.br Universidade Estadual de Maringá Brasil Bolfarine, Mariana A (des)integração da África pós-colonial em A Bend in the River de V. S. Naipaul Acta Scientiarum. Language and Culture, vol. 32, núm. 1, 2010, pp. 61-66 Universidade Estadual de Maringá .jpg, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=307426643015 Como citar este artigo Número completo Mais artigos Home da revista no Redalyc Sistema de Informação Científica Rede de Revistas Científicas da América Latina, Caribe , Espanha e Portugal Projeto acadêmico sem fins lucrativos desenvolvido no âmbito da iniciativa Acesso Aberto Sistema de Informação Científica Rede de Revistas Científicas da América Latina, Caribe , Espanha e Portugal Projeto acadêmico sem fins lucrativos desenvolvido no âmbito da iniciativa Acesso Aberto DOI: 10.4025/actascilangcult.v32i1.4737 Acta Scientiarum. Language and Culture A (des)integração da África pós A (des)integração da África pós A (des)integração da África pós A (des)integração da África pós----colonial em colonial em colonial em colonial em A Bend in the River A Bend in the River A Bend in the River A Bend in the River de V. de V. de V. de V. S. Naipaul S. Naipaul S. Naipaul S. Naipaul Mariana Bolfarine Introdução Introdução Introdução Introdução Segundo Naipaul, Mobutu passou a usar um chapéu de pele de leopardo e um cajado de madeira em aparições públicas, ambos símbolos de liderança, pois somente o chefe poderia matar o leopardo, a fim de defender sua tribo e demonstrar seu poder e autoritarismo (NAIPAUL, 1980). Na narrativa, o presidente compartilha a mesma trajetória de Mobutu em direção ao poder e, do mesmo modo, troca o terno, símbolo da influência ocidental, por trajes africanos, adotando o chapéu de pele de leopardo e o cajado de madeira. Tal mudança de hábitos e de identidade é descrita pelo protagonista de A Bend in the River: “The president I had only seen in photographs – first in army uniform, then in the stylish short-sleeved jacket and cravat, and then, with his leopard-skin chief´s cap and his carved stick, emblem of his chieftaincy” (NAIPAUL, 1979, p. 125, tradução nossa). Tanto no ensaio quanto no romance, o rio tem uma importância fundamental para a sobrevivência dos habitantes da região. Pela decadência dos meios de transporte rodoviário e aeroviário no Zaire, o rio, em 1975, ficou conhecido como a grande rodovia do país, “[...] the great highway of the country” (NAIPAUL, 1980, p. 98, tradução nossa), no qual o tráfego de barcos a vapor era intenso. Há, na narrativa, diversas referências às comunidades ribeirinhas, a exemplo da de Zabeth, mãe de Ferdinand, que passavam dias de jornada em barcos a vapor, para comprar utensílios domésticos simples, apenas para facilitar-lhes as tarefas diárias, visto que sua subsistência era extraída da natureza, sobretudo do rio. A diversidade étnica e cultural foi uma consequência da vinda dos europeus e dos imigrantes indianos e asiáticos, cuja interação com os africanos nativos resultou em um caleidoscópio de povos. A Bend in the River revela que as comunidades formadas pelos imigrantes e sua convivência com povos nativos da África formaram grupos quase independentes, mas que dividiam o mesmo espaço geográfico e eram subordinados aos colonizadores britânicos (BRAH, 1996). Grande parte da opinião crítica sobre a África está vinculada à política posta em prática pelos colonizadores britânicos de dividir para governar (divide and rule policy). As consequências de tal postura perduraram sob a forma de conflitos vividos pelos Estados africanos pós-coloniais, que emergiram no século XX, por meio da rude amalgamação de povos e de terras realizada pelos colonialistas fundadores (EMEZUE, 2006). Introdução Introdução Introdução Introdução A imagem recorrente em A Bend in the River atua como um símbolo dos danos causados pelas transformações realizadas em virtude do imperialismo, pois, ao crescer demais, a planta enroscava no motor dos barcos e ameaçava o fluxo do rio: Mobutu Sesé Seko, com o intuito de reconstruir a África pós-independente. Tanto no romance quanto no texto crítico, o autor versa sobre o destino da população imigrante descendente dos trabalhadores contratados, após o confisco dos seus comércios pelo governo de Mobutu, em 1973 (NAIPAUL, 1980). O romance é narrado em primeira pessoa pelo protagonista Salim, um muçulmano de origem indiana, que sai da costa leste para se estabelecer como comerciante, no interior da África. Por meio da narrativa de Salim, podemos ter acesso ao destino dessa cidade após a busca do presidente, inspirado em Mobutu, por uma autenticidade política, econômica e cultural, como tentativa de rejeitar os valores impostos pelos colonizadores e retornar à África anterior ao imperialismo (NAIPAUL, 1980). […] the water hyacinth was the fruit of the river alone. The tall lilac-coloured flower had appeared only a few years before, and in the local language there was no word for it. The people still called it ‘the new thing’ or ‘the new thing in the river’, and to them it was another enemy. Its rubbery vines and leaves formed thick tangles of vegetation that adhered to the river bank and clogged up the waterways (NAIPAUL, 1979, p. 46, grifo nosso, tradução nossa). […] the water hyacinth was the fruit of the river alone. The tall lilac-coloured flower had appeared only a few years before, and in the local language there was no word for it. The people still called it ‘the new thing’ or ‘the new thing in the river’, and to them it was another enemy. Its rubbery vines and leaves formed thick tangles of vegetation that adhered to the river bank and clogged up the waterways (NAIPAUL, 1979, p. 46, grifo nosso, tradução nossa). Em A New King for the Congo (1975), Naipaul menciona que Joseph Mobutu, apesar de sua origem humilde, teve acesso à escolaridade e atuou como jornalista antes de tornar-se general do exército em 1960. Em 1965, Mobutu tomou o poder e iniciou o processo de revalorização da África, adotando o nome Mobutu Sesé Seko. Introdução Introdução Introdução Introdução da atualidade. De ancestralidade indiana, Vidiadhar Surajprasad Naipaul nasceu em Trinidad no ano de 1932. Nos seus primeiros romances, o espaço retratado com maior recorrência correspondia ao Caribe de sua infância, porém, aos poucos, o pano de fundo de suas narrativas foi transposto do Caribe para a região metropolitana de Londres e, em seguida, para países da África e da Ásia. O presente artigo investiga, em um primeiro momento, o processo de formação das diferentes esferas sociais na cidade africana fictícia representada em A Bend in the River, a partir da imigração de indianos e asiáticos como trabalhadores contratados (indentured workers), os quais dividiam o mesmo território com europeus e africanos. Em um segundo momento, o trabalho é dirigido à falta de integração entre essas esferas sociais, por meio da análise de trechos do romance que possuem como foco as consequências dos relacionamentos vividos entre personagens de diferentes etnias e, por conseguinte, de diferentes classes sociais. A Bend in the River, que data de 1979, passa-se em uma cidade africana fictícia, supostamente inspirada no porto de Kisangani, situado no Zaire. No ensaio não-ficcional de Naipaul, A New King for the Congo (NAIPAUL, 1980), o paralelo entre o fato histórico e a ficção fica claro, na medida em que o ensaio propõe uma reflexão sobre a política nacionalista colocada em prática pelo presidente do Zaire, Vencedor do Prêmio Nobel em 2001, V. S. Naipaul é um dos maiores escritores caribenhos pós-coloniais Maringá, v. 32, n. 1, p. 61-66, 2010 62 Bolfarine região, a exemplo do jacinto aquático, que Salim descreve como “another enemy” (NAIPAUL, 1979, p. 46, tradução nossa), um outro inimigo presente na cidade retratada no romance, além dos colonizadores. A imagem recorrente em A Bend in the River atua como um símbolo dos danos causados pelas transformações realizadas em virtude do imperialismo, pois, ao crescer demais, a planta enroscava no motor dos barcos e ameaçava o fluxo do rio: região, a exemplo do jacinto aquático, que Salim descreve como “another enemy” (NAIPAUL, 1979, p. 46, tradução nossa), um outro inimigo presente na cidade retratada no romance, além dos colonizadores. Acta Scientiarum. Language and Culture A reflexão de Salim continua: […] I thought a lot about my refusal to say ‘The white men’ when Ferdinand asked me to define the ‘they’ who were working on the new telephone. And I saw that, in my wish to give him political satisfaction, I had indeed said what I intended to say. I didn’t mean the white men. I didn’t mean, I couldn’t mean, people like those I knew in our town, the people who had stayed behind after independence. I really did mean the scientists; I meant people far away from us in every sense. Além dos indianos como Salim, os demais representantes da camada intermediária, gregos, asiáticos, portugueses e belgas, tinham consciência da existência desse tipo de conhecimento e a quem ele pertencia. Muitos possuíam diferentes graus de escolaridade e, assim, poderiam ter contato com veículos de comunicação, como revistas, que divulgavam a produção desse conhecimento. Como detentores de capital, eles também estavam entre os que poderiam adquirir as ‘bênçãos’, como o binóculo e máquina fotográfica que Salim exibia orgulhosamente a Ferdinand. They! […] When we wanted to speak of the doers and makers and the inventors, we all - whatever our race - said ‘they’. We separated these men from their groups and countries and in this way attached them to ourselves. [...] The ‘they’ we spoke of in this way were very far away as to be hardly white. They were impartial, up in the clouds, like good gods. We waited for their blessings, and showed off those blessings - as I had shown off my cheap binoculars and my fancy camera to Ferdinand - as though we had been responsible for them. [...] In fact, I - and all the others like me in our town, Asian, Belgian, Greek - were as far away from they as he was (NAIPAUL, 1979, p. 44 e 45, grifo nosso, tradução nossa). Maringá, v. 32, n. 1, p. 61-66, 2010 Os africanos, como Ferdinand (nesse trecho), ocupavam a posição mais baixa da hierarquia social, pois, como não tinham acesso à educação, eram incapazes de produzir conhecimento dominante; a maioria, portanto, sobrevivia à custa da agricultura de subsistência e, de tempos em tempos, comprava objetos de pouco valor que facilitavam a sua vida, como refratários esmaltados, seringas, pilhas e lâminas de barbear, vendidos, sobretudo, pelos representantes da esfera social intermediária, os indianos e asiáticos. Introdução Introdução Introdução Introdução A sociedade africana foi tornando-se determinista, pois o status social era determinado pela pigmentação da pele e, com base nisso, as pessoas passaram a ser dividas em diferentes esferas sociais, consolidando um abismo racial e cultural entre os povos. Uma das implicações disso, para a sociedade pós-colonial africana, foi a criação de estereótipos que se calcificaram sob a forma de um “prisma sólido e transparente através do qual as diferentes raças se observavam mutuamente” (EMEZUE, 2006, p. 136). Dentre as intervenções perpetradas pelos europeus, estão as profundas transformações na paisagem natural e no quadro humano, tanto durante a época colonial quanto logo após a independência. Os europeus e os próprios africanos importaram elementos da flora e da fauna, como animais domésticos, cereais e plantas não-nativas da Maringá, v. 32, n. 1, p. 61-66, 2010 Maringá, v. 32, n. 1, p. 61-66, 2010 A (des)integração da África pós-colonial A (des)integração da África pós-colonial 63 abolido em 1916; porém, no leste da África, perdurou até 1922, quando a maior parte dos imigrantes retornou às suas terras e os que permaneceram ficaram limitados ao pequeno comércio (BRAH, 1996). Origem das esferas sociais Origem das esferas sociais Origem das esferas sociais Origem das esferas sociais Salim, de origem indiana, durante uma conversa com seu protegido, o africano Ferdinand, não só aponta para a existência das diferentes esferas sociais como também para a situação de subordinação cultural e econômica da África pós-colonial de A Bend in the River ao antigo colonizador: “I couldn't help myself: the teacher in me and the guardian came out. I said, ‘You should look at this. They are working on a new telephone. It works by light impulses rather than on electric current’” (NAIPAUL, 1979, p. 44, grifo nosso, tradução nossa). A constituição das esferas sociais na África pós- independente pode ser representada pelo conceito de colonial sandwich proposto por Brah (1996). Segundo essa definição, os europeus representariam a parte superior do sanduíche, os Asiáticos estariam no meio e os Africanos estariam abaixo de todos. Os indianos atuavam como um elo entre os bancos e os negros, pois transitavam entre esses dois grupos étnicos e ocupavam uma posição social intermediária, abaixo do colonizador branco, mas acima do negro africano. O poder político era restrito aos colonizadores e foi passado aos africanos somente após a independência. Ferdinand pergunta, “Who are they?”, e Salim responde a ele, após uma breve reflexão: I thought: We are here already, after only a few months at the lyceé. He's just out of the bush; I know his mother; I treat him like a friend; and already we're getting this political nonsense. I didn't give the answer I thought he was expecting. I didn´t say ‘the white men’. Though with half of myself I felt like saying it, to put him in his place. I said instead, ‘The scientists’ (NAIPAUL, 1979, p. 44, tradução nossa). Em A Bend in the River, os europeus eram os detentores dos meios de produção, os doers and makers, pois faziam parte do grupo dos criadores e inventores. Quando Salim referia-se a este grupo, ele mencionava que todas as pessoas pertencentes ao seu meio social aludiam a eles como they (eles), a fim de separá-los dos demais grupos étnicos. A reflexão do protagonista demonstra que os europeus, como detentores dos meios de produção, representavam o único grupo capaz de produzir e proferir suas ‘bênçãos’: descobertas, criações e inventos tecnológicos aos demais. Sendo assim, a cultura, representada pelo conhecimento científico dominante, não era igualmente acessível às diferentes esferas sociais. A reflexão de Salim continua: Acta Scientiarum. Language and Culture A (des)integração entre as esferas sociais A (des)integração entre as esferas sociais A (des)integração entre as esferas sociais A (des)integração entre as esferas sociais Nesse segundo momento, a análise é voltada para a falta de integração entre as esferas sociais de A Bend in the River e, também, para como esse fato reflete a construção de uma sociedade desigual, hierárquica e fragmentada. Há, no romance, dois relacionamentos inter-raciais que ratificam a impossibilidade de integração entre as diferentes etnias e a manutenção das camadas do ‘sanduíche colonial’ de Brah (1996): o de Metty com uma ‘mulher africana’ e o de Salim com Yvette. Salim, por sua vez, reencontrou Indar, um amigo de infância que, por pertencer a uma família abastada na costa da África, foi estudar em Londres. Após seu regresso, Indar ocupou um cargo de professor no Domain, uma cidade universitária construída pelo presidente, com o intuito de modernizar a África. Nas festas do Domain, Salim conheceu Yvette, vinda da Bélgica e casada com Raymond, o braço direito do presidente, conhecido como “o homem branco do grande Homem”, “Big Man’s White man” (NAIPAUL, 1979, p. 184, tradução nossa), o homem branco do presidente. Assim que Indar retornou à Londres, Salim e Yvette iniciaram um relacionamento amoroso extraconjugal. Salim estava realizado, pois, além de ter sido a primeira mulher que ele possuiu sem pagar, ela representava um meio de ascensão social para a vida europeia repleta de glamour. Metty, que significa mestiço, do francês métis, ganhou este apelido ao mudar-se da costa da África para o interior. Ele era um escravo pertencente à família de Salim, que o herdara. Desenvolto, Metty relacionava-se muito bem com as mulheres africanas e, com uma delas, teve um filho. Salim, ao descobrir, sentiu-se traído e manifestou sua decepção, Oh, Metty, why didn’t you tell me? Why did you do this to me? […] Ali, Ali-wa! We lived together. I took you under my roof and treated you as a member of my family. And now you do this […]. ‘I will leave her, patron. She’s an animal’. Apesar de sua satisfação para com sua nova vida, Salim passou a enxergar Metty, que no início parecia bem adaptado ao interior Africano, como alguém deslocado: ‘How can you leave her? You’ve done it. You can’t go back on that. You’ve got that child out there. Oh, Ali, what have you done? Don’t you think it’s disgusting to have a little African child running about in somebody’s yard with its toto swinging from side to side? Aren’t you ashamed? A reflexão de Salim continua: Os bens produzidos pelos Esse excerto revela a hierarquia da sociedade em que os personagens estavam inseridos, pois remete a um momento histórico posterior ao recrutamento de indianos e chineses para atuar como indentured workers, trabalhadores contratados, na construção de ferrovias. Este tipo de regime de semiescravidão foi Maringá, v. 32, n. 1, p. 61-66, 2010 Maringá, v. 32, n. 1, p. 61-66, 2010 Acta Scientiarum. Language and Culture 64 Bolfarine brancos eram-lhes inacessíveis, visto que não possuíam o capital para adquiri-los. de fundo, um cenário e uma atmosfera ao romance. Não é possível diferenciá-los uns dos outros, pois eles não possuem um nome, uma voz ou um papel significativo; eles são a black woman, an African woman. Logo, durante o imperialismo, a política colonial visava a restringir as atividades dos negros à agricultura e dos indianos e demais imigrantes ao pequeno comércio e troca; havia o cuidado de manter esses dois grupos subordinados aos ocupantes brancos de origem europeia, representantes da burguesia comercial. Esta política foi essencial para a formação das classes sociais no leste da África (BRAH, 1996). A situação de Metty não era das melhores, pois apesar de ter frequentado a escola quando morava com a família de Salim, ele não deixou de ser seu empregado e dependia daquele trabalho para sobreviver, pois como não recebia um salário, o patron oferecia-lhe comida e moradia. No entanto, apesar de mestiço, Metty considerava-se superior à mãe de seu filho que, para ele, não passava de uma ‘mulher africana’. O desejo de garantir seu sustento e manter sua posição social privilegiada fez com que Metty abandonasse a família, cujos membros, para ele, não passavam de animais. Acta Scientiarum. Language and Culture O pessimismo de Salim: eco da voz de Naipaul O pessimismo de Salim: eco da voz de Naipaul O pessimismo de Salim: eco da voz de Naipaul O pessimismo de Salim: eco da voz de Naipaul???? Para Naipaul, os diferentes povos integrados pelo império não se tornaram um único povo, pois eles fracassaram em formar uma cultura comum. Houve uma ‘colisão’ entre o ocidente, a Índia e a África, mas sem que houvesse uma “coerência” (GORRA, 1997, p. 80). Para Naipaul, justamente essa ‘colisão’ é a origem dos embates dos países pós-coloniais, em que o povo subjugado não tem escolha a não ser aspirar à cultura dominante imposta pelo colonizador. Salim já não via mais nada de positivo em seu relacionamento, pois Yvette passou a ser como as prostitutas com quem ele saía e, a seu ver, ela passou a tratá-lo como Raymond, seu marido fracassado. Uma noite em que Yvette o procurou, com roupa de festa e um pouco embriagada, Salim a agrediu fisicamente e cuspiu entre suas pernas. Convém explicitar que o romance é narrado em primeira pessoa pelo protagonista Salim e que a sua narrativa pode ser considerada um “eco do posicionamento do autor” (GORRA, 1997, p. 106), a respeito do pessimismo em relação ao destino dos países pós-coloniais. O título do ensaio no qual A Bend in the River foi baseado, A New King for the Congo: Mobutu and the Nihilism of Africa (NAIPAUL, 1980), contém em si a visão do autor sobre o fracasso das sociedades pós-coloniais. Para Naipaul, a saída dos europeus corresponderia ao fim da civilização e ao retorno à barbárie, a um momento em que não haveria nada além de “bush”, mato, “I had to talk to myself – just to drive through bush and more bush [...] Sun and rain and bush had made the site look old, like the site of a dead civilization” (NAIPAUL, 1979, p. 3 e 27, tradução nossa). Tal pessimismo, presente na maioria dos seus romances, demonstra que, para Naipaul, “a separação dos diferentes povos de suas origens, provocada pelo imperialismo não resultou em nada além de um estranhamento e um anseio por uma terra idealizada” (GORRA, 1997, p. 78). When she was almost at the door she turned and smiled and Said, ‘You don’t have a woman hidden in the cupboard, do you?’ […] It was so much the kind of thing I heard from whores who pretended to be jealous in order to please. It blasted the moment. Opposites: again this communication by opposites. That woman in the cupboard: that other person outside […] Affection, just before betrayal. O pessimismo de Salim: eco da voz de Naipaul O pessimismo de Salim: eco da voz de Naipaul O pessimismo de Salim: eco da voz de Naipaul O pessimismo de Salim: eco da voz de Naipaul???? And I had been in tears. [...] ‘Do you think I’m Raymond?’ This time she had no chance to reply. She was his so hard and so often about the face, even through raised, protecting arms, that she staggered back and allowed herself to fall on the floor (NAIPAUL, 1979, p. 218-219, tradução nossa). O fracasso dos relacionamentos inter-raciais analisados leva a refletir sobre a inscrição latina entalhada em um bloco de mármore que sobrou das ruínas de um monumento que celebrava os 60 anos de uma empresa europeia de barcos a vapor, destruído após o fim do colonialismo, com o qual Salim deparou- se logo após a mudança para o interior. De acordo com o padre Huismans, diretor do Liceu da cidade, a frase Micerique probat populos et foedera jungi é oriunda de um poema sobre a fundação de Roma e significa He approves of the mingling of the peoples and their bonds of union (NAIPAUL, 1979, p. 62), que o grandioso deus romano aprova o estabelecimento dos romanos na África, a mistura dos povos e tratados de união entre eles. A (des)integração da África pós-colonial 65 tradução nossa). Quando Salim descobriu que a inscrição era falaciosa, ficou indignado com o modo pelo qual os poderes imperiais manipularam seu governo, o que, para o protagonista, significava que aquela cidade havia sido construída sobre uma mentira. discursos elaborados por Raymond. Yvette refletia o fracasso do marido e Salim passou a vê-la como uma pessoa tão desencontrada quanto ele próprio. And failure like that wasn’t what I would have chosen to be entangled with. My wish for an adventure with Yvette was a wish to be taken up to the skies, to be removed from the life I had – the dullness, the pointless tension, the situation of the country. It wasn’t a wish to be involved with people as trapped as myself (NAIPAUL, 1979, p. 183, tradução nossa). A (des)integração entre as esferas sociais A (des)integração entre as esferas sociais A (des)integração entre as esferas sociais A (des)integração entre as esferas sociais A boy like you?’ In the flat I saw that Metty’s light was on. I said, ‘Metty?’ He said through the door, ‘Patron’. He stopped calling me Salim; we had seen little of one another outside the shop for some time. And going on to my own room, considering my own luck, I thought: poor Metty. How will it end for him? So friendly and yet in the end always without friends. He should have stayed on the coast. He had his place there. He had people like himself. Here he is lost (NAIPAUL, 1979, p. 178, tradução nossa). ‘It is disgusting, Salim.’ He came and put his hand on my shoulder. ‘And I am very ashamed. She’s only an African woman. I will leave her’ (NAIPAUL, 1979, p. 105-106, grifo nosso, tradução nossa). ‘It is disgusting, Salim.’ He came and put his hand on my shoulder. ‘And I am very ashamed. She’s only an African woman. I will leave her’ (NAIPAUL, 1979, p. 105-106, grifo nosso, tradução nossa). Esse trecho revela que na cidade africana retratada no romance, dentre as classes não-brancas, existia uma hierarquia, segundo a qual os africanos negros estavam situados por último. A mulher referida pelo empregado não tem nome, ela representa, de modo pejorativo, as ‘mulheres africanas’, comparadas a animais, cujos filhos perambulavam nus pela vizinhança. No entanto, não tardou para Salim descobrir que logo voltaria a estar tão deslocado quanto Metty. O protagonista percebeu que se havia enganado a respeito de Yvette, pois ela não ocupava mais a posição de poder que ele havia pensado. Em virtude da política nacionalista, o presidente dependia menos dos Segundo Gorra (1997), os nativos retratados em A Bend in the River, limitam-se a fornecer um pano Maringá, v. 32, n. 1, p. 61-66, 2010 A (des)integração da África pós-colonial Acta Scientiarum. Language and Culture License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i No livro de Michael Gorra (1997), a referência original ao trecho de Robinson Crusoe é “learn to shape...where nothing was / the language of a race”, Derek Walcott, “Crusoe’s Journal”, in Collected Poems, 94 (apud GORRA, 1997, p. 190). Bolfarine Por fim, o imperialismo pregava que a independência era para brancos e europeus, pois para que houvesse dominação, “os povos inferiores e subjugados tinham que ser governados, na medida em que a ciência, o aprendizado e a história emanavam do ocidente” (SAID, 1993, p. 94-95), conforme demonstra a presente análise de A Bend in the River. A exemplo da hierarquia social e determinista existente na sociedade retratada no romance, as sociedades ocidentais modelavam e estabeleciam os limites na representação dos que eram considerados essencialmente seres subordinados. Para o crítico Said (1993, p. 94-95), tal representação “caracteriza a manutenção do subordinado, subordinado e do inferior, inferior”. heterogeneidade inevitável, por outro, o advento do próprio progresso da humanidade surge como resultado da mistura e comunicações culturais, por meio das quais as realizações culturais de uma sociedade enxertam-se em outra” (apud YOUNG, 1995, p. 49). Logo, o Nativismo proposto por Herder (apud GORRA, 1997), que defende a homogeneização das culturas, e posto em prática pelo presidente de A Bend in the River, por meio da política nacionalista, sugere que seria impossível recuperar o passado, isto é, regressar à África anterior à colonização, pois “a ilusão de reconquistar o sentimento de auto- valorização que o imperialismo destruiu, não passaria da nostalgia por um tempo de segurança, anterior à grande desordem causada pelo imperialismo” (GORRA, 1997, p. 78). Gorra (1997) faz referência à figura de Robinson Crusoé que, para Naipaul, serve como um emblema para as ilhas do Caribe que pode ser transposto para a África de A Bend in the River: ambas são sociedades que devem “aprender a construir, onde nada havia, [...] a língua de uma raça” (GORRA, 1997, p. 190, tradução nossa), uma raça na qual todos os representantes são náufragos, apartados das suas origens.1 Por conseguinte, tomando como base os estudos de Herder, podemos inferir que o Nativismo, em detrimento da heterogeneidade proporcionada pela mistura racial, não só é um ideal racista, que defende a segregação do Outro, mas é também uma utopia. Além de a mistura racial ser inevitável, o progresso e a evolução cultural somente ocorrerão por meio do contato entre as civilizações. i Referências Referências Referências Referências BRAH, A. Cartographies of diaspora. London: Routledge, 1996. Nativismo x evolução cultural Nativismo x evolução cultural Nativismo x evolução cultural Nativismo x evolução cultural Acerca do embate entre as diferentes culturas colocadas em contato, por meio do imperialismo, que é o tema central de A Bend in the River, o filósofo e crítico literário Johann Herder, afirma que “uniões híbridas e forçadas entre as nações estão fadadas à desintegração” (apud YOUNG, 1995, p. 47). Enquanto os próprios colonos degenerarão em climas não-naturais, o mesmo ocorrerá com os africanos, ao entrarem em contato com a cultura ocidental, imposta pelos europeus. Herder propõe um paradoxo, pois, por um lado, considera que “a colonização e a mistura racial introduzem uma Todavia, o diretor do Liceu revelou-lhe que, na verdade, a inscrição era falsa, pois no contexto original significava que o grandioso deus romano poderia não aprovar o estabelecimento dos romanos na África, nem a mistura de povos e nem tratados de união entre eles: “[...] the great Roman god might not approve of a settlement in Africa, of a mingling of peoples there, of treaties of union between Africans and Romans” (NAIPAUL, 1979, p. 62, Maringá, v. 32, n. 1, p. 61-66, 2010 66 Maringá, v. 32, n. 1, p. 61-66, 2010 Received on September 26, 2008. Accepted on October 8, 2008. Considerações finais Considerações finais Considerações finais Considerações finais EMEZUE, G. M. T. Failed heroes and failed memories: between VS Naipaul's Biswas and Mongo Beti's Medza. In: RE-IMAGINING African Literature (2). Tampa: Progeny International, 2006. p. 133-142. (Journal of African Literature and Culture JALC, 3). O objetivo do presente artigo foi demonstrar, à luz dos posicionamentos de críticos pós-coloniais a respeito de V. S. Naipaul e de suas obras, que a falta de integração entre os povos, unidos sob a forma do ‘sanduíche colonial’ (BRAH, 1996), constituiu as esferas sociais fragmentadas presentes em A Bend in the River. Tal (des)integração revela um pessimismo por parte do autor em relação às sociedades que viveram o imperialismo, pois o romance aponta que qualquer tentativa de miscigenação entre as diferentes raças não passaria de uma frustração. A conclusão é que, para Naipaul, a ‘colisão’ sem ‘coerência’ (GORRA, 1997) entre as culturas reflete a manutenção da ideologia racista do colonialismo, que prega a pressuposição pelo negro africano da superioridade do branco europeu, reafirmando o seu direito de oprimir e dominar os povos colonizados. Para Said, tal ideologia hierarquizante inclui a noção de que “certos povos exigem e procuram dominação, bem como formas de conhecimento associadas à mesma” (SAID, 1993, p. 8). GORRA, M. After empire: Scott, Naipaul, Rushdie. Chicago: The University of Chicago Press, 1997. NAIPAUL, V. S. A bend in the river. New York: Vintage, 1979. NAIPAUL, V. S. A New king for the Congo: Mobutu and the Nihilism of Africa, 1975. In: VINTAGE Naipaul. New York: Vintage, 1980. p. 88-128. SAID, E. Culture and imperialism. New York: Vintage, 1993. YOUNG, R. J. C. Desejo colonial: hibridismo em teoria, cultura e raça. São Paulo: Perspectiva, 1995. Received on September 26, 2008. Accepted on October 8, 2008. Received on September 26, 2008. Accepted on October 8, 2008. Maringá, v. 32, n. 1, p. 61-66, 2010 Acta Scientiarum. Language and Culture
https://openalex.org/W2160267609	https://www.scielo.br/j/bjmbr/a/f6XZgv6s5rdKV9V44ydtZVv/?lang=en&format=pdf	English	null	Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor	Brazilian Journal of Medical and Biological Research	2,000	cc-by	5,803	Expression of recombinant IFNAR 1 polypeptides Brazilian Journal of Medical and Biological Research (2000) 33: 771-778 ISSN 0100-879X Expression of recombinant IFNAR 1 polypeptides Brazilian Journal of Medical and Biological Research (2000) 33: 771-778 ISSN 0100-879X Abstract Correspondence S. Yoon Departamento de Análises Clínicas e Toxicológicas, FCF, USP Av. Lineu Prestes, 580, Bloco 17 05508-900 São Paulo, SP Brasil Fax: +55-11-813-2197 E-mail: sunok@zipmail.com.br Research supported by CAPES and FAPESP. Received April 28, 1999 Accepted March 22, 2000 Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the devel- opment of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have ex- pressed two polypeptides encoding amino acids 93-260 (P1) and 261- 410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 poly- peptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatogra- phy with 8 M urea and refolded by dialysis. Under reducing SDS- PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibod- ies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentra- tions (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus. Correspondence S. Yoon Departamento de Análises Clínicas e Toxicológicas, FCF, USP Av. Lineu Prestes, 580, Bloco 17 05508-900 São Paulo, SP Brasil Fax: +55-11-813-2197 E-mail: sunok@zipmail.com.br Research supported by CAPES and FAPESP. Received April 28, 1999 Accepted March 22, 2000 Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor S. Yoon1, R.D.C. Hirata1, N.Y. Nguyen2, R. Curi3, M. Russo4 and M.H. Hirata1 S. Yoon1, R.D.C. Hirata1, N.Y. Nguyen2, R. Curi3, M. Russo4 and M.H. Hirata1 1Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil 2Facility for Biotechnology Resources, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA Departamentos de 3Fisiologia e Biofísica, and 4Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil 1Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil 2Facility for Biotechnology Resources, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA Departamentos de 3Fisiologia e Biofísica, and 4Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil Introduction One approach to the inhibition of the action of IFN-a would be the production of anti-IFNAR antibodies that can block the effects of all IFN-a subclasses. Uzé et al. (19) showed that polyclonal anti- bodies raised against cells expressing the w share a common receptor called IFN-a receptor (IFNAR), while IFN-g interacts with a distinct receptor (IFN-g receptor). IFN-a comprises at least 13 subclasses of structur- ally similar glycoproteins encoded by re- lated genes, whereas IFNs-ß, -g and -w have only 1 subclass (3). Abnormal production of IFN-a has been reported in systemic lupus erythematosus (SLE), type I diabetes and in graft-versus-host disease (GVHD) (8-10). Preble et al. (8) have shown that an acid- labile type of IFN-a is specifically elevated in about 50% of SLE patients. Increased levels of IFN-a were observed in the pan- creases of patients with type I diabetes, while other cytokines (TNF-a, IL-1ß, IL-2, IL-4, and IL-6) were not increased (9). The devel- opment of autoimmune diseases and severe GVHD complication has been reported to occur after IFN-a therapy in patients with carcinoid tumors or chronic hepatitis C (10- 14). It was shown that irradiated mice under- going GVHD after allogeneic bone marrow transplant develop high levels of IFN-a ac- tivity. In contrast, mice given T-cell-depleted allogeneic bone marrow failed to develop GVHD and had no detectable serum IFN-a activity (15). This evidence indicates that IFN-a may contribute to the pathogenesis of autoimmune diseases and plays an important role in allograft rejection of bone marrow transplants (16-18). Therefore, the develop- ment of agents antagonistic to IFN-a would greatly contribute to new alternatives for the trreatment of these patients. Indeed, injec- tion of polyclonal anti-mouse IFN-a/ß anti- bodies has reduced the resistance to alloge- neic bone marrow transplantation in mice, demonstrating their potential inhibition of IFN-a activity in vivo. However, anti-IFN-a antibodies were able to neutralize only some subclasses of IFN-a. One approach to the inhibition of the action of IFN-a would be the production of anti-IFNAR antibodies that can block the effects of all IFN-a subclasses. Uzé et al. (19) showed that polyclonal anti- subunit 1 of IFNAR (IFNAR 1) can neutral- ize the activity of several IFN-a subclasses. Similarly, Benoit et al. Introduction (20) observed that monoclonal antibodies directed against the extracellular domain of the IFNAR, which was expressed in Escherichia coli and COS 7 cells, neutralize the biological activity of rIFN-a2, rIFN-aB, rIFN-ß and rIFN-w and of IFN-a purified from leukocytes or Namalwa cells, as well as the IFN present in the serum of AIDS patients. Nguyen et al. (21) have also expressed the extracellular domain of the IFNAR 1 in Escherichia coli as a fusion protein with glutathione-S-trans- ferase and have demonstrated that this re- combinant protein inhibits the antiviral and antiproliferative activities of rIFN-aB and competes with the cell surface receptor for IFN-a binding. This information led us to search for inhibitors of IFN-a activity by producing polypeptide fragments of IFNAR 1 and raising antibodies to the fragments. We report here the production of two polypeptide fragments encoding amino ac- ids 93-260 (P1) and 261-410 (P2) of the extracellular domain of IFNAR 1 expressed in Escherichia coli. The biological activity of these polypeptides and of their respective antibodies was evaluated using antiprolif- erative and antiviral assays. Introduction fects. These effects are mediated by the bind- ing of IFN to specific receptors on the cell surface, signaling transduction through the nucleus and activating specific genes (1-3). IFNs are assigned to two classes, type I and type II IFN, according to their interaction with specific receptors (4-7). IFNs a, ß and Interferons (IFNs) are biologically active proteins produced by cells in response to a variety of stimuli such as microbes, tumors and antigens. IFNs exert antiviral, antimicro- bial, antitumor, and immunomodulatory ef- Braz J Med Biol Res 33(7) 2000 Braz J Med Biol Res 33(7) 2000 772 S. Yoon et al. w share a common receptor called IFN-a receptor (IFNAR), while IFN-g interacts with a distinct receptor (IFN-g receptor). IFN-a comprises at least 13 subclasses of structur- ally similar glycoproteins encoded by re- lated genes, whereas IFNs-ß, -g and -w have only 1 subclass (3). Abnormal production of IFN-a has been reported in systemic lupus erythematosus (SLE), type I diabetes and in graft-versus-host disease (GVHD) (8-10). Preble et al. (8) have shown that an acid- labile type of IFN-a is specifically elevated in about 50% of SLE patients. Increased levels of IFN-a were observed in the pan- creases of patients with type I diabetes, while other cytokines (TNF-a, IL-1ß, IL-2, IL-4, and IL-6) were not increased (9). The devel- opment of autoimmune diseases and severe GVHD complication has been reported to occur after IFN-a therapy in patients with carcinoid tumors or chronic hepatitis C (10- 14). It was shown that irradiated mice under- going GVHD after allogeneic bone marrow transplant develop high levels of IFN-a ac- tivity. In contrast, mice given T-cell-depleted allogeneic bone marrow failed to develop GVHD and had no detectable serum IFN-a activity (15). This evidence indicates that IFN-a may contribute to the pathogenesis of autoimmune diseases and plays an important role in allograft rejection of bone marrow transplants (16-18). Therefore, the develop- ment of agents antagonistic to IFN-a would greatly contribute to new alternatives for the trreatment of these patients. Indeed, injec- tion of polyclonal anti-mouse IFN-a/ß anti- bodies has reduced the resistance to alloge- neic bone marrow transplantation in mice, demonstrating their potential inhibition of IFN-a activity in vivo. However, anti-IFN-a antibodies were able to neutralize only some subclasses of IFN-a. Preparation of polyclonal anti-P1 and anti-P2 antibodies The two cDNA fragments of IFNAR 1 encoding insert 1 from nucleotides 277 to 780 and insert 2 from nucleotides 781 to 1230 were produced by the polymerase chain reaction using cDNA of IFNAR 1 as tem- plate. Specific oligonucleotide primers con- taining the restriction enzyme sites EcoRI and BamHI for P1 and EcoRI and NdeI for P2 were used. cDNA fragments were in- serted into plasmid pRSET A (Invitrogen, San Diego, CA, USA) and the clones were sequenced with a SequenaseTM v2.0 instru- ment (Amersham, Buckinghamshire, Eng- land) (22). Expression of the two fragments of IFNAR 1 cDNA was achieved by trans- formation into E. coli BL21(DE3)pLysS cells, followed by IPTG induction for 3 h at 37oC in L-broth containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol. Polypeptide 1 was synthesized with a polyhistidine linker and an enterokinase cleavage site. Mice were immunized by sc injection with 200 µg of polypeptides P1 or P2 in complete Freund adjuvant. After 15 days, incomplete Freund adjuvant was also in- jected sc. Ten days later, the level of specific serum antibodies was assayed by ELISA and the good responders were boosted with poly- peptides by sc injection in incomplete Freund adjuvant. Five days later, the serum of the animals was obtained and used as polyclonal antibody after IgG purification through a protein A column. In vitro inhibition assay of antiproliferative activity of IFN-aB Antiproliferative assays were performed using Daudi cells. Daudi cells were col- lected by centrifugation and resuspended in warmed (37oC) RPMI 1640 medium at a density of 4 x 105 cells/ml. Cultures were allowed to equilibrate for 2 h at 37oC and 100 µl of cell culture was added to each well of a 96-well plate. For the inhibition assay of P1 and P2, 6.25 nM IFN-aB was pre-incu- bated at 37oC for 1 h with varying concentra- tions (0-250 µM) of the polypeptides. The mixtures were added to the Daudi cells and incubated at 37oC for 3 days. For the inhibi- tion assay of polyclonal antibodies, serial dilutions (10-1-10-5) of the polyclonal anti- bodies were pre-incubated with Daudi cells at 4oC for 1 h, followed by the addition of 6.25 nM IFN-aB and incubation at 37oC for 3 days. Cell proliferation was determined by an MTT colorimetric assay (24). Interferon 50 for P1 and P2, respectively. The purified polypeptides were refolded by dialysis against decreasing concentrations of urea (6, 4, 2, 1.5, 1, and 0 M) and against RPMI 1640 medium supplemented with 1% FBS at 4oC. The protein content of the purified fractions was determined by Lowry’s method (23). Recombinant IFN-aB, with a specific activity of 2 x 108 IU/mg, was provided by Dr. David Gangemi (Clemenson State Uni- versity, SC, USA). Cell lines Human Daudi lymphoblastoid cells were provided by Dr. Kath C. Zoon (Food and Drug Administration, Bethesda, MD, USA) and maintained in RPMI 1640 medium (Gibco) supplemented with 10% heat-inacti- vated fetal bovine serum (FBS). Hep 2/c cells of the epidermoid carcinoma cell line were provided by Dr. Consuelo L.F. Dias (Fundação Ezequiel Dias, Belo Hori- zonte, MG, Brazil) and maintained in RPMI 1640 medium supplemented with 5% heat- inactivated FBS. Braz J Med Biol Res 33(7) 2000 Braz J Med Biol Res 33(7) 2000 773 Expression of recombinant IFNAR 1 polypeptides Expression and purification of polypeptides P1 and P2 The cloned plasmids with the two cDNA fragments of the extracellular domain of IFNAR 1 were purified and cleaved with the restriction enzymes to check the presence of the inserted cDNA fragment. The high ex- pression of polypeptides P1 (22 kDa) and P2 (17 kDa) in E. coli BL21(DE3)pLysS was demonstrated by SDS-PAGE under reduc- ing conditions (Figure 1). Polypeptides P1 and P2, produced as in- clusion bodies in E. coli, were submitted to serial sonication steps for the isolation of recombinant polypeptides from bacterial pro- teins. As shown in Figure 1, the third sonica- tion removed most, but not all, contaminant bacterial proteins. Sonication partially puri- fied the polypeptides and facilitated the next purification step by gel filtration chromatog- raphy. The amount of purified and refolded polypeptides obtained by dialysis was 25 µg/ ml for P1 and 15 µg/ml for P2. Purification Polypeptides P1 and P2 produced in E. coli as insoluble inclusion bodies were extracted twice by sonication (30 s), using sonication buffer (1 mM EDTA, 100 mM NaCl, 1 mM PMSF, 2 mM DTT, and 100 mM Tris-HCl, pH 9.2, for P1 and pH 7.5 for P2) and once more with sonication buffer plus 1% Triton X-100. The final fraction was solubilized in 8 M urea containing 1 mM DTT, 1 mM EDTA and 100 mM Tris-HCl, pH 9.2, for P1 and pH 7.5 for P2. The urea- solubilized fractions containing polypeptides P1 or P2 were purified by gel filtration chro- matography using Sephadex G-100 and G- Braz J Med Biol Res 33(7) 2000 Braz J Med Biol Res 33(7) 2000 774 S. Yoon et al. set at P<0.05. In vitro inhibition assay of antiviral activity of IFN-aB In vitro inhibition assay of antiviral activity of In vitro inhibition assay of antiviral activity of IFN-aB Results Antiviral assays were performed using a combination of Hep 2/c cells and encephalo- myocarditis virus (EMCV). Polypeptides (0- 1,250 µM) were pre-incubated with 2.5 nM IFN-aB at 37oC for 1~2 h. The mixture was added to 100 µl of Hep 2/c cells (5 x 104 cells/100 µl/well) on a 96-well plate and incubated at 37oC for 1 day. Following incu- bation, 200 µl of viral suspension was added to all wells and incubated at 37oC for 25~26 h. The complete (100%) cytopathic effect (CPE) of EMCV on unprotected virus was used as control. The CPE was determined by staining with amido blue black and absorb- ance measured at 620 nm in a Microelisa reader (25). For the inhibition assay of poly- clonal antibodies, serial dilution and pre- incubation with Hep 2/c cells at 37oC for 2 h in a 96-well plate were performed. One IU/ 100 µl (2.5 nM) IFN-aB was added to this mixture and incubated at 37oC for 1 day. Addition of the viral suspension and deter- mination of the CPE were performed as men- tioned above. Expression and purification of polypeptides P1 and P2 Inhibition of the antiproliferative activity of IFN-aB The non-parametric Spearman correla- tion test and Student t-test were used to detect differences as indicated in the Results section (26). The level of significance was Based on the sensitivity of Daudi cells to IFN-aB we initially used 25 nM and 12.5 kDa MW C 1 2 3 4 5 6 7 8 A 97.0 97.0 kDa MW C 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 MW 66.2 31.0 21.0 14.0 6.5 66.2 31.0 21.0 14.0 6.5 kDa A B B Figure 1 - Expression and purifi- cation of P1 (A) and P2 (B) by serial sonication steps and gel filtration chromatography. MW: Protein molecular weight mark- er; lane C: bacterial cell suspen- sion before induction; lane 1: cell suspension after induction by IPTG; lanes 2, 4 and 6: superna- tants of the 1st, 2nd and 3rd sonication; lanes 3, 5 and 7: pel- lets of the 1st, 2nd and 3rd soni- cation; lane 8: purified and re- folded polypeptides (P1: 25 µg/ ml; P2: 15 µg/ml). Figure 1 - Expression and purifi- cation of P1 (A) and P2 (B) by serial sonication steps and gel filtration chromatography. MW: Protein molecular weight mark- er; lane C: bacterial cell suspen- sion before induction; lane 1: cell suspension after induction by IPTG; lanes 2, 4 and 6: superna- tants of the 1st, 2nd and 3rd sonication; lanes 3, 5 and 7: pel- lets of the 1st, 2nd and 3rd soni- cation; lane 8: purified and re- folded polypeptides (P1: 25 µg/ ml; P2: 15 µg/ml). Figure 1 - Expression and purifi- cation of P1 (A) and P2 (B) by serial sonication steps and gel filtration chromatography. MW: Protein molecular weight mark- er; lane C: bacterial cell suspen- sion before induction; lane 1: cell suspension after induction by IPTG; lanes 2, 4 and 6: superna- tants of the 1st, 2nd and 3rd sonication; lanes 3, 5 and 7: pel- lets of the 1st, 2nd and 3rd soni- cation; lane 8: purified and re- folded polypeptides (P1: 25 µg/ ml; P2: 15 µg/ml). B A Inhibition of the antiviral activity of IFN-aB IFN-aB inhibited the CPE of Hep 2/c cells challenged with EMCV in a dose-de- pendent manner. The CPE of the virus was less than 10% with 125 nM IFN-aB (50 IU/ 100 µl) and 15-25% with 2.5 nM (1 IU/100 µl) IFN-aB, showing the extremely high antiviral activity of IFN-aB on Hep 2/c cells challenged with EMCV. Figure 2 - - - - - Inhibition of antiproliferative activity of IFN-aB by polypeptide (A) or by polyclonal antibodies (B). Daudi cells (4 x 105/ml) were incubated for 3 days with 25 nM~250 µM polypeptides or serial dilutions of polyclonal antibodies in the presence of 6.25 nM IFN-aB. Cell growth was determined by an MTT colorimetric assay (595 nm). P<0.05 for inhibition of IFN-aB activity compared to the IFN control (Student t-test). As shown in Figure 3, there were no significant differences in CPE between controls and cells treated with the poly- peptides. Polypeptides P1 and P2 inhib- ited the antiviral activity of 2.5 nM IFN- aB by less than 3%. The polyclonal anti- P1 and anti-P2 antibodies did not block the antiviral activity of IFN-aB, showing the same CPE for IFN-aB on control cells and cells treated with antibodies. The in- creased CPE at 0.1 antibody dilution ob- served at the same dilution of a negative serum may have been due to the interfer- ence of serum with IFN-a activity. Figure 3 - Effect of polypeptides (A) and of their respective antibodies (B) on the antiviral activity of IFN-aB. Hep 2/c cells (3 x 105/ml) were incubated with 0.125~1250 µM polypep- tides or serial dilutions of polyclonal antibodies in the presence of 2.5 nM IFN-aB for 1 day before challenge with encephalomyocarditis virus. The cytopathic effect was determined by staining with amido blue black and absorbance reading at 620 nm. A 97.0 1 2 3 4 5 6 7 8 MW 66.2 31.0 21.0 14.0 6.5 kDa kDa MW C 1 2 3 4 5 6 7 8 97.0 kDa MW C 1 2 3 4 5 6 7 8 66.2 31.0 21.0 14.0 6.5 Braz J Med Biol Res 33(7) 2000 775 Expression of recombinant IFNAR 1 polypeptides Discussion * * ** * * * * * A B Control Cell growth (%) 130 110 90 70 50 Cell growth (%) 130 110 90 70 50 IFN Control (0 M) 25 nM 250 nM 2.5 µM 25 µM 250 µM Polypeptide concentration IFN 0.00001 0.0001 0.001 0.01 0.1 Antibody dilution P1 P2 Anti-P1 antibody Anti-P2 antibody * * * ** * * * * * A B Control Discussion nM for the inhibition assay of the antiprolif- erative activity of this cytokine. Polypep- tides P1 and P2 and their respective poly- clonal antibodies did not inhibit IFN-aB activity at these concentrations (data not shown). However, polypeptides P1 and P2 and their respective antibodies inhibited the antiproliferative activity of 6.25 nM IFN- aB. A significant inhibition of the antiprolif- erative activity of 6.25 nM IFN-aB was ob- served at 250 nM, 2.5, 25 and 250 µM P1 and at 250 µM P2 (Student t-test, P<0.05). This inhibition of IFN-aB activity was dose- dependent (Spearman test: r = 0.9856, P = 0.0028 for P1; r = 0.8857, P = 0.0333 for P2). Anti-P1 antibodies inhibited the antiprolif- erative activity of IFN-aB at dilutions of 10-3 and 10-1 and anti-P2 antibodies at dilutions of 10-3, 10-2 and 10-1 (Student t-test, P<0.05); this inhibition was also dose-dependent (Spearman test, r = 1.0000, P = 0.0028 for anti-P1 and anti-P2 antibodies) (Figure 2). Our main interest in the production of IFN-a inhibitors stems from the finding of the important role of IFN-a in the develop- ment of autoimmune diseases and GVHD. In this study, we have expressed two recombi- nant polypeptide fragments of the extracel- lular domain of IFNAR 1 in E. coli and their polyclonal antibodies in order to evaluate their in vitro inhibition activity on IFN-a. The recombinant polypeptides P1 and P2 were expressed in the E. coli BL21(DE3) pLysS strain, using the pRSET expression plasmid vector, a pET derivative (plasmid for expression by T7 RNA polymerase) Cell growth (%) 130 110 90 70 50 Cell growth (%) 130 110 90 70 50 IFN Control (0 M) 25 nM 250 nM 2.5 µM 25 µM 250 µM Polypeptide concentration IFN 0.00001 0.0001 0.001 0.01 0.1 Antibody dilution P1 P2 Anti-P1 antibody Anti-P2 antibody Figure 2 - - - - - Inhibition of antiproliferative activity of IFN-aB by polypeptide (A) or by polyclonal antibodies (B). Daudi cells (4 x 105/ml) were incubated for 3 days with 25 nM~250 µM polypeptides or serial dilutions of polyclonal antibodies in the presence of 6.25 nM IFN-aB. Cell growth was determined by an MTT colorimetric assay (595 nm). *P<0.05 for inhibition of IFN-aB activity compared to the IFN control (Student t-test). Inhibition of the antiviral activity of IFN-aB Cytopathic effect (%) 25 20 15 10 5 35 30 25 20 15 10 5 0 IFN Control (0 M) 125 nM 1.25 µM 12.5 µM 125 µM 1250 µM Polypeptide concentration IFN 0.00001 0.0001 0.001 0.01 0.1 Antibody dilution P1 P2 Anti-P1 antibody Anti-P2 antibody A B 0 Cytopathic effect (%) Control Cytopathic effect (%) 25 20 15 10 5 35 30 25 20 15 10 5 0 IFN Control (0 M) 125 nM 1.25 µM 12.5 µM 125 µM 1250 µM Polypeptide concentration IFN 0.00001 0.0001 0.001 0.01 0.1 Antibody dilution P1 P2 Anti-P1 antibody Anti-P2 antibody A B 0 Cytopathic effect (%) Control Figure 3 - Effect of polypeptides (A) and of their respective antibodies (B) on the antiviral activity of IFN-aB. Hep 2/c cells (3 x 105/ml) were incubated with 0.125~1250 µM polypep- tides or serial dilutions of polyclonal antibodies in the presence of 2.5 nM IFN-aB for 1 day before challenge with encephalomyocarditis virus. The cytopathic effect was determined by staining with amido blue black and absorbance reading at 620 nm. Braz J Med Biol Res 33(7) 2000 776 S. Yoon et al. which permits high levels of expression of heterologous gene products and easy purifi- cation of recombinant insoluble protein by centrifugation (27). The expression system BL21(DE3)pLysS/pRSET yielded a signifi- cant amount of polypeptides even when pro- tein was lost during the purification process. The purification of the recombinant poly- peptides involved three important steps: 1) isolation by serial sonication to separate the insoluble recombinant polypeptides from soluble bacterial proteins, 2) purification of the recombinant polypeptides by chroma- tography under denaturing conditions, and 3) refolding of the polypeptides by dialysis against decreasing concentrations of dena- turant in order to gradually remove the dena- turant. To avoid aggregation during dialysis, refolding experiments were performed with lower concentrations of the polypeptides (<100 µg/ml). nant polypeptide. In our experiments, each inhibition test of the antiproliferative activ- ity of IFN-aB was carried out by incubation for 3 days at 37oC. During this period, a reversible reaction of IFN-aB with recombi- nant polypeptides and natural IFNAR may occur. The higher affinity of IFN-aB for natural IFNAR on cells probably allowed IFN-aB to display the antiproliferative ac- tivity. Moreover, Raz et al. (31) have sug- gested the participation of a third factor (ac- cessory elements) encoded by a gene located on chromosome 21q22.2 for full IFN-a re- ceptor activity. Inhibition of the antiviral activity of IFN-aB Therefore, the absence of these factors in the assays could result in the low affinity of the recombinant polypeptides. Although the two recombinant polypep- tides showed low inhibitory activity on IFN- aB in our in vitro experiments, they open the possibility of practical antagonist produc- tion in the cytokine research area. In patho- logical processes associated with cytokine disorders, such as GVHD and autoimmune diseases, the production of a cytokine or antibody has been investigated for therapeu- tic purpose. P1 and P2 inhibited 6.25 nM IFN-aB in a dose-dependent manner in the antiprolifera- tive assays. P1 showed stronger inhibition than P2 at 250 nM, 2.5 µM, and 25 µM. However, the polypeptides did not inhibit the antiproliferative activity of higher con- centrations of IFN-aB (12.5 and 25 nM IFN- aB) (data not shown) although they were used at 10,000-fold molar excess over IFN- aB. The polyclonal anti-P1 and anti-P2 anti- bodies inhibited 6.25 nM IFN-aB in a dose- dependent manner in the antiproliferative assays. Anti-P1 and anti-P2 antibodies showed similar inhibition of IFN-aB activ- ity (Figure 2), but did not block IFN-aB activity at higher concentrations. These anti- bodies were able to bind IFNAR on the surface of Daudi cells competing with IFN- aB for receptor binding and proved to be useful in studies of IFNAR binding for the isolation of natural IFNAR and accessory components related to natural IFNAR on the cell surface. It has been reported that the two subunits of IFN-a receptor, IFNAR 1 and IFNAR 2, participate in the binding of IFN-a. The IFN- a binding affinity for the receptor was in- creased by the co-expression of IFNAR 1 and IFNAR 2, which itself binds weakly to IFN-a (28-30). Therefore, the need of a high concentration of recombinant polypeptides for inhibition of IFN-aB activity and the absence of response to high concentrations of IFN-aB in our experiments could be due to the lack of synergism of IFNAR 2 in the binding process. In other words, the natural IFNAR 1 on the surface of Daudi cells has more affinity for IFN-a because of the coop- erative action of IFNAR 2 than the recombi- In the inhibition assay of IFN-aB activity by an antiviral test, we used Hep 2/c cells and EMCV. Acknowledgments The authors gratefully acknowledge the support of IFN-aB in the person of Dr. David Gangemi. We also thank Dr. Kath C. Zoon and Dr. Consuelo Dias for providing Daudi cells and Hep 2/c cells. Inhibition of the antiviral activity of IFN-aB This combination showed high sensitivity to the antiviral activity of IFN- aB, being able to inhibit more than 80% of the viral activity at less than 6.25 nM (1 IU/ 100 µl) IFN-aB. IFN-aB (125 nM) inhibited Braz J Med Biol Res 33(7) 2000 Braz J Med Biol Res 33(7) 2000 777 Expression of recombinant IFNAR 1 polypeptides Daudi cell proliferation by 80% (50 IU/100 µl) and 80% of the viral CPE on Hep 2/c cells at only 6.25 nM, showing its powerful anti- viral activity. For this reason, the standard procedure for the identification of neutraliz- ing anti-IFN antibodies is the antiviral neu- tralization assay according to the recom- mendations of the WHO Expert Committee on Biological Standardization (32). Never- theless, it has been shown that the antiprolif- erative assay is easy to perform, reproduc- ible, and more sensitive than the antiviral neutralization assay for the detection of neu- tralizing IFN-a antibodies (33). In our ex- periments, the antiviral assay did not show good sensitivity for the inhibition assay of two recombinant polypeptides and their re- spective polyclonal antibodies. The block- ing activity of the recombinant polypeptides and polyclonal antibodies in the antiviral assay was lower when compared with the antiproliferative assay. This difference in sensitivity between the antiproliferative test and the antiviral test may be due to the different cell lines used presenting different IFNAR and consequently different IFNAR affinity for antibodies or IFN-aB. The possibility of new therapeutic ap- proaches to autoimmune diseases and GVHD is very important. We have studied the pro- duction of IFN-a inhibitors. However, the two recombinant polypeptides showed a low ability to act as IFN-a inhibitors. In further studies, we will investigate the role of IFNAR 2 and their antibodies. The anti-IFNAR 1 and anti-IFNAR 2 antibodies may be used together to totally inhibit the activity of all IFN-a subclasses. References 63: 1086-1090. 1. Branca A (1988). Interferon receptors. In Vitro Cellular and Developmental Biology, 24: 155-165. 7. Flores L, Mariano TM & Pestka S (1991). Human interferon omega (w) binds to the a/ß receptor. Journal of Biological Chem- istry, 266: 19875-19877. 12. 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https://openalex.org/W2157424552	https://figshare.com/articles/journal_contribution/Representing_Embedded_System_Sequence_Diagrams_As_A_Formal_Language/6625583/1/files/12123035.pdf	English	null	Representing Embedded System Sequence Diagrams as a Formal Language	Lecture notes in computer science	2,001	cc-by	5,245	Representing Embedded System Sequence Diagrams As A Formal Language Elizabeth Latronico and Philip Koopman , , Carnegie Mellon University Electrical and Computer Engineering Department D-202 Hamerschlag Hall, 5000 Forbes Avenue Pittsburgh, PA 15213 beth@cmu.edu, koopman@cmu.edu Abstract. Sequence Diagrams (SDs) have proven useful for describing transaction-oriented systems, and can form a basis for creating statecharts. However, distributed embedded systems require special support for branching, state information, and composing SDs. Actors must traverse many SDs when using a complex embedded system. Current techniques are insufficiently rich to represent the behavior of real systems, such as elevators, without augmentation, and cannot identify the correct SD to execute next from any given state of the system. We propose the application of formal language theory to ensure that SDs (which can be thought of as specifying a grammar) have sufficient information to create statecharts (which implement the automata that recognize that grammar). A promising approach for SD to statechart synthesis then involves `compiling` SDs represented in a LL(1) grammar into statecharts, and permits us to bring the wealth of formal language and compiler theory to bear on this problem area. UML 2001, Toronto Ontario, 3-5 Oct. 2001. UML 2001, Toronto Ontario, 3-5 Oct. 2001. 1 Introduction We examine systems with all three characteristics. We examine systems with all three characteristics. In this paper, we explore how to use UML sequence diagrams to support the needs of embedded systems designers. First, we review methods for composing sequence diagrams that support flexible embedded systems modeling. Then, we show how determining required information content can be represented as a grammar parsing problem to guarantee correct, cohesive diagrams. A generic approach is described, with supporting embedded systems examples incorporating data, state, and timing information. Finally, the more commonly discussed transaction processing model is revisited to illustrate system differences. Section 2 introduces terminology and examines related work. Section 3 presents a methodology for determining sufficient information in sequence diagrams, and illustrates this methodology with four examples (state information, message preconditions, timing information, and the standard transaction processing system.) Finally, we present our conclusions in section 4. 1 Introduction One of a designer’s toughest challenges is attaining the appropriate level of abstraction. Include too little information, and a design is under-specified, often resulting in an incorrect or incomplete implementation. Include too much information, and the design is overly constrained or exceeds time-to-market allowances. Distributed, embedded systems present particularly onerous design challenges, combining complex behavior with a need for quick development cycles. The Unified Modeling Language (UML) supplies an appraoch to express requirements and design decisions at various stages in the product development life cycle. The ideal level of abstraction provides the minimum sufficient amount of information required to create a set of correct, cohesive diagrams. UML offers two main diagrams for modeling system behavior: sequence diagrams and statecharts. While related, these diagrams often originate separately and serve diverse purposes. Sequence diagrams are easier for people to generate and discuss, while statecharts provide a more powerful and thorough description of the behavior of a system. We present an algorithm to ensure that sequence diagrams contain sufficient information to be translated into statecharts. Specifically, this algorithm determines whether or not a set of sequence diagrams produces deterministic statecharts. This allows the designer to start with a skeletal structure and add information only when necessary. 1 UML can be used to model a wide range of systems. To ensure that our results are applicable to actual embedded systems, the problem space needs to be carefully defined. Transaction processing systems are the traditional area of focus. Embedded systems differ from the transaction processing paradigm in the following major ways: · Multiple initial conditions · Multiple initial conditions p Distributed, embedded systems typically run continuously and handle many user requests concurrently. Therefore, the system may not necessarily be in the same initial state for each user. Additionally, users may have disjoint objectives and responsibilities, so a second user may finish what a first user started. · Same user action evokes different system response In transaction processing systems, there tends to be a one-to-one mapping from a user request to a system response. Embedded systems often have a limited user interface, so interface component functionality may depend on context. · Same user action evokes different system response · Timing sensitivity Embedded system functionality may depend on temporal properties such as duration, latency, and absolute time. · Timing sensitivity Embedded system functionality may depend on temporal properties such as duration, latency, and absolute time. 2.1.1 Sequence Diagrams and Message Sequence Charts A scenario describes a way to use a system to accomplish some function [5]. Scenarios can be expressed in many forms, both textual and graphical, informal and formal. UML supports two main ways of expressing scenarios: collaboration diagrams and sequence 2 diagrams. Sequence diagrams emphasize temporal ordering of events, whereas collaboration diagrams focus on the structure of interactions between objects [7]. Each may be readily translated into the other [7]. We concentrate on sequence diagrams because they elucidate temporal properties. A sequence diagram “presents an Interaction, which is a set of Messages between ClassifierRoles within a Collaboration to effect a desired operation or result” [14]. A sequence diagram models temporal and object relationships using two dimensions, vertical for time and horizontal for objects [14]. The notation of sequence diagrams is based on, and highly similar to, the Message Sequence Chart standard for the telecommunications industry [6]. 2.1.3 Finite State Machines and Statecharts Finite state automata describe the possible states of a system and transitions between these states. Unfortunately, properties of complex systems such as concurrent execution of components lead to extremely large state machines that challenge human comprehension. Statecharts were proposed by Harel [3] to control state explosion problems with finite state machines by introducing the concepts of hierarchy and orthogonal execution. The UML standard 1.3 defines a statechart as “a graph that represents a state machine”, which is used to “represent the behavior of entities capable of dynamic behavior by specifying its response to the receipt of event instances” [14]. 2.1.2 Composition of Scenarios A crucial challenge in describing distributed embedded systems is the composition of scenarios. In order to be adequately expressive, sequence diagrams must reflect the structures of the programs they represent. In this paper, we survey approaches to modeling execution structures and transfer of control, and select a method that lends itself to embedded systems. Our first objective is to refine a model that utilizes sequential, conditional, iterative, and concurrent execution. As many ideas exist, our task is to determine which are appropriate for embedded systems. [5] discusses a process for scenario analysis that includes conditional branching. [12] presents three ways of composing scenarios: sequential, alternative, and parallel. [2] includes iteration as well. [8] and [13] present a tool that handles “algorithmic scenario diagrams” - sequence diagrams with sequential, iterative, conditional and concurrent behavior. We use elements of each, for a combined model that allows sequential, conditional, iterative, and concurrent behavior. Our second objective is to model transfer of control through sequence diagram composition. The main decision to make is where to annotate control information. One approach is to include composition information in individual diagrams. [4] examines sequence diagram generation from use cases, and discusses the probe concept, a smaller piece of a scenario that can be inserted at a specified point in a larger scenario. [8] presents a similar approach using sub-scenarios. A second approach is to use a separate hierarchical diagram, instead of embedding control information in the constituent diagrams. The Message Sequence Chart (MSC) standard specifies a separate diagram to organize sub-diagrams [6]. [9] explores the usage of base MSCs and high-level MSCs. The high-level MSC graph describes how to compose base MSC graphs to obtain sequential, conditional, iterative, and concurrent execution. [10] presents an additional example of a high-level MSC graph, and applies this method to UML sequence diagrams to assess timing inconsistency. We use the hierarchical diagram approach. 3 2.1.4 Statechart Synthesis The second challenge in describing distributed, embedded systems is ensuring there is sufficient information for correct, cohesive diagrams. Sequence diagrams are often constructed first in the design life cycle; therefore, we addresses synthesis of statecharts from sequence diagrams. Existing work has two shortcomings. First, sufficiency of information for generating statecharts is not checked. Additional information is either absent or applied globally. Our goal is to provide an approach by which a designer can include a minimum amount of information, thereby reducing design time and guaranteeing a correct set of statecharts. We present a methodology to verify sufficiency, by applying well-established parsing theory. Second, systems with all three embedded system qualities of multiple initial conditions, mapping identical user actions to different system responses, and timing dependencies have not been scrutinized. Systems that lack one of these three qualities generally do not require additional information to produce correct statecharts; therefore, the sufficiency question seems to have not arisen. We present three embedded systems and show, by applying grammar parsing techniques, that these embedded systems do require additional information to produce correct statecharts. Additionally, we examine a transaction processing system, to illustrate that additional information is not required. Prior work contains a number of suggestions as to what information sequence diagrams should include to enable statechart synthesis. Information is used for various purposes, but deterministic translation to statecharts is not been emphasized. Further, added information is applied globally, which is effort intensive and potentially unnecessary for statechart synthesis. [5] gives a regular grammar for scenarios in order to construct a deterministic finite state machine. This grammar is similar to ours, but information sufficiency is assumed, not proven. Other work has proposed additional information, comprising three categories: state, preconditions and assertions, and timing information. [1] advocates the addition of state symbols to sequence diagrams to represent object state. [12] examines scenarios with pre-conditions and post-conditions 4 to define possible execution ordering. [15] discusses implications of repeated user actions as a motivation for incorporating pre-conditions and post-conditions. [8] annotates sequence diagrams with assertions, conditions guaranteed to be true at a specified execution point. Timing intervals between messages are included in [10]. Our examples examine these three categories, exposing situations where additional information is needed for statechart synthesis, and situations where it is not. 2.1.4 Statechart Synthesis A number of different systems have been explored and documented; however, these systems lack the combination of multiple initial conditions, same user action evoking different system responses, and timing criteria. Systems without one of these characteristics often fail to manifest sufficiency issues. A library checkout system is explored by [2] and [7]. In [2], scenarios have differing initial conditions, and system response depends on data attributes. However, statecharts are constructed directly from an informal textual description, not sequence diagrams. [7] synthesizes statecharts from UML collaboration diagrams, but these diagrams have identical initial conditions, one-to-one response mapping, and no timing criteria. The Automated Teller Machine (ATM) system is a common example, discussed in [12], [15], and [8]. [12] permits timeouts and global timed transitions, but all scenarios start with a single initial condition, and user actions are mapped one-to-one with system responses (aside from time-influenced transitions). In [15], the scenarios can have a one-to-many action-response mapping, but have identical initial conditions and no timing restrictions. [8] approaches the problem in an iterative fashion, generating partial statecharts from sequence diagrams and vice versa. Different subscenarios may handle the same user request; however, there is a single initial condition and timing information is not discussed. The methodology in [8] is extended in [13] for a File Dialog application with the same properties. We examine three embedded systems to provoke sufficiency questions, then apply our methodology to a traditional transaction processing system to show that these systems do not require additional information for sufficiency. 2.2 Embedded Systems View The hierarchical graph approach used by the Message Sequence Chart community [6, 9, 10] explicitly represents state and composition information not shown in standard UML sequence diagrams. Figure 1 shows a set of sequence diagrams for a television power switch. TV1 and TV2are regular sequence diagrams. The system has two objects - the user and the TV. The user can send one message, power. The TV can send two messages, turn_on and turn_off. TVmainexpresses the relationships between TV1 and TV2. The triangle indicates a possible initial condition - the system may start out in TV1 and TV2 . Arrows indicate legal compositions. TV1 and TV2 must alternate - the sequence TV1 TV1 is not allowed. Without TVmain , state and composition information would be lost. 5 5 TV2 User TV power turn on User TV power turn off TV1 TV1 TV2 TVmain Figure 1 : Sequence diagrams for a television power switch Figure 1 : Sequence diagrams for a television power switch Embedded system statechart synthesis typically requires more information than solely the messages an object receives. Three cases will be examined where sequence diagrams can be extended using state information, message pre-conditions, and timing information to generate a deterministic grammar. Finally, the widely used ATM example will be reviewed to show that the ATM sequence diagrams generate a deterministic grammar without additional information regarding state, pre-conditions, or timing. We note that the next revision of UML is expected to support additional information through state symbols, OCL constraints and timing marks. message response a ResponseA \| ResponseB \| ResponseC … (2) where , and are distinct sequences of messages. A grammar of the form where , and are distinct sequences of messages. A grammar of the form ResponseA \| ResponseB (3) (3) (3) message response ResponseA \| ResponseB does not produce a deterministic state machine. Upon receipt of , the object does not know whether to execute ResponseA or ResponseB. The sequence diagram set for this grammar is shown in Figure 2. The system may start in either Seq1 and Seq2, and execute any combination of Seq1 and Seq2. Seq2 User Object Response A User Object Response B Seq1 Seq1 Seq2 Seqmain Figure 2 : Sequence diagrams for a generic non-deterministic grammar Figure 2 : Sequence diagrams for a generic non-deterministic grammar Left factoring transforms the grammar in (3) to Left factoring transforms the grammar in (3) to message response A’ A’ ResponseA \| ResponseB (4) (4) This is equivalent to the sequence diagram set given in Figure 3. The sequence diagram Seqfactor is executed, followed by either Seq1 and Seq2. However, this only changes the composition of the diagrams. The problem of whether to execute ResponseA or ResponseB after receipt of remains. The backtracking method picks a random response to be executed, and backtracks if the incorrect response was selected. Say the grammar is message response a ResponseA \| a ResponseB (5) (5) 7 3.1 Deterministic Grammar The main challenge in statechart synthesis is generating correct statecharts from a set of sequence diagrams with minimum sufficient information. The statecharts do not necessarily need to be complete, but they should give an unambiguous representation of the system. Development time constraints often preclude annotating each message of every sequence diagram. Rather than attempt an exhaustive annotation, an alternate goal is to include the minimum sufficient amount of information. Correct statechart synthesis from sequence diagrams with minimal annotation can be posed as a context-free grammar parsing problem. A similar approach was used in [5] for text-based scenarios. To identify information gaps, we locate sequence diagram messages that translate into non-deterministic transitions in statecharts, as non-deterministic transitions often indicate information deficiencies. Standard methods for removing non-determinism, such as left factoring [11], and for implementing non-determinism, such as backtracking [11], cannot always be applied to embedded systems sequence charts because messages may have global side effects on the external environment. Therefore, the only guaranteed correct approach is to ensure that sequence diagrams form an LL(1) grammar without left factoring or backtracking. The context-free grammar for a sequence diagram may be defined as a set of message-response pairs. Given a message or set of messages, an object must produce a unique response or set of responses. A SD can be defined as a series of message-response events: 6 6 SD message response SD \| (1) (1) SD message response SD \| SD message response SD \| where indicates the absence of a message or response. The goal is to construct a SD with a context-free grammar of the form (2) 7 7 Upon receipt of , it is unclear whether ResponseA or ResponseB is the correct behavior. One must be selected, so assume ResponseA is selected. The next message is . It is clear that ResponseA was incorrect, so the system backtracks and ResponseB is chosen instead. However, this approach is inapplicable to real-time embedded systems, as many responses cannot be undone. For example, one cannot undo detonating a bomb. A greater difficulty emerges in scenarios where it is impossible to select a correct response based on messages alone. For instance, if a is the only message the user can generate, no amplifying information can be acquired; thus, the correct choice will never be known without querying existing system state. Seq2 User Object Response A User Object Response B Seq1 Seq1 Seq2 Seqmain User Object Seqfactor Seqfactor Figure 3 : Left-factored sequence diagrams for a generic non-deterministic grammar Figure 3 : Left-factored sequence diagrams for a generic non-deterministic grammar SD state message response SD \| SD state message response SD \| (8) (8) g p \| state message response off power turn_on \| on power turn_off (8) state message response off power turn_on \| on power turn_off message response turn_on \| turn_off (9) (9) and is therefore deterministic. 3.2 State Information Adding state information, as suggested by [1], is often sufficient to allow the generation of a deterministic set of sequence charts. Figure 1 represents the sequence diagram set for a television with a power button. The television either turns on or off in response to the power message. There are two possible initial conditions – the television may be on or off when the user walks in the room. The grammar for the television is SD message response SD \| message response power turn_on \| power turn_off (6) SD message response SD \| message response power turn_on \| power turn_off (6) (6) This is of the form (7) message response and therefore non-deterministic, per the discussion in section 3.1 and therefore non-deterministic, per the discussion in section 3.1 Adding state information can solve this non-determinism. The problem is that the state of the television is not represented in either sequence diagram, so the response to the power message is ambiguous. The television can be in two states, on or off. Appending this information to the sequence diagrams yields Figure 4. 8 User TV power turn on off on User TV power turn off on off TV1 TV2 TV1 TV2 TVmain Figure 4 : Sequence diagrams for a television power switch, including state information User TV power turn on off on TV1 Figure 4 : Sequence diagrams for a television power switch, including state information The new state information can be incorporated into the grammar. The template for constructing the grammar is now 3.3 Preconditions/assertions State information alone does not guarantee a deterministic statechart, however. Sometimes execution depends on the value of a stored piece of data that is not directly modeled as a state or transition. Preconditions and assertions have been used to represent this additional information (e.g, [8, 13, 15]). Preconditions and assertions are statements, usually in a formal language, that specify properties of variables. As an example, consider an elevator. The elevator contains a set of numbered car buttons, one per floor, that passengers use to select a destination floor. While inside the car, if a passenger pushes the button for floor the elevator is already on, the doors will open. This is required to allow passengers inside an idle elevator to disembark at the current floor. If the passenger pushes the button for a floor other than the current floor, the doors will close. This is a common, although not universal, set of elevator behaviors. The two basic sequence diagrams for car button behavior are shown in Figure 5. The grammar for this example is SD message response SD \| message response push(f) close \| push(f) open (10) SD message response SD \| message response push(f) close \| push(f) open (10) 9 This is of the form (11) message response close \| open (11) message response User Car Button Door push(f) push(f) open User Car Button Door push(f) push(f) close Elevatpr1 Elevator2 Elevator1 Elevator2 Elevatormain Figure 5 : Sequence diagrams for an elevator Figure 5 : Sequence diagrams for an elevator Preconditions for the messages can be added to make this example deterministic, as shown in Figure 6. The crucial piece of missing information is that the response of the elevator depends on the value of (f) in push(f) compared to current state. The value of (f) in push(f) can be either the same as the current floor or other than the current floor. 3.3 Preconditions/assertions User Car Button Door push(f) push(f) open User Car Button Door push(f) push(f) close Elevatpr1 Elevator2 Elevator1 Elevator2 Elevatormain push(f : Floor) precondition : f == CurrentFloor push(f : Floor) precondition : f != CurrentFloor Figure 6 : Sequence diagrams for an elevator, including preconditions User Car Button Door push(f) push(f) close Elevator2 Elevator1 Elevator2 Elevatormain push(f : Floor) precondition : f != CurrentFloor User Car Button Door push(f) push(f) open Elevatpr1 push(f : Floor) precondition : f == CurrentFloor Figure 6 : Sequence diagrams for an elevator, including preconditions The template for constructing the grammar with preconditions is SD precondition message response SD \| precondition message response (f == currentFloor) push(f) open \| (f != currentFloor) push(f) close (12) This is of the form message response close \| open (13) and is deterministic. (12) (13) and is deterministic. 10 message response and is deterministic. 3.4 Timing Information Finally, the response of an embedded system may depend on the duration of the stimulus. Consider a car radio with a set of buttons to allow users to save and switch to preferred stations. If the button is held for a short time, the radio will change stations to the button’s preset station when the button is released. If the button is held longer, the radio will save the current station as the value of the button. The basic sequence charts for this system are given in Figure 7. User Button Radio hold hold change station User Button Radio hold hold station set Radio1 Radio2 Radio1 Radio2 Radiomain release release Figure 7 : Sequence diagrams for a radio Figure 7 : Sequence diagrams for a radio The grammar for the car radio is The grammar for the car radio is SD message response SD \| message response hold release change_station \| hold station_set (14) SD message response SD \| message response hold release change_station \| hold station_set (14) This is of the form message response ® release change_station \| station_set (15) (14) release change_station \| station_set (15) (15) and therefore non-deterministic. At first glance, it may seem deterministic because of the release message. However, assume the system receives the hold message. Does it do nothing (waiting for release), or set the station? Timing information is needed to express which transition should be taken. Figure 8 illustrates the car radio sequence charts with timing information included. User Button Radio hold hold change station User Button Radio hold hold station set Radio1 Radio2 Radio1 Radio2 Radiomain release release [release - hold < 2 seconds] [hold == true for 2 seconds] Figure 8 : Sequence diagrams for a radio, including timing information User Button Radio hold hold change station Radio1 release release [release - hold < 2 seconds] Fi 8 S di Figure 8 : Sequence diagrams for a radio, including timing information The template for constructing the grammar with timing information is 11 SD message duration response SD \| message duration response hold (holdDuration < 2 seconds) release change_station \| hold (holdDuration reaches 2 seconds) station_set (16) SD message duration response SD \| message duration response hold (holdDuration < 2 seconds) release change_station \| hold (holdDuration reaches 2 seconds) station_set SD message duration response SD \| message duration response hold (holdDuration < 2 seconds) release change_station \| hold (holdDuration reaches 2 seconds) station_set (16) (16) This is of the form release change_station \| station_set (17) (17) 3.5 ATM example To demonstrate the distinction between embedded systems and transaction processing systems, the classic Automated Teller Machine (ATM) example will be analyzed. [15] synthesizes statecharts from a set of four scenarios for the ATM system. Figure 9 is the sequence diagram for the first scenario. There are four objects exchanging messages : the user, the ATM, the consortium, and the bank. In this example, statecharts are generated for the ATM object only. The scenarios share the same initial condition. We constructed grammar descriptions for the set of diagrams, provided in formulas 18-21. We will apply grammar parsing to identify any non-determinism present. User ATM Consortium Insert card Request password Bank Display main screen Enter password Verify account Verify card with bank Bad bank account Bad account Bad account message Print receipt Eject card Request take card Take card Display main screen Figure 9 : Interaction with an ATM (from [15]) Consortium Verify account Verify card with bank Bad bank account Bad account Figure 9 : Interaction with an ATM (from [15]) 12 12 SD message response SD \| message response Display_main_screen \| Insert_card Request_password \| Enter_password Verify_account \| Bad_account Bad_account_message Eject_card Request_take_card \| Take_card Display_main_screen (18) (18) (19) SD message response SD \| message response Display_main_screen \| Insert_card Request_password \| Cancel Canceled_message Eject_card Request_take_card \| Take_card Display_main_screen (20) (20) SD message response SD \| message response Display_main_screen \| Insert_card Request_password \| Enter_password Verify_account \| Cancel Canceled_message Eject_card Request_take_card \| Take_card Display_main_screen (21) SD message response SD \| message response Display_main_screen \| Insert_card Request_password \| Enter_password Verify_account \| Cancel Canceled_message Eject_card Request_take_card \| Take_card Display_main_screen (21) SD message response SD \| message response Display_main_screen \| Insert_card Request_password \| Enter_password Verify_account \| Cancel Canceled_message Eject_card Request_take_card \| Take_card Display_main_screen (21) (21) Table 1 lists all the message-response pairs observed in the grammar for the sequence diagram set. Note that each incoming message produces a unique set of system responses, with the exception of Cancel. In the first SD grammar (19), Cancel evokes Canceled_message and Eject_card. In the third and fourth SD grammars (20) and (21), cancel evokes Canceled_message, Eject_card, and Request_take_card. Upon reflection, this is probably an omission in the first sequence diagram, not a design decision. 3.5 ATM example The Display_main_screen message occurs before the receipt of any user messages, but does not cause non-determinism because the ATM has a single initial condition. If multiple initial conditions existed, this would pose a problem. [15] discusses a permutation of SD1, where Insert_card is repeated: (22) message response Insert_card \| Insert_card Request_Password (22) 13 This is non-deterministic and would mandate additional information for constructing statecharts (which the authors provide.) Table 1 : Message-response pairs for the ATM system Message ATM Response Used in SD# Display main screen All Insert card Request password All Bad account Bad account message Print receipt Eject card Request take card SD1 Bad password Canceled message Eject card SD2 Cancel Canceled message Eject card SD2 Cancel Canceled message Eject card Request take card SD3, SD4 Take card Display main screen All 4 Conclusions We presented a methodology that guarantees sufficient sequence diagram information to generate correct statecharts. We converted sequence diagrams to a context-free grammar, and applied parsing theory to locate non-deterministic behavior. When state, message preconditions, and timing information are included in the grammar, being LL(1) seems to be sufficient to guarantee determinism for the embedded systems we discussed. We showed how this approach illustrated what additional information was required to attain deterministic behavior, and provided examples incorporating state information, message preconditions, and timing information. Finally, we discussed a transaction processing example to show that transaction processing systems are inherently deterministic. We have also examined diagram composition and information content to assess adequacy for embedded systems. We advocate hierarchical diagrams [6, 9, 10] as the preferred format for sequence diagram composition for designing embedded systems. These diagrams work well for expressing sequential, conditional, iterative, and concurrent execution of sequence diagrams that are common in embedded systems. Further, they support multiple initial conditions, one-to-many action-response mapping, and timing dependencies. 14 5 Acknowledgements This research is supported by the General Motors Satellite Research Lab at Carnegie Mellon University and the United States Department of Defense (NDSEG/ONR). This research is supported by the General Motors Satellite Research Lab at Carnegie Mellon University and the United States Department of Defense (NDSEG/ONR). References [1] Douglass, B. Doing Hard Time. Addison-Wesley, 1999. [2] Glinz, M. An Integrated Formal Model of Scenarios Based on Statecharts. In Proceedings of the 5th European Software Engineering Conference (ESEC 95), Sitges, Spain, 1995, pp. 254-271. [3] Harel, D. Statecharts: A Visual Formalism for Complex Systems. Science of Computer Programming, vol.8, no.3, 1987, pp. 231-274. [4] Hitz, M., and G. Kappel. Developing with UML - Some Pitfalls and Workarounds. 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Synthesizing Software Architecture Descriptions from Message Sequence Chart Specifications. In Proceedings of the 13th IEEE International Conference on Automated Software Engineering, Honolulu, Hawaii, 1998, pp. 192-195. [10] Li, X. and J. Lilius. Checking Compositions of UML Sequence Diagrams for Timing Inconsistency. In Proceedings of the Seventh Asia-Pacific Software Engineering Conference (APSEC 2000), Singapore, 2000, pp. 154-161. [11] Louden, K. Compiler Construction : Principles and Practice. PWS Publishing Company, 1997. [12] Some, S., R. Dssouli, and J. Vaucher. From Scenarios to Timed Automata: Building Specifications from User Requirements. In Proceedings of the 1995 Asia-Pacific Software Engineering Conference, Australia, 1995, pp. 48-57. [13] Systa, T. Incremental Construction of Dynamic Models for Object-Oriented Software Systems. Journal of Object-Oriented Programming, vol.13, no.5, 2000, pp. 18-27. [14] Unified Modeling Language Specification, Version 1.3, 1999. 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https://openalex.org/W3189544665	https://zenodo.org/records/7104099/files/35%2025866.pdf	English	null	The effects of the cross-entropy stopping criterion and quadrature amplitude modulation on iterative turbo decoding performance	Indonesian journal of electrical engineering and computer science	2,021	cc-by	5,013	Indonesian Journal of Electrical Engineering and Computer Science Vol. 23, No. 3, September 2021, pp. 1557~1564 ISSN: 2502-4752, DOI: 10.11591/ijeecs.v23.i3.pp1557-1564 Indonesian Journal of Electrical Engineering and Computer Science Vol. 23, No. 3, September 2021, pp. 1557~1564 ISSN: 2502-4752, DOI: 10.11591/ijeecs.v23.i3.pp1557-1564  1557  1557 Keywords: Cross-entropy Iterative decoding Quadrature amplitude modulation Stopping criteria Turbo codes This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author: Roslina Mohamad Wireless High Speed Network Research Group (WHiSNet) School of Electrical Engineering, College of Engineering Universiti Teknologi MARA 40450 Shah Alam, Selangor, Malaysia Email: roslina780@uitm.edu.my The effects of the cross-entropy stopping criterion and quadrature amplitude modulation on iterative turbo decoding performance Roslina Mohamad1, Mohamad Yusuf Mat Nasir2, Nuzli Mohamad Anas3 1,2Wireless High Speed Network Research Group (WHiSNet), School of Electrical Engineering, College of Engineering, Universiti Teknologi MARA, Selangor, Malaysia 1,2College of Engineering, Universiti Teknologi MARA, Selangor, Malaysia 3Wireless Innovation, MIMOS Berhad, Wilayah Persekutuan, Malaysia Article Info Article history: Received Jun 1, 2021 Revised Aug 4, 2021 Accepted Aug 10, 2021 This is an open access article under the CC BY-SA license. ABSTRACT One of the most often-used stopping criteria is the cross-entropy stopping criterion (CESC). The CESC can stop turbo decoder iterations early by calculating mutual information improvements while maintaining bit error rate (BER) performance. Most research on iterative turbo decoding stopping criteria has utilised low-modulation methods, such as binary phase-shift keying. However, a high-speed network requires high modulation to transfer data at high speeds. Hence, a high modulation technique needs to be integrated into the CESC to match its speed. Therefore, the present paper investigated and analysed the effects of the CESC and quadrature amplitude modulation (QAM) on iterative turbo decoding. Three thresholds were simulated and tested under four situations: different code rates, different QAM formats, different code generators, and different frame sizes. The results revealed that in most situations, the use of CESC is suitable only when the signal-to-noise ratio (SNR) is high. This is because the CESC significantly reduces the average iteration number (AIN) while maintaining the BER. The CESC can terminate early at a high SNR and save more than 40% AIN compared with the fixed stopping criterion. Meanwhile, at a low SNR, the CESC fails to terminate early, which results in maximum AIN. 1. INTRODUCTION Error-correcting coding, also known as channel coding, is critical to achieving optimum efficiency in information transmission by lowering the likelihood of errors in digital communications systems [1], [2]. A channel encoder systematically adds redundant data and transforms bit sequences of information into encoded bit sequences. A channel decoder can correct a received signal’s redundant bits that have been distorted by the receiver’s noisy channel [3], [4]. This channel coding technique is called forward error correction (FEC). Turbo codes are a leading candidate for FEC codes when superior output is required near the Shannon capacity limit [5], [6]. Turbo codes have been used widely in various areas, including digital video broadcasting, deep-space and satellite communications, to improve the chance of obtaining error-free data Journal homepage: http://ijeecs.iaescore.com  1558  ISSN: 2502-4752 [7], [8]. The maximum a posteriori (MAP) probability algorithm and iterative turbo decoding are two important components in the success of turbo codes [9], [10]. However, using a MAP algorithm to accomplish iterative turbo decoding necessitates additional computations, significant memory costs, and decoding system delay [11], [12]. g y y Researchers solve this problem by developing stopping criteria and increasing turbo codes’ performance [13]-[16]. Specifically, stopping criteria help reduce the iteration number in iterative turbo decoding while preserving turbo code efficiency [17], [18]. Most stopping criteria have been developed based on the frame level [7], [9], [14], and bit/window level [11], [12], [16]. Soft-decision-based stopping criteria have become a popular choice for output decisions at the frame level [10], [19], [20]. The average number of bits required to identify an event selected from the set is determined by the cross-entropy between two probability distributions A and B, over the same underlying set of events, according to information theory. This is especially true when the set’s coding method is optimised for an estimated probability distribution A rather than the genuine distribution B. Cross-entropy is used in many applications, such as for determining stopping criteria in iterative decoding scenarios [13]-[15], defining loss functions in machine learning [21], [22], and optimising classification models [23]. Fowdur et al. [14], [24] proposed the cross-entropy stopping criterion (CESC) to calculate improvements in mutual information to cease turbo decoder iterations. Most of the research on iterative turbo decoding stopping criteria have utilised the binary phase-shift keying (BPSK) modulation technique [6], [7], [11], [15], which is the simplest form of phase-shift keying. 1. INTRODUCTION High-speed networks such as long-term evolution (LTE) networks and those based on fifth-generation (5G) technology require high modulation (For example, eight phase-shift keyings (8PSK), 16 phase-shift keyings (16PSK), 16-QAM, 32-QAM, and 64-QAM) to transfer data at a sufficient speed [14], [16], [25]. A higher modulation needs to be integrated into stopping criteria to meet the speed requirements of current LTE applications [14], [16]. This paper is intended to address the lack of information about the performance of CESC and high modulation via iterative turbo decoding [26]. Hence, this paper describes the effects of CESC and QAM on iterative turbo decoding performance. Three predefined thresholds of CESC were tested under four situations: different code rates, different QAM formats, different code generators, and different frame sizes. The CESC’s performance was assessed using the bit error rate (BER) and average iteration number (AIN). The content of this paper is organised in the following manner. The CESC and its algorithm are discussed in Section 2. In Section 3, the simulation parameters and the methodology used in the research are described. In Section 4, the effects and performances of the CESC with QAM on iterative turbo decoding are analysed and compared. Finally, conclusions are drawn in Section 5. 2. CROSS-ENTROPY STOPPING CRITERION Figure 1 illustrates the process for the early termination of an iterative turbo decoder using the CESC. The CESC is between decoder two and the deinterleaver. The outputs from the first turbo encoder are decoded by decoder 1, which are the systematic input bit of the decoder, and the initial parity input bit of the decoder ,1 , s p b b a a . The following is an explanation of the iterative turbo decoder with a CESC: At the i th iteration and the b th discrete-time index, a posteriori log-likelihood ratio (LLR), , ( ) i l n b L x , and extrinsic information LLR , ( ) i e n b L x are delivered by the n th component decoder. Here, 1,2 n  is given by (1) and (2). 1 ,1 ,2 ,1 ( ) ( ) ( ) i i i l b e b b e b L x L x a L x     (1) ,2 ,1 ,2 ( ) ( ) ( ) i i i l b e b b e b L x L x a L x    (2) 1 ,1 ,2 ,1 ( ) ( ) ( ) i i i l b e b b e b L x L x a L x     ,2 ,1 ,2 ( ) ( ) ( ) i i i l b e b b e b L x L x a L x    (1) (2) (2) The functions ,2( ) i e b L x and ,2( ) i l b L x are input into the CESC. In CESC, the ith iteration is compared to the maximum number of iterations ( max i ). If the condition is not fulfiled, the online CE threshold ( ( ) T i ) is calculated, ,1 2 ,2 ( ) ( ) ( ) i l b i e b b L x L x T i e   (3) ,1 2 ,2 ( ) ( ) ( ) i l b i e b b L x L x T i e   (3) (3) Indonesian J Elec Eng & Comp Sci, Vol. 23, No. 3, September 2021: 1557 - 1564 Indonesian J Elec Eng & Comp Sci, Vol. 23, No. 2. CROSS-ENTROPY STOPPING CRITERION 3, September 2021: 1557 - 1564 ISSN: 2502-4752  1559 ISSN: 2502-4752 1559 Indonesian J Elec Eng & Comp Sci where ,2( ) i e b L x  , where ,2( ) i e b L x  , ere ,2( ) i e b L x  , 1 ,2 ,2 ,1 ,2 ,2 ( ) ( ) ( ) ( ) ( ) i i i i i e b l b l b e b e b L x L x L x L x L x       (4) (4) the iteration is stopped if ( ) T i is in the thresholds predefined in (5), he iteration is stopped if ( ) T i is in the thresholds predefined in (5), 2 4 ( ) (10 10 (1) T i T    (5) 2 4 ( ) (10 10 (1) T i T    (5) if the condition is not fulfiled, then ,2( ) i e b L x is returned to decoder 1, and the process is repeated until max i is achieved. Otherwise, when max i is met, the deinterleaver/final output ( bx ) can be estimated from ,2( ) i l b L x , as shown by (6), ,2 ,2 ,2 0 ( ) 0 ( ( )) 1 ( ) 0 i l b i b l b i l b if L x x sign L x if L x        (6) (6) he final turbo decoder output can be written in the following sequence, the final turbo decoder output can be written in the following sequence, nal turbo decoder output can be written in the following sequence,  1 2 1 , ,..., N b N b x x x x    x (7)  1 2 1 , ,..., N b N b x x x x    x (7) Figure 1. Iterative turbo decoder with a CESC Figure 1. Iterative turbo decoder with a CESC 4. RESULTS AND DISCUSSION The effects and performances of CESC and QAM with three predefined thresholds were compared and analysed under four situations: different code rates, different QAM formats, different code generators, and different frame sizes as shown in Table 1. Each threshold’s performance was analysed based on the BER and AIN. 3. RESEARCH METHOD Table 1 shows the parameters used in the present simulation. The thresholds for CESC were set at 0.1, 0.01, and 0.001. One million random binary data with three different modulations (4-QAM, 8-QAM, and 16-QAM) were applied in the simulation. Meanwhile, the frame size (N) was set to either 1000, 5000, or 10,000 for each data transmission. The turbo code generator (in octal) and code rate values were (7,5), (17,15), (37,21), and (1/2, 1/3). The codes were simulated under the AWGN channel, and the maximum iteration number max ( ) I was set to 8. The flowchart in Figure 2 outlines the simulation. The simulation was started with setting up the turbo decoder parameters, such as code generator (g), code rate (R), and frame size (N) as shown in Table 1. Next, threshold and QAM formats were selected based on the data in Table 1. Then, the turbo decoder with QAM was integrated and simulated and repeated for other turbo code parameters and thresholds for all CESC and QAM formats. Finally, the performances of the turbo decoders with QAM were analysed. The effects of the cross-entropy stopping criterion and quadrature amplitude… (Roslina Mohamad)  1560 ISSN: 2502-4752 Table 1. Simulation parameters Parameters Name/ Value CESC threshold 0.1, 0.01, 0.001 Modulation 4-QAM, 16-QAM, 32-QAM Frame size 1000 (1K), 5000 (5K), 10000 (10K) Turbo code generator (7,5), (17,15), (37,21) Turbo code rate 1/2, 1/3 Channel model AWGN Maximum iteration number 8 Figure 2. Flowchart for the simulation Table 1. Simulation parameters Parameters Name/ Value CESC threshold 0.1, 0.01, 0.001 Modulation 4-QAM, 16-QAM, 32-QAM Frame size 1000 (1K), 5000 (5K), 10000 (10K) Turbo code generator (7,5), (17,15), (37,21) Turbo code rate 1/2, 1/3 Channel model AWGN Maximum iteration number 8 Figure 2. Flowchart for the simulation Name/ Value 0.1, 0.01, 0.001 4-QAM, 16-QAM, 32-QAM 1000 (1K), 5000 (5K), 10000 (10K) (7,5), (17,15), (37,21) 1/2, 1/3 AWGN 8 Figure 2. Flowchart for the simulation 4.1. Performance of the CESC with different turbo code rates The CESC’s AIN and BER performance for coding rates is shown in Figure 3. For the 1/3 code rate, the AIN for threshold values of 0.01, 0.001, and 0.0001 started to decrease at 2 dB. At 4 dB Eb/No, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 2.8, 2.9, and 3 AIN, respectively. These values represent decreases of around 65%, 63.75%, and 62.5% compared to the fixed stopping criterion (FISC). Moreover, the BER output of the CESC and FISC are nearly the same for threshold values of 0.01, 0.001, and 0.0001. For the 1/2 code rate, the AIN for threshold values of 0.01, 0.001, and 0.0001 started to decrease at 3 dB. At 4 dB, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 3.7, 3.9, and 4.1, respectively. These values represent decreases of 53.75%, 51.25%, and 48.75% compared to FISC. Regarding BER performance, it can be observed that for 0.01, there is a slight difference in BER starting at 3.5 dB, which increases until 4 dB at BER = 10-4.5. For thresholds of 0.001 and 0.0001, there are slight differences in BER, starting at 3.5 dB and increasing until 4 dB at BER = 10-4.7. Indonesian J Elec Eng & Comp Sci, Vol. 23, No. 3, September 2021: 1557 - 1564 ISSN: 2502-4752 Indonesian J Elec Eng & Comp Sci  1561 1561  156  (a) (b) Figure 3. (a) AIN and (b) BER performances of the CESC with 16-QAM, g = (37,21), and N = 10K for different turbo code rates (a) (b) (b) (a) Figure 3. (a) AIN and (b) BER performances of the CESC with 16-QAM, g = (37,21), and N = 10K for different turbo code rates 4.2. Performance of the CESC with different QAM formats 4.2. Performance of the CESC with different QAM formats The AIN and BER for CESC for QAM formats of 4-QAM, 16-QAM, and 32-QAM are shown in Figure 4. The 4-QAM graph shows that the AIN start to reduce at 0 dB for all three threshold values. At 4 dB Eb/No, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at two iterations, around 75% less than FISC. Moreover, slight differences in BER occurred for all three threshold values, starting at 0 dB and increasing until 0.5 dB at BER = 10-5. Meanwhile, for 16-QAM, the AIN for threshold values of 0.01, 0.001, and 0.0001 started to reduce at 2 dB. At 4 dB, the threshold value of 0.01, 0.001, and 0.0001 can terminate early at 2.8, 2.9, and 3 AIN, respectively, signifying reductions of around 65%, 63.75%, and 62.5% compared to FISC. The BER performance of the CESC was the same as FISC for all three threshold values. The 32-QAM indicates that the AIN for threshold values of 0.01, 0.001, and 0.0001 started to reduce at 3 dB. At 4 dB, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 4.5, 4.6, and 4.8, respectively—these values are 43.75%, 42.5%, and 40% lower than for FISC. The BER performance of the CESC is quite similar to the performance of the FISC at thresholds of 0.01, 0.001, and 0.0001. (a) (b) Figure 4. (a) AIN and (b) BER performances of the CESC with g = (37,21) R = 1/3, N = 10K for different QAM formats (a) (b) (a) (b) Figure 4. (a) AIN and (b) BER performances of the CESC with g = (37,21) R = 1/3, N = 10K for different QAM formats The effects of the cross-entropy stopping criterion and quadrature amplitude… (Roslina Mohamad) 4.3. Performance of the CESC with different turbo code generators The AIN and BER performances of the CESC at different threshold values for different turbo code generators are depicted in Figure 5. The graphs for g = (7,5), (17,15) and (37,21) show that the AIN for threshold values of 0.01, 0.001 and 0.0001 started to reduce at 2 dB. At 4 dB Eb/No, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 3 AIN, representing savings of around 62.5% from a FISC. Also, the BER performances of the CESC and FISC are the same for the threshold values of 0.01, 0.001, and 0.0001. 4.3. Performance of the CESC with different turbo code generators 4.3. Performance of the CESC with different turbo code generators The AIN and BER performances of the CESC at different threshold values for different turbo code generators are depicted in Figure 5. The graphs for g = (7,5), (17,15) and (37,21) show that the AIN for threshold values of 0.01, 0.001 and 0.0001 started to reduce at 2 dB. At 4 dB Eb/No, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 3 AIN, representing savings of around 62.5% from a FISC. Also, the BER performances of the CESC and FISC are the same for the threshold values of 0.01, 0.001, and 0.0001. g The AIN and BER performances of the CESC at different threshold values for different turbo code generators are depicted in Figure 5. The graphs for g = (7,5), (17,15) and (37,21) show that the AIN for threshold values of 0.01, 0.001 and 0.0001 started to reduce at 2 dB. At 4 dB Eb/No, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 3 AIN, representing savings of around 62.5% from a FISC. Also, the BER performances of the CESC and FISC are the same for the threshold values of 0.01, 0.001, and 0.0001.  1562  ISSN: 2502-4752 For g = (17,15), the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 2.9, 3, and 3 AIN, respectively, representing reductions of around 65%, 65%, and 63.75% compared to FISC. The BER performances of the CESC and FISC were the same at thresholds of 0.01, 0.001, and 0.0001. Meanwhile, for g = (37,21), at 4 dB, the threshold value of 0.01, 0.001, and 0.0001 can terminate early at 2.8, 2.9, and 3 AIN, respectively, representing savings of around 65%, 63.75%, and 62.5% compared to the FISC. Once again, the CESC’s and FISC’s BER performances are the same for all three thresholds. (a) (b) Figure 5. (a) AIN and (b) BER performances of the CESC with 16-QAM, R = 1/3, N = 10K for different turbo code generators (a) (b) (b) (a) Figure 5. (a) AIN and (b) BER performances of the CESC with 16-QAM, R = 1/3, N = 10K for different turbo code generators 5. CONCLUSION In most situations, the use of the CESC is suitable only when Eb/No or SNR is high. In such cases, the CESC yields significant reductions in AIN while maintaining a desirable BER performance. The CESC can terminate early when Eb/No is high and can save more than 40% AIN compared with FISC. However, at a low Eb/No, the CESC fails to terminate early and increases AIN. The CESC’s BER performance remains unchanged at BER >10-4. However, for the cases of R = 1/2, 16-QAM and R = 1/3, 4-QAM, minor degradations (i.e. of less than 0.2 dB) occur in the high Eb/No region and increase to 0.5 dB at BER≤10-4. The same situation was apparent for R = 1/3, N = 1K, and threshold = 0.01, as the CESC’s BER performance started to decrease after BER = 10-4. For future works, researchers can use SNR estimations to estimate the low Eb/No region for early CESC termination. In addition, suitable thresholds and enhanced stopping criteria should be calculated and implemented to improve the performance of CESC with QAM for iterative turbo decoding. ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS The authors gratefully acknowledge the support of the Universiti Teknologi MARA (UiTM) for this study, which was funded by grant 600-RMC/MYRA 5/3/LESTARI (039/2020). The authors gratefully acknowledge the support of the Universiti Teknologi MARA (UiTM) for this study, which was funded by grant 600-RMC/MYRA 5/3/LESTARI (039/2020). Indonesian J Elec Eng & Comp Sci Finally, for N = 10K, the AIN for threshold values of 0.01, 0.001, and 0.0001 start to reduce at 2 dB. At 4 dB Eb/No, the threshold values of 0.01, 0.001 and 0.0001 can terminate early at 2.8, 2.9 and 3.0 AIN, respectively. This early termination represents reductions of around 65%, 63.75%, and 62.5% when compared to the FISC. The BER performances of the CESC and FISC are the same for all three threshold values. REFERENCES [1] M. AlMahamdy and J. Dill, “Early Termination of Turbo Decoding by Identification of Undecodable Blocks,” in 2017 IEEE Wireless Communications and Networking Conference (WCNC), 2017, pp. 1-6, doi: 10.1109/WCNC.2017.7925644. [1] M. AlMahamdy and J. 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Lentmaier, “Threshold Computation for Spatially Coupled Turbo-Like Codes on the AWG Channel,” Entropy, vol. 23, no. 2, p. 240, 2021, doi: 10.3390/e23020240. [6] A. Ouardi, “Effect of Interleaving on The Performances of The MAP Turbo Decoder Controlled by The IHDA and CE Stopping Criteria,” Journal of Physics: Conference Series, vol. 1812, no. 1, p. 012006, 2021, doi: 10.1088/1742-6596/1812/1/012006. [7] R. Mohamad, Harlisya Harun, Makhfudzah Mokhtar, Wan Azizun Wan Adnan and Kaharudin Dimyati, “Threshold-Based Bit Error Rate for Stopping Iterative Turbo Decoding in a Varying SNR Environment,” Frequenz, vol. 71, no. 1-2, pp. 83-94, 2017, doi: 10.1515/freq-2016-0018. [8] A. Hadji, et al., “Shaping duo binary turbo-coded BICM scheme for JPWL image transmission using a link adaptation strategy over wireless channels,” Eurasip Journal on Wireless Communications and Networking, vol. 64, no. 1, pp. 1-26, 2021, doi: 10.1186/s13638-021-01949-9. pp [9] H. Tawfeek, Gamal M. Abdel-Hamid and Ashraf Mahran, “A Reliability-based Stopping Criterion for Turbo Product Codes,” in 2018 14th International Computer Engineering Conference (ICENCO), 2018, pp. 141-145, doi: 10.1109/icenco.2018.8636122. [10] S. Yoon, Byungkyu Ahn and Jun Heo, “An Advanced Low-Complexity Decoding Algorithm for Turbo Product Codes Based on the Syndrome,” Eurasip Journal on Wireless Communications and Networking, vol. 2020, no. 1, pp. 1-31, 2020, doi: 10.1186/s13638-020-01740-2. [11] R. Mahdi and A. A. Hamad, “Implementation of Efficient Stopping Criteria for Turbo Decoding,” Journal of Physics: Conference Series, vol. 1804, no. 1, pp. 1-13, 2021, doi: 10.1088/1742-6596/1804/1/012015. 4.4. Performance of the CESC with different frame sizes The CESC’s AIN and BER performances for frame sizes of 1K, 5K, and 10K are illustrated in Figure 6. The N = 1K graph shows that the AIN for threshold values of 0.01, 0.001, and 0.0001 starts to decline at 1.5 dB. At 4 dB Eb/No, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 2.2, 2.3, and 2.4 AIN, respectively, thus saving around 72.5%, 71.25%, and 70% compared to the FISC. For threshold values of 0.001 and 0.0001, the BER performances of the CESC and FISC are quite close. For threshold = 0.01, there is a slight indifference in BER it starts at 3 dB and increases until 4 dB at BER = 10-4.8. g Meanwhile, for N = 5K, the AIN for threshold values of 0.01, 0.001, and 0.0001 starts to reduce at 2 dB. At 4 dB, the threshold values of 0.01, 0.001, and 0.0001 can terminate early at 2.6, 2.8, and 2.9, respectively, thereby saving around 67.5%, 65%, and 63.75% compared to the FISC. Further, the BER performances of the CESC and FISC are the same for thresholds of 0.01, 0.001, and 0.0001. I d i J El E & C S i V l 23 N 3 S b 2021 1557 1564 (a) (b0 Figure 6. (a) AIN and (b) BER performances of the CESC with 16-QAM, g = (37,21), R = 1/3 for different frame sizes (a) (b0 (b0 (a) Figure 6. (a) AIN and (b) BER performances of the CESC with 16-QAM, g = (37,21), R = 1/3 for different frame sizes Indonesian J Elec Eng & Comp Sci, Vol. 23, No. 3, September 2021: 1557 - 1564 1563  ISSN: 2502-4752 Indonesian J Elec Eng & Comp Sci REFERENCES 2] C.-H. Lin, Shu-Wei Guo, Li-An Ou, “Analysis and Power Evaluation of Window-Stopped Parallel Turbo Decodi for LTE Rate Matching,” Iet Communications, vol. 12, no. 9, pp. 1148-1154, 2018, doi: 10.1049/iet-com.2017.1078. g, pp [13] M. Ferrari, S. Bellini and A. Tomasoni, “Safe Early Stopping for Layered LDPC Decoding,” IEEE Communications Letters, vol. 19, no. 3, pp. 315-318, 2015, doi: 10.1109/LCOMM.2014.2386853. [14] T. Fowdur, Y. Beeharry and K. M. S. Soyjaudah, “A Novel Scaling and Early Stopping Mechanism for LTE Turbo Code Based on Regression Analysis,” Annals of Telecommunications, vol. 71, no. 7, pp. 369-388, 2016, doi: 10.1007/s12243-016-0514-y. The effects of the cross-entropy stopping criterion and quadrature amplitude… (Roslina Mohamad)  1564 ISSN: 2502-4752 [15] R. Xue, Yanbo Sun and Qiang Wei, “Dynamic Iteration Stopping Algorithm for Non-Binary LDPC-Coded High- Order Prcpm in The Rayleigh Fading Channel,” Eurasip Journal on Wireless Communications and Networking, vol. 2016, no. 1, pp. 1-11, 2016, doi: 10.1186/s13638-016-0562-z. , , pp , , [16] Y. Beeharry, Tulsi Pawan Fowdur and Krishnaraj M. S Soyjaudah, “Performance of Bit Level Decoding Algorithms for Binary LTE Turbo Codes with Early Stopping,” Istanbul University-Journal of Electrical & Electronics Engineering, vol. 17, no. 2, pp. 3399-3415, 2017. [17] M. Zhu, David G. M. Mitchell; Michael Lentmaier; Daniel J. Costello; Baoming Bai, “Braided Convolutional Codes with Sliding Window Decoding,” IEEE Transactions on Communications, vol. 65, no. 9, pp. 3645-3658, 2017, doi: 10.1109/TCOMM.2017.2707073. [18] M. AlMahamdy and J. Dill, “Half-Iteration Early Termination of Turbo Decoding,” in 2017 IEEE 7th Annual Computing and Communication Workshop and Conference (CCWC), 2017, pp. 1-5, doi: 10.1109/CCWC.2017.7868365. [19] L. O. Mataveli and C. De Almeida, “Complexity Reduction of Convolutional and Turbo Decoding Based on Reliability Thresholds,” Wireless Personal Communications, vol. 82, no. 3, pp. 1279-1290, 2015, doi: 10.1007/s11277-015-2282-9. [20] C. L. Li, Gangfeng Liu, Jie Zhao, “Adaptive Iteration Stopping Criterion for AMSS Equations,” in International Conference on Computer Science and Electronic Technology (CSET 2016), 2016, pp. 7-10, doi: 10.2991/cset-16.2016.2. [21] O. Kocadagli and R. Langari, “Classification of EEG Signals for Epileptic Seizures Using Hybrid Artificial Neural Networks Based Wavelet Transforms and Fuzzy Relations,” Expert Systems with Applications, vol. 88, pp. 419- 434, 2017, doi: 10.1016/j.eswa.2017.07.020. j [22] N. Gkalelis and V. Mezaris, “Subclass Deep Neural Networks: Re-enabling Neglected Classes in Deep Network Training for Multimedia Classification ” in Multimedia Modeling vol 11961 Y M Ro et al Eds (Lecture Notes in Computer j [22] N. Gkalelis and V. REFERENCES Mezaris, “Subclass Deep Neural Networks: Re-enabling Neglected Classes in Deep Network Training for Multimedia Classification,” in Multimedia Modeling, vol. 11961, Y. M. Ro et al. Eds., (Lecture Notes in Computer Science. Cham: Springer International Publishing Ag, 2020, pp. 227-238, doi: 10.1007/978-3-030-37731-1_19. [23] M. Bee, Giuseppe Espa,Diego Giuliani and Flavio Santi, “A Cross-Entropy Approach to the Estimation of Generalized Linear Multilevel Models,” Journal of Computational and Graphical Statistics, vol. 26, no. 3, pp. 695- 708, 2017, doi: 10.1080/10618600.2016.1278003. [24] F. Alberge, “On Some Properties of the Mutual Information Between Extrinsics With Application to Iterative Decoding,” IEEE Transactions on Communications, vol. 63, no. 5, pp. 1541-1553, 2015, doi: 10.1109/tcomm.2015.2422293. [25] M. Krasicki, “Labeling-based Recipient Identification for 16-QAM BICM-ID,” Eurasip Journal on Wi Communications and Networking, vol. 2019, no. 1, pp. 1-9, 2019, doi: 10.1186/s13638-019-1500-7. [26] M. Y. M. Nasir, Roslina Mohamad, Murizah Kassim, Nooritawati Mat Tahir and Ezmin Abdullah, “Performance Analysis of Cross-Entropy Stopping Criterion for Quadrature Amplitude Modulation,” in 2019 IEEE 9th International Conference on System Engineering and Technology (ICSET), 2019, pp. 273-276, doi: 10.1109/ICSEngT.2019.8906345. BIOGRAPHIES OF AUTHORS Roslina Mohamad obtained a B. Eng. degree in Electrical Engineering and M. Eng. Science degree from Universiti Malaya, Kuala Lumpur, in 2003 and 2008. She later received a PhD in Aerospace Engineering from Universiti Putra Malaysia in 2016. Since 2006, she has worked at the School of Electrical Engineering, Universiti Teknologi MARA, as a senior lecturer. Her research interests include computing algorithms and digital signal processing for deep space communication, channel coding, information-theoretic security, computation theory, and wireless communication. Mohamad Yusuf Mat Nasir obtained a B. Eng. degree in Electronics Engineering from Universiti Teknologi MARA, Shah Alam, in 2017. He is currently pursuing his master’s degree at Universiti Teknologi MARA. His research interests include channel coding and wireless communication. Nuzli Mohamad Anas (SM’14) graduated from Universiti Teknologi Malaysia with a B. Eng. in Electrical Engineering in 2000 and an M. Eng. in Electrical Engineering in 2013. Since 2008, he has worked as a senior researcher at the wireless communication lab at MIMOS Berhad. His research interests include wireless mobile communications and signal processing, especially as it relates to cognitive radio and physical-MAC layer communication design. Indonesian J Elec Eng & Comp Sci, Vol. 23, No. 3, September 2021: 1557 - 1564 Indonesian J Elec Eng & Comp Sci, Vol. 23, No. 3, September 2021: 1557 - 1564
https://openalex.org/W4254420157	https://research.monash.edu/files/248849458/246459238_oa.pdf	English	null	Stable isotopes of nitrate reveal different nitrogen processing mechanisms in streams across a land use gradient during wet and dry periods	null	2,017	cc-by	11,489	Correspondence: Wei Wen Wong (weiwen.wong@monash.edu) Received: 8 June 2017 – Discussion started: 4 October 2017 Revised: 22 May 2018 – Accepted: 1 June 2018 – Published: 2 July 2018 Abstract. Understanding the relationship between land use and the dynamics of nitrate (NO− 3 ) is the key to constrain sources of NO− 3 export in order to aid effective manage- ment of waterways. In this study, isotopic compositions of NO− 3 (δ15N–NO− 3 and δ18O–NO− 3 ) were used to elucidate the effects of land use (agriculture in particular) and rain- fall on the major sources and sinks of NO− 3 within the West- ern Port catchment, Victoria, Australia. This study is one of the very few studies carried out in temperate regions with highly stochastic rainfall patterns, enabling a more compre- hensive understanding of the applications of NO− 3 isotopes in catchment ecosystems with different climatic conditions. Longitudinal samples were collected from ﬁve streams with different agriculture land use intensities on ﬁve occasions – three during dry periods and two during wet periods. At the catchment scale, we observed signiﬁcant positive relation- ships between NO− 3 concentrations (p < 0.05), δ15N–NO− 3 (p < 0.01) and percentage agriculture (particularly during the wet period), reﬂecting the dominance of anthropogenic nitro- gen inputs within the catchment. Different rainfall conditions appeared to be major controls on the predominance of the sources and transformation processes of NO− 3 in our study sites. Artiﬁcial fertiliser was the dominant source of NO− 3 during the wet periods. In addition to artiﬁcial fertiliser, ni- triﬁed organic matter in sediment was also an apparent source of NO− 3 to the surface water during the dry periods. Denitri- ﬁcation was prevalent during the wet periods, while uptake of NO− 3 by plants or algae was only observed during the dry periods in two streams. The outcome of this study suggests that effective reduction of NO− 3 load to the streams can only be achieved by prioritising management strategies based on different rainfall conditions. Biogeosciences, 15, 3953–3965, 2018 https://doi.org/10.5194/bg-15-3953-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Biogeosciences, 15, 3953–3965, 2018 https://doi.org/10.5194/bg-15-3953-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Stable isotopes of nitrate reveal different nitrogen processing mechanisms in streams across a land use gradient during wet and dry periods Wei Wen Wong1, Jesse Pottage1, Fiona Y. Warry1, Paul Reich1,2, Keryn L. Roberts1, Michael R. Grace1, and Perran L. M. Cook1 Jesse Pottage1, Fiona Y. Warry1, Paul Reich1,2, Keryn L. Roberts1, Michael R. Grace1, and ok1 1Water Studies Centre, School of Chemistry, Monash University, Clayton, 3800, Australia 2Arthur Rylah Institute for Environmental Research, Department of Environment, Land Water and Planning, Heidelberg, 3084, Australia 1Water Studies Centre, School of Chemistry, Monash University, Clayton, 3800, Australia 2Arthur Rylah Institute for Environmental Research, Department of Environment, Land Water and Planning, Heidelberg, 3084, Australia 1Water Studies Centre, School of Chemistry, Monash University, Clayton, 3800, Australia 2Arthur Rylah Institute for Environmental Research, Department of Environment, Land Water and Planning, Heidelberg, 3084, Australia 1 Introduction Anthropogenic sources of NO− 3 from catchments can pose substantial risk to the quality of freshwater ecosystems (Vi- tousek et al., 1997; Galloway et al., 2004). Over-enrichment of NO− 3 in freshwater systems is a major factor in devel- opment of algal blooms which often promote bottom water hypoxia and anoxia. Such anoxia intensiﬁes nutrient recy- cling and can lead to disruption of ecosystem functioning and ultimately loss of biodiversity (Galloway et al., 2004; Car- mago and Alonso, 2006). Freshwater streams are often sites for enhanced denitriﬁcation (Peterson et al., 2001; Barnes and Raymond, 2010). However, when NO− 3 loading from the catchment exceeds the removal and retention capacity of the streams, NO− 3 is transported to downstream receiving wa- ters including estuaries and coastal embayments, which are often nitrogen-limited, further compounding the problem of eutrophication. Understanding the sources, transport and sinks of NO− 3 is critical, particularly in planning and setting guidelines for better management of the waterways (Xue et al., 2009). Es- tablishing the link between land use and the biogeochemistry of NO− 3 provides fundamental information to help develop NO− 3 reduction and watershed restoration strategies (Kaushal Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3954 et al., 2011). To date, the most promising tool to investigate the sources and sinks of NO− 3 are the dual isotopic com- positions of NO− 3 at natural abundance level (expressed as δ15N–NO− 3 and δ18O–NO− 3 in ‰). Preferential utilisation of lighter isotopes (14N and 16O) over heavier isotopes (15N and 18O) leads to distinctive isotopic signatures that differenti- ate the various NO− 3 sources/end members (e.g. inorganic and organic fertiliser, animal manure, atmospheric deposi- tion) and the predictable kinetic fractionation effect when NO− 3 undergoes different biological processes (e.g. nitrogen ﬁxation and denitriﬁcation). For instance, numerous previ- ous culture-based experiments revealed that denitriﬁcation and phytoplankton assimilation fractionate N and O isotopes equally (1 : 1 pattern), leaving behind NO− 3 that is enriched in both 15N and 18O (Fry, 2006). Simultaneous measurement of δ15N–NO− 3 and δ18O–NO− 3 also provides complementary in- formation on the cycling of NO− 3 in the environment. δ18O– NO− 3 is a more effective proxy of internal cycling of NO− 3 (i.e. assimilation, mineralisation and nitriﬁcation) compared to δ15N–NO− 3 . This is because during NO− 3 assimilation and mineralisation, N atoms are recycled between ﬁxed N pools and the O atoms are removed and replaced by nitriﬁcation (Sigman et al., 2009; Buchwald et al., 2012). thus different fractionation effects to the residual NO− 3 pool (Chien et al., 1977; Billy et al., 2010). However, the lack of NO− 3 isotope studies in the Southern Hemisphere (Ohte, 2013) impedes a more thorough understanding of NO− 3 dy- namics within catchment ecosystems. y Most previous studies investigating the relationship be- tween land use and NO− 3 export using δ15N–NO− 3 and δ18O– NO− 3 have either focused on the seasonal or spatial varia- tions in one stream, or used multiple streams with one site per stream (i.e. Mayer et al., 2002; Yevenes et al., 2016). Far fewer studies have incorporated longitudinal sampling of multiple streams over multiple seasons. Nitrate concen- trations and concomitant isotopic signatures can change sub- stantially, not only spatially but temporally. Published by Copernicus Publications on behalf of the European Geosciences Union. Changes in hy- drological and physicochemical (notably temperature) con- ditions of a river can affect the relative contribution of dif- ferent sources of NO− 3 and the seasonal predominance of a speciﬁc source (Kaushal et al., 2011; Panno et al., 2008). In some studies (e.g. Riha et al., 2014; Kaushal et al., 2011), denitriﬁcation and assimilation by plants and algae have been reported to be more prominent during the dry seasons com- pared to the wet seasons, but in other studies (e.g. Murdiyarso et al., 2010; Enanga et al., 2016) denitriﬁcation appeared to be more prevalent during the wet seasons as precipita- tion induces saturation of soils, resulting in oxygen depletion and thereby low redox potentials that favour denitriﬁcation. As such, if spatial and temporal variations of δ15N–NO− 3 and δ18O–NO− 3 are not considered thoroughly in a sampling regime, it can lead to misinterpretation of the origin and fate of NO− 3 . Proper consideration of the temporal variability of NO− 3 isotope signatures and transformation are particularly pertinent in catchments with highly stochastic rainfall pat- terns, such as Australia. ( g ) In addition to constraining NO− 3 budget and N cycling in various environmental settings, previous studies have also utilised the dual isotopic signatures of NO− 3 to study the ef- fects of different land uses on the pool of NO− 3 in headwater streams (Barnes and Raymond, 2010; Sebilo et al., 2003), creeks (Danielescu and MacQuarrie, 2013) and large rivers (Voss et al., 2006; Battaglin et al., 2001). Barnes and Ray- mond (2010) for example found that both δ15N–NO− 3 and δ18O–NO− 3 varied signiﬁcantly between urban, agricultural and forested areas in the Connecticut River watershed, USA. Several other investigators (Mueller et al., 2016; Mayer et al., 2002) showed positive relationships between δ15N–NO− 3 and the percent of agricultural land in their study area, indicat- ing the applicability of δ15N–NO− 3 and δ18O–NO− 3 to distin- guish NO− 3 originating from different land uses. Danielescu and MacQuarrie (2013) and Chang et al. (2002) on the other hand found no correlations between NO− 3 isotopes and land use intensities in the Trout River catchment and the Missis- sippi River basin, respectively. These studies attributed the lack of correlation to catchment size (Danielescu and Mac- Quarrie, 2013) and the homogeneity of land use (Chang et al., 2002). Published by Copernicus Publications on behalf of the European Geosciences Union. In this study, we examine both spatial and temporal vari- ations of NO− 3 concentrations and isotopic compositions within and between ﬁve streams in ﬁve catchments span- ning an agricultural land use gradient, enabling us to eval- uate (1) the effects of agriculture land use on the sources and transformation processes of NO− 3 and (2) the effects of rain- fall on the predominance of the sources and fate of NO− 3 in the catchments. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3955 Bass Strait WPB Watson Lang Lang Lower Bunyip Bass Toomuc km 0 10 20 N Upper Bunyip (63) (56) (43) (9) (2) (24) (13) (81) (75) (74) (92) (92) (96) (96) (74) (78) (47) (84) (83) (81) (73) Figure 1. Map of Western Port Bay (WPB) in southern Victoria, Australia, and major rivers discharging into WPB. Closed circles represent sampling sites where surface water samples were ob- tained. Values in parentheses represent the % agriculture area in the catchment. (see Fig. S1 in the Supplement), thus enabling comparisons within and between the streams. Bass Strait WPB Watson Lang Lang Lower Bunyip Bass Toomuc km 0 10 20 N Upper Bunyip (63) (56) (43) (9) (2) (24) (13) (81) (75) (74) (92) (92) (96) (96) (74) (78) (47) (84) (83) (81) (73) In this study, catchment-intensive agriculture was used as a predictor of land use intensity in the catchment. These data were obtained from the National Environmental Stream At- tributes database v1.1 (Stein et al., 2014), Bureau of Rural Sciences’ 2005/06 Land Use of Australia V4 maps (http:// www.agriculture.gov.au/abares/aclump, last access: 30 April 2016) and Victorian Resources Online (VRO). In the con- text of this study, the catchment-intensive agriculture vari- able is termed “percentage agriculture”. This term repre- sents the percentage of the catchment subject to intensive animal production, intensive plant production (horticulture and irrigated cropping) and grazing of modiﬁed pastures. This variable also reﬂects the integrated diffuse sources of nutrients derived from intense agriculture including animal manure and inorganic fertilisers. The percentage agricul- ture for the sampling sites ranged between 2 and 96 % with the Bass River (94±2 %) > Lang Lang (79±5 %) > Watsons (76 ± 4 %) > Toomuc (71 ± 16 %) > Bunyip (upper Bunyip: 12 ± 9 %; lower Bunyip: 54 ± 10 %; Fig. 2). For the pur- pose of this study, Bunyip is divided into two sectors (up- per and lower Bunyip) based on the proximity of the sam- pling sites (Fig. 1) and the percentage of land use. All the sampling sites in the upper Bunyip are situated in areas with > 30 % forestation (see Fig. S1). In general, the percentage agriculture decreases with increased distance from Western Port Bay (WPB) for all the streams except Bass River. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale There is an increase of about 2 % in percentage agriculture for Bass River with increased distance from WPB. Watsons Creek has the largest percentage of market gardens (∼91 %). Figure 1. Map of Western Port Bay (WPB) in southern Victoria, Australia, and major rivers discharging into WPB. Closed circles represent sampling sites where surface water samples were ob- tained. Values in parentheses represent the % agriculture area in the catchment. catchments cover an area of 3721 km2 with land uses rang- ing from semi-pristine/state forest to high-density residen- tial and intense agricultural activities. The area experiences a temperate climate with average annual rainfall ranging from 750 mm along the coast to 1200 mm in the northern high- lands. Mean monthly rainfall was about 20 and 53 mm in 2014 and 2015, respectively (Australian Bureau of Meteo- rology 2014 – http://www.bom.gov.au/, last access: 30 Apri 2016). 2.1 Study area Despite the extensive application of NO− 3 isotopes to study the transport of terrestrial NO− 3 to the tributaries in the catch- ment, the majority of these studies were carried out in the United States and western Europe where climatic conditions, for example temperature and rainfall patterns, are different compared to that in the Southern Hemisphere. The Southern Hemisphere tends to have more sporadic and variable rain- fall patterns compared to the Northern Hemisphere, and Aus- tralia is an example of this. The variable rainfall patterns can modulate different efﬁciencies of denitriﬁcation in soils and This study was undertaken using ﬁve major streams (Bass River, Lang Lang River, Bunyip River, Watsons Creek and Toomuc Creek) draining into Western Port (Fig. 1) which lies approximately 75 km south-east of Melbourne, Australia. Western Port is a nitrogen-limited coastal embayment recog- nised as a Ramsar site for migratory birds. The catchments in Western Port contain three marine national parks, high- lighting its environmental and ecological signiﬁcance. The www.biogeosciences.net/15/3953/2018/ Biogeosciences, 15, 3953–3965, 2018 2.2 Sample collection and preservation Water quality parameters (pH, electrical conductivity, turbid- ity, dissolved oxygen (DO) concentration and water temper- ature) were measured using a calibrated Horiba U-10 multi- meter. Stream samples were collected for the analyses of dis- solved inorganic nutrients (DIN) (ammonium, NH+ 4 ; NO− 3 and nitrite, NO− 2 ), dissolved organic carbon (DOC) and NO− 3 isotopes (δ15N–NO− 3 and δ18O–NO− 3 ). These samples were ﬁltered on site using 0.2 µm Pall Supor® membrane disc ﬁl- ters. Filtered DOC samples were acidiﬁed to pH < 2 with concentrated hydrochloric acid. Samples for δ18O–H2O were collected directly from the streams without ﬁltering. Sedi- ment samples were collected from the bottom of the rivers and were kept in zip-lock bags. All samples were stored and transported on ice until they were refrigerated (nutrient sam- ples were frozen) in the laboratory. In addition to stream wa- ter and sediment, we also collected four samples of artiﬁ- cial/inorganic fertiliser (from the fertiliser distributor in the area) and ﬁve of cow manure (from local farmers). The catchment overlies a multi-layered combined aquifer system. The main aquifer consists of Quaternary alluvial and dune deposit (average thickness of < 7 m) as well as Bax- ter, Sherwood and Yallock formations (average thickness be- tween 20 and 175 m). These aquifers are generally uncon- ﬁned with radial groundwater ﬂow direction from the basin edge towards Western Port Bay. The hydrogeology of West- ern Port can be found in Carillo-Rivera (1975). Five longitudinal surveys were carried out between April 2014 and May 2015, two during wet periods (14 April 2014; 15 May 2015 – the total rainfall for 5 days before sampling was between 45 and 65 mm) and three during dry periods (8 April 2014; 22 May 2014; 21 March 2015 – the total rain- fall for 5 to 10 days before sampling was < 5 mm). A total of 21 sampling sites indicated in Fig. 1 were selected across a gradient of catchment land use intensity. The ﬁve streams were selected based on the extent and distribution of land use types between and within each stream sub-catchment Biogeosciences, 15, 3953–3965, 2018 www.biogeosciences.net/15/3953/2018/ Biogeosciences, 15, 3953–3965, 2018 3 Results The samples for δ15N–NO− 3 and δ18O–NO− 3 were analysed using the chemical azide method based on the procedure out- lined in McIlvin et al. (2005). In brief, NOx (NO− 3 + NO− 2 ) was quantitatively converted to NO− 2 using cadmium reduc- tion and then to N2O using sodium azide. The initial NO− 2 concentrations were insigniﬁcant, typically < 1 % relative to NO− 3 . Hence, the inﬂuence of δ15N–NO− 2 was negligible and the measured δ15N–N2O represents the signature of δ15N– NO− 3 . The resultant N2O was then analysed on a Hydra 20– 22 continuous ﬂow isotope ratio mass spectrometer (CF- IRMS; Sercon Ltd., UK) interfaced to a cryoprep system (Sercon Ltd., UK). Nitrogen and oxygen isotope ratios are reported in per mil (‰) relative to atmospheric air (AIR) and Vienna Standard Mean Ocean Water (VSMOW), respec- tively. The external reproducibility of the isotopic analyses lies within ±0.5 ‰ for δ15N and ±0.3 ‰ for δ18O. The inter- national reference materials used were USGS32, USGS 34, USGS 35 and IAEA-NO− 3 . Lab-internal standards (KNO− 3 and NaNO− 2 ) with pre-determined isotopic values were also processed the same way as the samples to check on the efﬁ- ciency of the analytical method. The δ18O–H2O values were measured via equilibration with He–CO2 at 32 ◦C for 24 to 48 h in a Finnigan MAT Gas Bench and then analysed using The streams were oxic throughout the course of our study period with % DO saturation between 60 and 110 % (see Fig. S2 in the Supplement). There was no apparent spa- tial and temporal variation in DO; however, % DO satu- ration was slightly lower during the dry periods (average of 73 ± 20 %) compared to the wet periods (average of 82 ± 12 %). Temperature was also relatively consistent, with an average of 13 ± 2 ◦C. Ammonium concentration was gen- erally low (< 4 µM) except during the wet periods in Bunyip (∼7 µM), Lang Lang (∼21 µM) and Bass (∼29 µM). DOC concentrations were typically 0.8 ± 0.4 mM. Nitrite concen- trations were also low in all the streams, ranging between 0.1 and 0.4 µmol L−1. The spatial and temporal variations of NO− 3 concentration, δ15N and δ18O across the sites are shown in Fig. 3. 2.5 Data analysis The relationships between percentage agriculture and sur- face water NO− 3 concentrations were assessed using linear regression. Percentage agriculture was the predictor variable. NO− 3 concentration and δ15N–NO− 3 were response variables. Relationships between δ15N–NO− 3 and NO− 3 concentration as well as δ18O–NO− 3 and δ15N–NO− 3 were assessed using Pearson’s correlation. The NO− 3 isotopes’ response variables were assessed at two spatial scales – individual stream and catchment scale. The catchment scale integrates data from all ﬁve studied streams. Any graphical patterns or relationships derived from using these scales represent processes that oc- cur somewhere in the catchment either in the streams or prior to entering the streams with data from the individual stream likely to represent more localised processes to that particular stream. Figure 2. The percent agriculture upstream for each of the sampling sites. Figure 2. The percent agriculture upstream for each of the sampling sites. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3956 0 20 40 60 80 100 0 20 40 60 80 Percent agriculture (%) Distance from WPB (km) Bunyip Lang Lang Toomuc Watson Bass Figure 2. The percent agriculture upstream for each of the sampling sites. CF-IRMS. The δ18O–H2O values were referenced to internal laboratory standards, which were calibrated using VSMOW and Standard Light Antarctic Precipitation. Measurement of two sets of triplicate samples in every run showed a precision of 0.2 ‰ for δ18O–H2O. Sediment samples for the analysis of δ15N of total nitrogen were dried at 60 ◦C before being analysed on the 20–22 CF-IRMS coupled to an elemental analyser (Sercon Ltd., UK). The precision of the elemental analysis and δ15N was 0.5 µg and ±0.2 ‰ (n = 5), respec- tively. 2.3 DIN and DOC concentration measurements All chemical analyses were performed within 1–2 weeks of sample collection except for isotope analyses (within 2 months). The concentrations of NO− 3 , NO− 2 , and NH+ 4 were determined spectrophotometrically using a Lachat QuikChem 8000 Flow Injection Analyzer (FIA) follow- ing standard procedures (APHA 2005). DOC concentrations were determined using a Shimadzu TOC-5000 Total Organic Carbon analyser. Analysis of standard reference materials in- dicated the accuracy of the spectrophotometric analyses and the TOC analyser was always within 2 % relative error. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale Based on the assumptions that ammonia was fully oxidised to NO− 3 (as no accumulation of NO− 2 was observed during our study period) and there was complete exchange of oxygen isotope between nitrite and H2O during ammonia oxidation (xAO = 1), which likely characterises most freshwater sys- tems (Casciotti et al., 2007; Snider et al., 2010; Buchwald and Casciotti, 2013); we calculated the δ18O of produced NO− 3 from nitriﬁcation to be between −2.03 and −0.23 ‰. Table 1. The isotopic compositions of potential sources of NO− 3 in the catchment. Table 1. The isotopic compositions of potential sources of NO− 3 in the catchment. (Fig. 3). During the wet periods, NO− 3 concentrations typi- cally followed an increasing trend heading downstream ex- cept for the Bass River which exhibited the opposite NO− 3 trend with lower concentrations at downstream sites. Dur- ing the dry periods, only the Bunyip and Bass rivers showed apparent longitudinal patterns in NO− 3 concentrations, with decreasing concentrations moving downstream in both. Sites with high-percentage agriculture generally also exhibited high NO− 3 concentrations (Fig. 4), particularly during the wet periods. p Overall, δ15N of the riverine NO− 3 spanned a wide range (+4 to +33 ‰). Approximately 62 % of the obtained δ15N– NO− 3 values fell below +10 ‰. More enriched δ15N–NO− 3 values (> +10 ‰) were typically observed during the dry pe- riods and were coincident with a high-percentage agriculture (Fig. 4). Among all sites, δ15N–NO− 3 values in the Bunyip and Bass were relatively depleted (+4 to +12 ‰ for Bunyip and +10 to 12 ‰ for Bass), with the lower range found at up- per Bunyip (+4 to +8 ‰). There was no discernible pattern spatially or temporally in δ18O–NO− 3 , except that higher val- ues were found in Lang Lang and Bass during the wet peri- ods, with +4 to +6 and +5 to +9 ‰, respectively, compared to the dry periods (< +4 ‰). For other sampling sites, δ18O– NO− 3 ranged between +2 and +13 ‰. The isotope compo- sitions of sediment, water, artiﬁcial fertiliser and cow ma- nure/organic fertiliser are presented in Table 1. The δ15N-TN of three potential sources – artiﬁcial fertiliser, organic fer- tiliser and soil organic matter – ranged from −0.5 to +0.7, +6 to +13 and +4 to +5 ‰, respectively. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale δ18ONO− 3 ﬁnal = 2 3 + 1 3xAO δ18OH2O + 1 3 h δ18OO2 −18εk,O2 −18εk,H2O,1 (1 −xAO) −18εk,H2O,2 i + 2 3 18εeqxAO (1) (1) W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale W. W. Wong et al.: Nitrate isotopes in streams on a catchm Table 1. The isotopic compositions of potential sources of NO− 3 in the catchment. Sample δ15N-TN δ18O–H2O (‰) (‰) Artiﬁcial/inorganic fertiliser −0.5 to +0.7 – Cow manure/organic fertiliser +6 to +13 – Sediment (SOM) +4 to +5 – Stream water – −5.5 to −4.9 monium from organic matter) and nitriﬁcation (oxidation of ammonium to NO− 3 ). The δ18O of NO− 3 generated by nitri- ﬁcation of these sources (δ18O–NO− 3 ﬁnal) is, however, de- coupled from δ15N–NO− 3 . As shown in Eq. (1), which is adapted from Buchwald et al. (2012), δ18O–NO− 3 ﬁnal relies on the oxygen isotope of water (δ18O–H2O), oxygen isotope of dissolved oxygen (δ18O–O2), kinetic isotope fractiona- tion associated with incorporation of oxygen during ammo- nia oxidation (18εk-O2), kinetic isotope fractionation asso- ciated with incorporation of oxygen from water during am- monia oxidation (18εk-H2O,1) and nitrite oxidation (18εk- H2O,2), equilibrium isotope effect associated with oxygen isotope exchange between nitrite and water (18εeq) as well as the fraction of nitrite oxygen atoms exchanged with H2O dur- ing ammonia oxidation (xAO) (Casciotti et al., 2010; Buch- wald et al., 2012). To date, 18εk-O2 and 18εk-H2O cannot be separated. Previous culture studies have reported the over- all 18εk-O2 + 18εk-H2O,1 to range between 17.9 and 37.6 ‰ (Casciotti et al., 2010), while 18εk-H2O,2 ranged from 12.8 to 18.2 ‰ (Buchwald and Casciotti, 2010). These values to- gether with a 18εeq value of 14 ‰, average δ18O–H2O of −5.3 ‰ and δ18O–O2 of 23.5 ‰ were used to calculate the maximum and minimum estimates of the δ18O of newly pro- duced NO− 3 from nitriﬁcation. The minimum estimate of δ18O–NO− 3 ﬁnal was calculated using the lower range of 18εk- O2 + 18εk-H2O,1 (17.9 ‰) and 18εk-H2O,2 (12.8 ‰), while the maximum estimate was calculated using the upper range of 18εk-O2 + 18εk-H2O,1 (37.6 ‰) and 18εk-H2O,2 (18.2 ‰). 4 Discussion The δ15N-TN of cow manure (+6 to +13 ‰) was most variable compared to other end members. This variation re- ﬂects the extent of volatilisation, a highly fractionating pro- cess. Volatilisation can cause a fractionation effect of up to 25 ‰ in the residual NH+ 4 (Hübner, 1986). As such, the lower value of +6 ‰ indicates a relatively fresh manure sample and is assumed to represent the initial δ15N of the cow manure before undergoing any extensive fractionation. 3 Results NO− 3 concentrations ranged between 7 and 790 µM with averages of 21 ± 15, 50 ± 130, 64 ± 43, 71 ± 43 and 190 ± 280 µM for Toomuc, Bunyip, Bass, Lang Lang and Watsons, respec- tively. The lowest NO− 3 concentration was observed in the lower Bunyip (4 µM), while the highest NO− 3 concentration was observed in Watsons Creek (790 µM) at the most down- stream site. Nitrate concentrations were generally higher dur- ing the wet periods compared to the dry periods in all streams www.biogeosciences.net/15/3953/2018/ Biogeosciences, 15, 3953–3965, 2018 3957 4.1 Potential sources of NO− 3 There are three major potential sources of NO− 3 in the catch- ments – artiﬁcial fertiliser, cow manure/organic fertiliser and soil organic matter (SOM) – see Table 1 for the δ15N-TN values. The average δ15N-TN value of soils is used to di- rectly represent the soil organic portion as most of the nitro- gen in soils is generally bound in organic forms. Nitrogen isotope of the NO− 3 produced from the potential end mem- bers usually retains the signature of the δ15N-TN as a result of tight coupling between mineralisation (production of am- Atmospheric deposition did not appear to be an impor- tant source of NO− 3 in this study based on the relatively de- pleted δ18O–NO− 3 values (ranged from +2 to +8 ‰ dur- www.biogeosciences.net/15/3953/2018/ W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3958 [NO3 -] (µM) 0 200 400 600 800 0 20 40 60 80 Wet Dry Bunyip 0 200 400 600 800 0 10 20 30 40 Lang Lang 0 200 400 600 800 0 10 20 30 40 Toomuc 0 200 400 600 800 0 10 20 30 40 Watson 0 200 400 600 800 0 20 40 60 Bass 0 5 10 15 0 20 40 60 80 0 5 10 15 0 10 20 30 40 0 5 10 15 0 10 20 30 40 0 5 10 15 0 10 20 30 40 0 5 10 15 0 20 40 60 δ 18O-NO3 - (‰) 0 12 24 36 0 20 40 60 80 0 12 24 36 0 10 20 30 40 0 12 24 36 0 10 20 30 40 0 12 24 36 0 10 20 30 40 0 12 24 36 0 20 40 60 δ 15N-NO3 - (‰) Distance from WPB (km) Lower Upper 3. Spatial and temporal variations of nitrate concentrations and isotope values. Closed circles represent data obtained during the wet s. Open circles represent data obtained during the dry periods. Figure 3. Spatial and temporal variations of nitrate concentrations and isotope values. Closed circles represent data obtained during the wet periods. Open circles represent data obtained during the dry periods. www.biogeosciences.net/15/3953/2018/ www.biogeosciences.net/15/3953/2018/ Biogeosciences, 15, 3953–3965, 2018 W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale In addition to min- eralisation and nitriﬁcation, volatilisation and assimila- tion by plant and algae were highly likely to occur in the stream, further reducing the NO− 3 concentration and further fractionating the isotopic signature of NO− 3 . Percentage agriculture (%) Figure 4. Relationship between (a) NO− 3 concentration, (b) δ15N– NO− 3 and the percentage of agricultural land use. In (b) the solid line represents the relationship between the variables during dry pe- riods; the dotted line represents wet periods. These processes are conceptualised in Fig. 5 and are cor- roborated in the following discussion using two graphical methods: the Keeling plot and the isotope biplot. In an agri- cultural watershed, the co-existence of multiple sources and transformation processes can potentially complicate the use of NO− 3 isotopes as tracers of its origin. Keeling plots (δ15N– NO− 3 vs. 1/[NO− 3 ]) are generally very useful to distinguish between mixing and fractionation (i.e. assimilation and bac- terial denitriﬁcation) processes (Kendall et al., 1998). The latter typically results in progressively increasing δ15N–NO− 3 values as NO− 3 concentrations decrease and yields a curved Keeling plot. Meanwhile, mixing of NO− 3 from two or more sources can result in a concomitant increase in both δ15N– NO− 3 and NO− 3 concentrations and results in a straight line on the Keeling plot (Kendall et al., 1998). A biplot (δ18O– NO− 3 vs. δ15N–NO− 3 ) on the other hand is a proven diagnos- tic method to elucidate the presence of two isotope fraction- ating processes: assimilation and denitriﬁcation. NO− 3 in the stream originated from agricultural activities. In fact, the most enriched δ15N–NO− 3 values (> 30 ‰) were ob- served at the most downstream site of Watsons Creek which has the largest percentage of market gardens (although the total agricultural area is not the highest amongst all the stud- ied sites). We also observed a signiﬁcant positive relation- ship between δ15N–NO− 3 and percentage agriculture during the wet periods (r2 = 0.39, p < 0.01; Fig. 4b). This further supports the contention that agricultural activities were the main control of the δ15N–NO− 3 in the streams. Other re- searchers (e.g. Mayer et al., 2002; Voss et al., 2006) have also documented similar trends of enriched δ15N–NO− 3 with increasing percentage agriculture. For example, Harrington et al. (1998), Mayer et al. (2002) and Voss et al. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3959 δ15N-NO3 - (‰) 0 5 10 15 20 25 30 35 0 20 40 60 80 100 [NO3 -] (µM) 0 40 80 120 160 200 Wet Dry 0 300 600 900 0 50 100 Percentage agriculture (%) Dry: r = 0.20 (n = 42, p < 0.01) 2 Wet: r = 0.39 (n = 55, p < 0.01) 2 (a) (b) δ15N-NO3 - (‰) 0 5 10 15 20 25 30 35 0 20 40 60 80 100 [NO3 -] (µM) 0 40 80 120 160 200 Wet Dry 0 300 600 900 0 50 100 Percentage agriculture (%) Dry: r = 0.20 (n = 42, p < 0.01) 2 Wet: r = 0.39 (n = 55, p < 0.01) 2 (a) (b) Figure 4. Relationship between (a) NO− 3 concentration, (b) δ15N– NO− 3 and the percentage of agricultural land use. In (b) the solid line represents the relationship between the variables during dry pe- riods; the dotted line represents wet periods. Given that none of the predicted sources of NO− 3 in the Western Port catchment exhibited an initial δ15N–NO− 3 of more than +6 ‰ (see Table 1), the isotopically enriched NO− 3 as well as the variability of NO− 3 concentrations ob- served in this study were consequences of a series of transfor- mation processes. Hence, we propose the following factors to explain the heavy isotopes and the different NO− 3 concentra- tions across different periods observed in our study. 1. During the wet period when surface runoff was con- spicuous and residence time of the water column was low, in-stream NO− 3 was comprised mainly of exter- nally derived NO− 3 (i.e. fertilisers, manure and soil or- ganic matter) and there was limited in-stream process- ing of these NO− 3 . The high NO− 3 concentrations and the heavy δ15N–NO− 3 values reﬂect the occurrence of mineralisation, nitriﬁcation and subsequent preferential denitriﬁcation of the isotopically lighter NO− 3 source/s in either the waterlogged soil or in the soil zone under- neath the market gardens before transport to the streams through surface runoff. (b) 2. During the dry periods when surface runoff was negli- gible and residence time of the water column was high, there was minimal introduction of external NO− 3 into the streams and in-stream processing of NO− 3 was more apparent than during the wet periods. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale (2006) ob- served highly signiﬁcant positive relationships between per- centage agriculture land area and δ15N–NO− 3 with r2 ∼0.7. However, these studies showed comparatively narrower and more depleted ranges of δ15N–NO− 3 , with 2.0 to 7.3, 4 to 8 and −0.1 to 8.3 ‰, respectively, suggesting more subtle changes in δ15N–NO− 3 over a large span of agriculture land areas in these studies compared to our study (see Table 2). 4.2 General characteristics of NO− 3 in the streams ing the wet periods, and from +1.5 to +13 ‰ during the dry periods) of the riverine samples. The δ18O–NO− 3 of at- mospheric deposition were reported to range from +60 to +95 ‰ in the literature (Kendall, 2007; Elliott et al., 2007; Pardo et al., 2004). Similarly, groundwater was not consid- ered as an important source of NO− 3 to the streams based on the low NO− 3 concentrations (∼0.7 to 7.0 µM) reported in previous studies (Water Information System Online; http: //data.water.vic.gov.au/monitoring.htm, last access: 29 April 2016). Agricultural land use (i.e. market gardens and cattle rear- ing) appeared to inﬂuence NO− 3 concentrations in our study sites. As shown in Fig. 4a, during the wet periods, high NO− 3 concentrations (> 40 µM) were particularly observed at sites with more than 70 % agricultural land use. During the dry periods, although NO− 3 concentrations were generally lower than 36 µM, the outliers were observed at sites with more than 70 % agricultural land use. Similarly, enriched δ15N– NO− 3 in the streams were mainly found at sites with high- percentage agricultural land use (between 75 and 85 %) for both dry and wet periods, suggesting that enriched δ15N– Biogeosciences, 15, 3953–3965, 2018 www.biogeosciences.net/15/3953/2018/ 4.3 Key controlling processes of nitrate during the wet periods In-stream processing of NO− 3 was not evident during the wet periods based on the lack of relationships between δ18O– www.biogeosciences.net/15/3953/2018/ Biogeosciences, 15, 3953–3965, 2018 W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3960 Figure 5. Conceptual diagram illustrating the sources and processes of NO− 3 during the wet and dry periods in the Western Port catchment The values of the enrichment factor (ε) were obtained from the literature (Kendall et al., 2007) to indicate the relative contribution of th transformation processes to the isotopic compositions of the residual NO− 3 . Figure 5. Conceptual diagram illustrating the sources and processes of NO− 3 during the wet and dry periods in the Western Port catchment. The values of the enrichment factor (ε) were obtained from the literature (Kendall et al., 2007) to indicate the relative contribution of the transformation processes to the isotopic compositions of the residual NO− 3 . 0 10 20 30 0 10 20 0 10 20 30 0 5 10 0 10 20 30 0 10 20 0 10 20 30 0 10 20 0 10 20 30 0 10 20 0 10 20 30 0 10 20 0 5 10 15 20 25 0 5 10 Upper Bunyip y-intercept: 7.1 ‰ r = 0.52, p < 0.001 2 0 5 10 15 20 25 0 5 10 Bunyip y-intercept: 8.8 ‰ r = 0.62, p < 0.001 2 0 5 10 15 0.0 0.5 1.0 1.5 Lang Lang y-intercept: 15.5 ‰ r = 0.68, p < 0.001 2 0 5 10 15 20 25 0 1 2 3 Toomuc y-intercept: 10.4 ‰ r = 0.69, p < 0.001 2 0 5 10 15 20 0 5 10 15 Watson y-intercept: 12.9 ‰ r = 0.62, p < 0.001 2 0 5 10 15 0.0 0.5 1.0 1.5 Bass y-intercept: 7.2 ‰ r = 0.52, p < 0.001 2 0 10 20 30 0 2 4 6 Bass y-intercept: 16.3 ‰ r 2 = 0.86, p < 0.001 1/[NO3 -] δ 15N-NO3 - (‰) Wet period Dry period 0 5 10 15 20 25 0 2 4 Lower Bunyip y-intercept: 9.1 ‰ r = 0.37, p < 0.001 2 Dry period Wet period 1/[NO3 -] 1/[NO3 -] 1/[NO3 -] Figure 6. Relationship between δ15N–NO− 3 and 1/[NO− 3 ] for individual streams during the wet and dry periods. gure 6. www.biogeosciences.net/15/3953/2018/ W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3961 Nitrified cow manure Nitrified SOM Inorganic fertiliser δ15N-TN of potential end members -2 0 2 4 6 8 10 12 14 16 18 0 20 40 60 80 100 ry Wet D Upper Bunyip Lower Bunyip Toomuc Watson Lang Lang Bass Bass δ15N-NO3 - (‰) Percentage agriculture (%) Figure 7. Relationship between δ15N–NO− 3 of the dominant initial source (indicated by the y-intercept of the Keeling plots; Fig. 6) and percentage agriculture during wet periods. Data for the Bass-dry pe- riod were also presented because only the Keeling plot for the Bass- dry period indicates mixing between different sources. The shaded area represents the δ15N–NO− 3 of the potential end members. Nitrified cow manure Nitrified SOM Inorganic fertiliser δ15N-TN of potential end members -2 0 2 4 6 8 10 12 14 16 18 0 20 40 60 80 100 ry Wet D Upper Bunyip Lower Bunyip Toomuc Watson Lang Lang Bass Bass δ15N-NO3 - (‰) Percentage agriculture (%) NO− 3 and [NO− 3 ] as well as between δ18O–NO− 3 and δ15N– NO− 3 for the individual streams (shown in Supplement Fig. S3). If denitriﬁcation was dominant, both δ15N–NO− 3 and δ18O–NO− 3 values are expected to increase in a 1 : 1 pattern at low NO− 3 concentration – a trend which has been proven by numerous culture-based experiments to indicate the oc- currence of denitriﬁcation (Granger and Wankel, 2016). In addition, high DO in the water column ruled out the possibil- ity of pelagic denitriﬁcation. Careful examination of the Keeling plots for individual streams (Fig. 6) revealed that during the wet periods, NO− 3 concentrations were signiﬁcantly and linearly correlated with 1/[NO− 3 ] in all the streams. These relationships strongly sug- gest mixing between two sources (with distinctive isotopic signatures) as the dominant process regulating the isotopic composition of the residual NO− 3 in the streams during the wet periods. The different trends in the Keeling plots (Fig. 6) for individual streams indicate that the isotopic signature of the dominant NO− 3 source varied temporally and spatially across the catchments. Negative trends on the Keeling plots for Bunyip, Lang Lang and Toomuc (Fig. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale Based on the multi-process model developed by Granger and Wankel (2016), the negative de- ﬂation of the denitriﬁcation trend (1 : 1) is strongly driven by concurrent NO− 3 production catalysed by nitriﬁcation and/or anammox (Granger and Wankel, 2016) when the rate of NO− 3 reduction to NO− 2 (via denitriﬁcation) is higher than the rate of NO− 2 oxidation to NO− 3 (via nitriﬁcation and/or anam- mox). A higher reduction rate of NO− 3 to NO− 2 tends to cre- ate a NO− 2 pool with enriched δ15N due to isotopic fraction- ation (0 to 20 ‰) during the reduction of NO− 2 to N2 (the last step of denitriﬁcation). The subsequent oxidation of the δ15N-enriched NO− 2 leads to the production of NO− 3 which is isotopically more enriched than denitriﬁed NO− 3 owing to inverse kinetic fractionation effects (−35 to 0 ‰), driving the negative deviation of δ18O–NO− 3 : δ15N–NO− 3 from the 1 : 1 trend (Granger and Wankel, 2016). During the wet periods, simultaneous occurrence of these three processes (nitriﬁca- tion, annamox and denitriﬁcation) was plausible due to the redox dynamics in the waterlogged soil zone. Downward per- colation of oxygenated rain water could induce nitriﬁcation while denitriﬁcation and anammox could be promoted in the anoxic interstitial spaces of the waterlogged soil zone. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 6) clearly show that the dominant NO− 3 source was isotopically enriched (above +10 ‰ for Bunyip and Toomuc and +14 ‰ for Lang Lang), while the positive trends on the Keeling plots for Bass and Watsons show that the dominant NO− 3 source was more iso- topically depleted (less than +8 ‰ for Bass and less than +9 ‰ for Watsons). Nevertheless, the isotopic signatures of the dominant source indicated by the y-intercepts of the Keeling plots were a lot more enriched than the initial δ15N– NO− 3 of all three pre-identiﬁed NO− 3 end members. Inter- estingly, these δ15N–NO− 3 values increased with percentage agriculture, except for Bass (Fig. 7). The fact that there was a clear fractionation pattern (∼2 : 1) when integrating the isotope values of all the streams (catchment scale) suggests that denitriﬁcation was still prevalent during the wet peri- ods (Fig. 8a), but this process was more likely to occur prior to NO− 3 entering the streams via surface runoff. We explain these observations on the basis that increased rainfall created a “hot moment” in the soil whereby organic matter mineral- isation and nitriﬁcation were stimulated, followed by deni- triﬁcation within the waterlogged soil. Waterlogging can re- sult in root anoxia and increased denitriﬁcation, leading to signiﬁcant isotopic enrichment of the residual NO− 3 (Chien et al., 1977; Billy et al., 2010), which was then washed into the streams. The extent of this process (mineralisation– nitriﬁcation–denitriﬁcation) was greatest at Bass and Wat- sons, sites with the highest agricultural activity (Fig. 8a). Based on Fig. 8a, the isotope enrichments of the riverine NO− 3 followed the denitriﬁcation trend of the artiﬁcial fer- tiliser and the NO− 3 isotopes were distributed in between the denitriﬁcation ranges of both artiﬁcial fertiliser and SOM, suggesting the important contributions of these two sources during the wet periods. I f h d i i f h δ15N NO δ18O NO f Figure 7. Relationship between δ15N–NO− 3 of the dominant initial source (indicated by the y-intercept of the Keeling plots; Fig. 6) and percentage agriculture during wet periods. Data for the Bass-dry pe- riod were also presented because only the Keeling plot for the Bass- dry period indicates mixing between different sources. The shaded area represents the δ15N–NO− 3 of the potential end members. processes in our system (i.e. nitriﬁcation and/or anammox) in addition to denitriﬁcation. 4.3 Key controlling processes of nitrate during the wet periods Relationship between δ15N–NO− 3 and 1/[NO− 3 ] for individual streams during the wet and dry periods. Figure 6. Relationship between δ15N–NO− 3 and 1/[NO− 3 ] for individual streams during the wet and dry periods. Biogeosciences, 15, 3953–3965, 2018 www.biogeosciences.net/15/3953/2018/ Biogeosciences, 15, 3953–3965, 2018 www.biogeosciences.net/15/3953/2018/ www.biogeosciences.net/15/3953/2018/ 4.4 Key controlling processes of nitrate during the dry periods Unlike the wet periods, only NO− 3 in the Bass River showed an apparent relationship with δ15N–NO− 3 (Fig. 6) during the dry periods. There was no obvious relationships between δ15N–NO− 3 and 1/[NO− 3 ] for all other systems during the dry periods limiting the interpretation available from the Keeling plots. This also suggests that mixing between two end mem- bers alone is inadequate to explain the variability of δ15N– NO− 3 during the dry periods. In general, during the dry peri- In fact, the deviation of the δ15N–NO− 3 : δ18O–NO− 3 from the 1 : 1 trend to 2 : 1 corroborates the co-existence of other www.biogeosciences.net/15/3953/2018/ W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale Assimilation rather than denitriﬁcation was considered a more plausible process controlling the distribu- tion pattern for the group B dataset as the water column was oxic throughout the study period. Figure 9. Biplot of δ15N–NO− 3 vs. δ18O–NO− 3 for Bunyip and Toomuc (group B data in Fig. 8b). Shaded areas represent theo- retical assimilation trends for cow manure, SOM and inorganic fer- tiliser. The maximum and minimum starting values for δ18O–NO− 3 were estimated from Eq. (1). The starting δ15N–NO− 3 is the δ15N- TN value of respective end member. Solid and dotted lines represent the assimilation trends for Bunyip (both lower and upper Bunyip) and Toomuc, respectively. Assimilation rather than denitriﬁcation was considered a more plausible process controlling the distribu- tion pattern for the group B dataset as the water column was oxic throughout the study period. Figure 8. Biplot of δ15N–NO− 3 vs. δ18O–NO− 3 for (a) wet and (b) dry periods. The blue shaded area represents possible iso- topic compositions of denitriﬁed NO− 3 originated from SOM (δ15N: +4.5 ‰). The grey shaded area represents the possible isotopic composition of denitriﬁed NO− 3 originated from inorganic fer- tiliser (δ15N–NO− 3 : +0.1 ‰). The δ18O–NO− 3 used were −2.3 and ±0.23 ‰ representing the minimum and maximum estimates of δ18O of nitriﬁed NO− 3 , respectively. The shaded areas were plotted based on the theoretical 1 : 1 and 2 : 1 denitriﬁcation relationships between δ15N–NO− 3 and δ18O–NO− 3 (Kendall et al., 2007). group A dataset is indicative of nitriﬁed “aged” animal ma- nure. Because of the huge variability in the fractionation ef- fect of ammonia volatilisation, it is difﬁcult to afﬁx an aver- age δ15N value to represent the isotopic signature of this end member. As such, apportioning the relative contribution of nitriﬁed manure versus other sources (nitriﬁed organic mat- ter in the sediment and inorganic fertiliser) is not possible. g p NO− 3 in group B has variable δ15N and δ18O values as shown by Bunyip and Toomuc. This could be attributed to isotopic fractionation during plant and/or algae uptake of NO− 3 as substantiated by the parallel increase in δ18O–NO− 3 vs. δ15N–NO− 3 (Fig. 9). Denitriﬁcation was ruled out due to high levels of dissolved oxygen in the water column. Close convergence of the linear relationships onto the theoreti- cal assimilation trends of the nitriﬁed artiﬁcial fertiliser and SOM (Fig. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3962 0 2 4 6 8 10 12 14 0 10 20 30 (b) Dry period δ18O-NO3 - (‰) δ15N-NO3 - (‰) C B A (a) Wet period δ18O-NO3 - (‰) 0 2 4 6 8 10 0 5 10 15 20 Lower Bunyip Lang Lang Toomuc Watson Bass Upper Bunyip Denitrification of inorganic fertiliser (1:1) Denitrification of SOM (1:1) Denitrification of SOM (2:1) Denitrification of inorganic fertiliser (2:1) y = 0.3x + 2.3 r2 = 0.39, p < 0.01 Figure 8. Biplot of δ15N–NO− 3 vs. δ18O–NO− 3 for (a) wet and (b) dry periods. The blue shaded area represents possible iso- topic compositions of denitriﬁed NO− 3 originated from SOM (δ15N: +4.5 ‰). The grey shaded area represents the possible isotopic composition of denitriﬁed NO− 3 originated from inorganic fer- tiliser (δ15N–NO− 3 : +0.1 ‰). The δ18O–NO− 3 used were −2.3 and ±0.23 ‰ representing the minimum and maximum estimates of δ18O of nitriﬁed NO− 3 , respectively. The shaded areas were plotted based on the theoretical 1 : 1 and 2 : 1 denitriﬁcation relationships between δ15N–NO− 3 and δ18O–NO− 3 (Kendall et al., 2007). 0 2 4 6 8 10 12 14 0 10 20 30 (b) Dry period δ18O-NO3 - (‰) δ15N-NO3 - (‰) C B A (a) Wet period δ18O-NO3 - (‰) 0 2 4 6 8 10 0 5 10 15 20 Denitrification of inorganic fertiliser (1:1) Denitrification of SOM (1:1) Denitrification of SOM (2:1) Denitrification of inorganic fertiliser (2:1) y = 0.3x + 2.3 r2 = 0.39, p < 0.01 δ18O-NO3 - (‰) δ15N-NO3 - (‰) 0 2 4 6 8 10 12 2 4 6 8 10 12 14 16 18 Group B data Bunyip Toomuc Assimilation of SOM Assimilation of inorganic fertiliser Assimilation of organic fertiliser Slope = 0.75 r2 = 0.48, p<0.01 Slope = 0.78 r2 = 0.71, p<0.01 δ15N-NO3 - (‰) Figure 9. Biplot of δ15N–NO− 3 vs. δ18O–NO− 3 for Bunyip and Toomuc (group B data in Fig. 8b). Shaded areas represent theo- retical assimilation trends for cow manure, SOM and inorganic fer- tiliser. The maximum and minimum starting values for δ18O–NO− 3 were estimated from Eq. (1). The starting δ15N–NO− 3 is the δ15N- TN value of respective end member. Solid and dotted lines represent the assimilation trends for Bunyip (both lower and upper Bunyip) and Toomuc, respectively. www.biogeosciences.net/15/3953/2018/ www.biogeosciences.net/15/3953/2018/ Biogeosciences, 15, 3953–3965, 2018 Wong et al.: Nitrate isotopes in streams on a catchment scale δ18O-NO3 - (‰) δ15N-NO3 - (‰) 0 2 4 6 8 10 12 2 4 6 8 10 12 14 16 18 Group B data Bunyip Toomuc Assimilation of SOM Assimilation of inorganic fertiliser Assimilation of organic fertiliser Slope = 0.75 r2 = 0.48, p<0.01 Slope = 0.78 r2 = 0.71, p<0.01 Figure 9. Biplot of δ15N–NO− 3 vs. δ18O–NO− 3 for Bunyip and Toomuc (group B data in Fig. 8b). Shaded areas represent theo- retical assimilation trends for cow manure, SOM and inorganic fer- tiliser. The maximum and minimum starting values for δ18O–NO− 3 were estimated from Eq. (1). The starting δ15N–NO− 3 is the δ15N- TN value of respective end member. Solid and dotted lines represent the assimilation trends for Bunyip (both lower and upper Bunyip) and Toomuc, respectively. Assimilation rather than denitriﬁcation was considered a more plausible process controlling the distribu- tion pattern for the group B dataset as the water column was oxic throughout the study period. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 9) reiterate the dominant contribution of these sources to the riverine NO− 3 during the dry periods. It is worth noting that the initial δ18O of nitriﬁed NO− 3 was es- timated assuming full O isotopic equilibration between NO− 2 and H2O. Partial O isotope disequilibrium which was possi- ble could affect the initial δ18O signature of nitriﬁed NO− 3 . If this happened, the minimum estimate of δ18O of nitri- ﬁed NO− 3 could be more depleted and the overall linear re- lationship of δ18O–NO− 3 : δ15N–NO− 3 would shift, resulting in more obvious contribution of artiﬁcial fertiliser, SOM and possibly organic fertiliser (Fig. 9). This scenario emphasises the sensitivity of the initial δ18O of nitriﬁed NO− 3 in deter- mining the relative contribution of multiple sources in the catchment. ods, none of the samples show a noticeable pattern of denitri- ﬁcation on a biplot of δ18O vs. δ15N (Fig. 8b). The isotopic composition of the riverine NO− 3 appeared to be clustered into three groups (A, B and C in Fig. 8b). NO− 3 in group A showed consistent δ18O but variable δ15N. This is demonstrated by the Lang Lang and Bass, co- incident with the highest percentage of agriculture. The con- sistent δ18O (δ18O of ∼2.5 ‰) shows the importance of ni- triﬁcation (δ18O of ∼−2.03 to −0.23 ‰) and at the same time ruled out the occurrence of denitriﬁcation and assimila- tion. In the absence of the removal processes, the heavy and variable δ15N–NO− 3 values (+6 to +20 ‰) imply that ani- mal manure was an apparent source of NO− 3 during the dry periods for Lang Lang and Bass. This is because volatilisa- tion of 14N ammonia from the animal manure over time can lead to enrichment of 15N in the residual NH+ 4 to > +20 ‰ (Bateman and Kelly, 2007) which can subsequently be nitri- ﬁed to produce isotopically enriched NO− 3 without affecting its δ18O–NO− 3 . Tight coupling between mineralisation and nitriﬁcation results in NO− 3 retaining the isotopic signature of the residual NH+ 4 (Deutsch et al., 2009) in the manure. Hence, it is not surprising that δ15N–NO− 3 > +13 ‰ in the www.biogeosciences.net/15/3953/2018/ Biogeosciences, 15, 3953–3965, 2018 W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3963 Table 2. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale Comparison of NO− 3 concentrations and isotopes across different systems reported in the literature. Study area Percentage [NO− 3 ] δ15N–NO− 3 δ18O–NO− 3 Reference agriculture (µM) (‰) (‰) (%) Mississippi River basin, USA 0 to 100 3.6 to 1290 −1.4 to +12.3 +3.1 to +43.3 Chang et al. (2002) Connecticut River watershed, USA 0.8 to 52 0 to 360 0∗to +14.5 −2∗to +14 Barnes et al. (2010) New York, USA 0 to 72 5∗to 640 0∗to +9 −8∗to +40 Burns et al. (2009) Mid-Atlantic and New England states of the USA 2 to 38 7.9 to 184 +3.6 to +8.4 +11.7 to +18.5 Mayer et al. (2002) Baltic Sea catchment 1 to 81 3 to 216 −1.5 to +14 +10 to +25 Voss et al. (2006) Trout River catchment, Atlantic Canada ∼39.7 32 to 170 +2.13 to +6.35 +1.51 to +7.07 Danielescu and MacQuarrie (2013) Skuterud catchment, Norway 0 to 100 21 to 1850 +3 to +18 +10 to +24 Kaste et al. (2006) Mørdre catchment, Norway 74 to 100 120 to 2320 +8 to +15 +5 to +20 Kaste et al. (2006) Pearl river drainage basin ∼86 41 to 110 +1.9 to +17.6 +5.6 to +17.3 Chen et al. (2009) Western Port catchment, Australia 2 to 96 4 to 790 +5.7 to +33 +1.4 to +12.7 This study ∗Values estimated from presented ﬁgures might not accurately represent the actual data. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale 3963 ∗Values estimated from presented ﬁgures might not accurately represent the actual data. catchments. During the wet periods, inorganic fertiliser ap- peared to be the primary source of NO− 3 to the streams while SOM, in addition to inorganic fertiliser was also a domi- nant source of NO− 3 during the dry periods. Denitriﬁcation in the catchment appeared to be the more active removal pro- cess during the wet periods in contrast to a greater impor- tance of in-stream assimilation during the dry periods. How- ever, these removal processes were insufﬁcient to remove the agricultural-derived NO− 3 inferring that the streams were un- reactive conduits of NO− 3 which might pose a potential NO− 3 enrichment threat to downstream ecosystems. W. W. Wong et al.: Nitrate isotopes in streams on a catchment scale To the best of our knowledge, this is the ﬁrst study in Australia and also one of the very few targeted studies in the Southern Hemi- sphere investigating the origin and sink of NO− 3 on a catch- ment scale using both δ15N and δ18O of NO− 3 . The applica- tion of NO− 3 isotopes in a region with highly variable and un- predictable rainfall patterns such as the Western Port catch- ments, although challenging, is imperative particularly in set- ting guidelines for sustainable land use management actions. NO− 3 in group C comprised the most enriched δ15N and δ18O in the entire dataset (Fig. 8). These isotope values were observed in Watsons Creek which has the highest percent- age of market gardens. These samples were collected when the creek was not ﬂowing, hence the enriched δ15N and δ18O values could be indications of repeated cycles of internal pro- cesses (i.e. volatilisation, nitriﬁcation, denitriﬁcation and as- similation) in the same pool which enriched the N isotope but had slight effects on the O isotope of NO− 3 . 3 Although the isotope values during the dry periods ap- peared to be more likely controlled by artiﬁcial fertiliser and SOM, the contribution from organic fertiliser cannot be excluded. As mentioned in the preceding text, most of the fertiliser-derived NO− 3 was denitriﬁed in the catchment dur- ing the wet periods creating an artefact of heavy NO− 3 iso- topes in the streams. This NO− 3 could exhibit a similar en- riched isotopic composition as the volatilised manure (par- ticularly if there was disequilibrium of O isotope between NO− 2 and H2O). 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https://openalex.org/W4318977048	https://www.frontiersin.org/articles/10.3389/fpls.2023.1115874/pdf	English	null	Comprehensive analysis of cucumber RAV family genes and functional characterization of CsRAV1 in salt and ABA tolerance in cucumber	Frontiers in plant science	2,023	cc-by	9,838	TYPE Original Research PUBLISHED 02 February 2023 DOI 10.3389/fpls.2023.1115874 OPEN ACCESS Li J, Song C, Li H, Wang S, Hu L, Yin Y, Wang Z and He W (2023) Comprehensive analysis of cucumber RAV family genes and functional characterization of CsRAV1 in salt and ABA tolerance in cucumber. Front. Plant Sci. 14:1115874. doi: 10.3389/fpls.2023.1115874 COPYRIGHT © 2023 Li, Song, Li, Wang, Hu, Yin, Wang and He. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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No use, distribution or reproduction is permitted which does not comply with these terms. abiotic stresses, cucumber, expression patterns, RAV family, salt tolerance, transcription factor Introduction OPEN ACCESS EDITED BY Jihong Hu, Northwest A&F University, China REVIEWED BY Yongguang Li, Northeast Agricultural University, China Yong Zhou, Jiangxi Agricultural University, China CORRESPONDENCE Wenxing He 163.x@163.com Zenghui Wang 374141645@qq.com SPECIALTY SECTION This article was submitted to Plant Bioinformatics, a section of the journal Frontiers in Plant Science RECEIVED 04 December 2022 ACCEPTED 25 January 2023 PUBLISHED 02 February 2023 CITATION Li J, Song C, Li H, Wang S, Hu L, Yin Y, Wang Z and He W (2023) Comprehensive analysis of cucumber RAV family genes and functional characterization of CsRAV1 in salt and ABA tolerance in cucumber. Front. Plant Sci. 14:1115874. doi: 10.3389/fpls.2023.1115874 Jialin Li 1, Chunying Song 2, Hongmei Li 1, Siqi Wang 1, Linyue Hu 1, Yanlei Yin 3, Zenghui Wang 3 and Wenxing He 1* 1School of Biological Science and Technology, University of Jinan, Jinan, China, 2Xilin Gol League Agricultural and Animal Product Quality and Safety Monitoring Center, Xilinhot, China, 3Shandong Institute of Pomology, Tai’an, Shandong, China 1School of Biological Science and Technology, University of Jinan, Jinan, China, 2Xilin Gol League Agricultural and Animal Product Quality and Safety Monitoring Center, Xilinhot, China, 3Shandong Institute of Pomology, Tai’an, Shandong, China The RAV (related to ABI3 and VP1) transcription factors are speciﬁc and exist in plants, which contain a B3 DNA binding domain and/or an APETALA2 (AP2) DNA binding domain. RAVs have been extensively studied in plants, and more and more evidences show that RAVs are involved in various aspects of plant growth and development, stress resistance and hormone signal transduction. However, the systematic analysis of RAV family in cucumber is rarely reported. In this study, eight CsRAV genes were identiﬁed in cucumber genome and we further comprehensively analyzed their protein physicochemical properties, conserved domains, gene structure and phylogenetic relationships. The synteny analysis and gene duplications of CsRAV genes were also analysed. Cis-element analysis revealed that the CsRAVs promoter contained several elements related to plant hormones and abiotic stress. Expression analysis showed that NaCl and ABA could signiﬁcantly induce CsRAV genes expression. Subcellular localization revealed that all CsRAVs were localized in the nucleus. In addition, 35S:CsRAV1 transgenic Arabidopsis and cucumber seedlings enhanced NaCl and ABA tolerance, revealing CsRAV1 may be an important regulator of abiotic stress response. In conclusion, comprehensive analysis of CsRAVs would provide certain reference for understanding the evolution and function of the CsRAV genes. abiotic stresses, cucumber, expression patterns, RAV family, salt tolerance, transcription factor Frontiers in Plant Science Introduction Meanwhile, members of the RAV family can be induced by some plant hormones and have been shown to be involved in brassinosteroid and ethylene responses (Alonso et al., 2003; Hu et al., 2004). The expression of all soybean RAV genes increases dramatically under ABA treatment (Zhao et al., 2017). In cotton, GhRAV4, GhRAV9 and GhRAV20 genes are signiﬁcantly induced by BL, JA and IAA hormones (Kabir et al., 2021). Therefore, it clearly shows that members of the RAV gene family play important roles in the development and stress response of different plant species. Cucumber (Cucumis sativus L.) is one of the most important vegetable crops in the world (Huang et al., 2009). Despite the important roles of RAV genes in plant growth, development and stress tolerance, the functions of CsRAVs in cucumber are largely unknown. In this study, we identiﬁed eight CsRAV genes and classiﬁed them into four clades. The gene structures, conserved motifs, phylogenetic analysis, synteny analysis and gene duplications were further performed. In addition, the expression patterns of CsRAV genes under different abiotic stresses were also measured. Furthermore, overexpression of CsRAV1 increased salt tolerance and ABA resistance. These results lay a foundation for the evolutionary and functional characterization of RAVs in cucumber. Increasing evidence has shown that RAV transcription factors play key roles in a few aspects of plant growth and development. Arabidopsis RAV1 negatively regulates ﬂowering time, hypocotyl elongation, and seed development (Hu et al., 2004; Woo et al., 2010; Shin and Nam, 2018). Similarly, ectopic expression of soybean RAV1 in tobacco results in slow development and delayed ﬂowering time (Zhao et al., 2008; Lu et al., 2014). It was reported that members of RAV family TEMPRANILLO 1 and 2 (TEM1 and TEM2) negatively regulate ﬂowering time by inhibiting FLOWERINGLOCUS T (FT) expression and the production of gibberellins in Arabidopsis (Castillejo and Pelaz, 2008; Marı́n-González et al., 2015; Kabir et al., 2021). The NGATHA genes (AtNGA1-AtNGA4), members of the RAV family in Arabidopsis thaliana, have been shown to play key roles in lateral organs development. Over-expression of AtNGA1 to AtNGA4 present small and narrow leaf and ﬂower, whereas the nga1/ nga2/nga3/nga4 quadruple mutant reveals large and wide lateral organs (Alvarez et al., 2009; Lee et al., 2015). Genome-wide identiﬁcation of CsRAVs in cucumber To identify the CsRAV genes from cucumber (Chinese Long) v3 genome database (http://cucurbitgenomics.org/organism/20), 13 AtRAV proteins were used as query sequences and Blastp was used to search for the predicted cucumber proteins. All candidate genes were further conﬁrmed by the existence of B3 (PF02362.21) and/or AP2 (PF00847.20) domains using the Pfam and Simple Modular Architecture Research Tool (SMART) datebase (http://smart.embl- heidelberg.de). The RAV protein sequences in Arabidopsis and tomato were downloaded from the Arabidopsis Information Resource database (https://www.Arabidopsis.org) and the Solanaceae Genomics Network (https://solgenomics.net), respectively. It is also reported that RAV transcription factors are involved in biotic and abiotic stress responses, such as salt, drought, plant pathogens and plant hormones. The ectopic expression of soybean GmRAV3 in Arabidopsis thaliana can improve the resistance of transgenic lines to high salt and drought, and lead to the insensitivity of transgenic plants to exogenous ABA (Lu et al., 2014; Zhao et al., 2017). The OsRAV2 expression can be signiﬁcantly induced by salt in rice, indicating its important roles in salt response (Duan et al., 2016). Melon RAV1 (CmRAV1) can be induced by NaCl treatment and ectopic overexpression of CmRAV1 in Arabidopsis enhances salt tolerance at the seed germination and seedling stages (Zhao et al., 2019a). It has been reported that MeRAV1 and MeRAV2 play important roles in resistance to cassava bacterial Introduction Besides, overexpressing of Brassica rapa NGA1 (BrNGA1) in Arabidopsis thaliana gives rise to signiﬁcantly smaller and narrower lateral organs such as roots, ﬂowers, leaves and cotyledons due to a reduction in cell numbers compared to the wild type (Kwon et al., 2009). In rice, OsRAV9/OsTEM1 was identiﬁed as a negative regulator of ﬂoral transition. Moreover, the down-regulation of OsRAV11 gene was associated with ovary enlargement and seed weight increase (Osnato et al., 2020). The results of functional characterization of RAVs support the notion that RAVs are negative growth regulators in plant (Shin and Nam, 2018). Introduction The RELATED TO ABI3/VP1 (RAV) family belongs to one of the plant-speciﬁc B3 superfamily, which also contains three other families encompassing the auxin response factor (ARF) family, leafy cotyledon2 (lec2)-abscisic acid insensitive3 (abi3)-val (LAV) family, and reproductive meristem arf (REM) family (Swaminathan et al., 2008; Wang et al., 2012a). All Frontiers in Plant Science 01 frontiersin.org 10.3389/fpls.2023.1115874 Li et al. 10.3389/fpls.2023.1115874 members of the B3 superfamily contain a region of about 110 amino acids called the B3 domain, which is a DNA binding domain named because it is the third basic domain in the maize gene VIVIPAROUS1 (VP1) (Kagaya et al., 1999). Most B3 genes in the ARF and LAV families have been extensively studied, but B3 genes in the RAV family are rarely unknown. The RAV proteins contain B3 domain and/or AP2 (APETALA2) domain (Lu et al., 2014). Therefore, RAV family members can reasonably be classiﬁed as B3 superfamily members or AP2/EREBP family members (Matı́as-Hernández et al., 2014). In Arabidopsis, there are 13 RAV genes, of which seven contain only the B3 domain and the other six contain both B3 and AP2 domains (Magnani et al., 2004; Romanel et al., 2009; Fu et al., 2014). Of these, AtRAV1 and AtRAV2, containing both B3 domain and AP2 domain, were the earliest discovered members of the RAV family (Kagaya et al., 1999). blight through activation of melatonin biosynthesis genes in cassava (Wei et al., 2018). In addition, over-expression of tomato RAV2 (SlRAV2) gives rise to increased bacterial wilt (BW) tolerance, whereas knockdown SlRAV2 signiﬁcantly decreases tomato resistance to BW (Li et al., 2011). Meanwhile, members of the RAV family can be induced by some plant hormones and have been shown to be involved in brassinosteroid and ethylene responses (Alonso et al., 2003; Hu et al., 2004). The expression of all soybean RAV genes increases dramatically under ABA treatment (Zhao et al., 2017). In cotton, GhRAV4, GhRAV9 and GhRAV20 genes are signiﬁcantly induced by BL, JA and IAA hormones (Kabir et al., 2021). Therefore, it clearly shows that members of the RAV gene family play important roles in the development and stress response of different plant species. blight through activation of melatonin biosynthesis genes in cassava (Wei et al., 2018). In addition, over-expression of tomato RAV2 (SlRAV2) gives rise to increased bacterial wilt (BW) tolerance, whereas knockdown SlRAV2 signiﬁcantly decreases tomato resistance to BW (Li et al., 2011). Frontiers in Plant Science Tolerance of transgenic plants to abiotic stress The seeds of 35S:CsRAV1 T3-generation homozygous lines and WT were seeded in vermiculite soil and cultured at 22°C for 3 weeks under normal conditions. For salt treatment, the 3-week-old seedlings were irrigated with 200 mM NaCl solution every two days, and the growth of different lines was observed every three days. Under ABA treatment, the transgenic lines and WT were watered 100 mM ABA solution every two days, respectively, and phenotypic evaluation was performed every three days. To investigate the seed germination rate of transgenic and WT seeds under salt stress and ABA treatment, the seeds of transgenic lines and WT were sown on 1/2 MS medium containing 100 mM NaCl or 2 mM ABA, respectively, and cultured under normal conditions. Germination rate was measured after 7 days of culture. The cotyledons of 8-d-old transgenic cucumber seedlings with transient transformation of 35S and 35S:CsRAV1 were subjected to salt and ABA tolerance by hydroponic method. Transgenic cucumber seedlings with equivalent growth were selected and transferred to 2 L Hoagland nutrient solution for hydroponics. After two days of hydroponics, NaCl and ABA were added into the nutrient solution, and the ﬁnal concentrations of NaCl and ABA in nutrient solution were 100 mM and 100 mM, respectively. To obtain accurate experimental results, the cucumber seedlings transfected with 35S and 35S:CsRAV1 were cultured in the same tank. The Analysis of CsRAV gene expression under different abiotic stresses replicates; random seed) (Li et al., 2022). The corresponding cDNA and DNA sequences of RAV genes were downloaded from corresponding genomes, and the gene structures were analyzed as described by Li et al. (2022). The conserved motifs in RAVs were identiﬁed using Multiple Expectation Maximization for Motif Elicitation (MEME) online program (http://meme-suite.org/index. html). CsRAV genes were classiﬁed according to its phylogenetic relationship with RAVs in other species. The visual evolutionary tree, conserved motif and gene structure maps were completed using TBtools (Li et al., 2020). The cucumber inbred line (Xintaimici) was used as materials to undergo stress treatments and transient transformation. The leaves of vigorous two-week-old cucumber seedlings were sprayed with 100 mM NaCl and 100 mM ABA, respectively. Leaves were taken at 0, 0.5, 1, 3, 6, 9, 12 and 24h for quantitative analysis. The two-week-old cucumber seedlings were cultured in a 4°C incubator with 16 h light/ 8 h dark for low temperature treatment, and sampled at the same time point for storage. Each sample was taken from six plants and each treatment had three biological replicates. Cis-element analysis on CsRAVs promoter in cucumber The relevant data of cucumber genome were downloaded from cucumber genome database (Chinese Long 9930: http:// cucurbitgenomics.org/), and we used TBtools to extract the 2 kb sequence of CsRAVs gene promoter. The cis-elements on the promoter regions of CsRAV genes were analysed using PlantCARE website (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Li et al., 2020). Transient transformation of cucumber cotyledons All CsRAVs were mapped to cucumber chromosomes based on physical location information from the database of cucumber genome using Circos (Krzywinski et al., 2009). Multiple Collinearity Scan toolkit (MCScanX) was used to analyze the gene duplication events, with the default parameters (Wang et al., 2012b). To show the synteny relationship of the orthologous RAVs in cucumber, Arabidopsis and tomato, we used TBtools to constructed the syntenic analysis maps (https://github.com/CJ-Chen/TBtools) (Chen et al., 2020). The coding sequence of CsRAV1 was recombined into the pCAMBIA1300 vector (universal vector: stored in our laboratory), which was then transformed into Agrobacterium tumefaciens LBA4404. The A. tumefaciens LBA4404 cells containing the recombinant vector was incubated in liquid medium overnight until the optical density (600 nm) was about 0.6-0.8. Then the Agrobacterium solution was centrifuged at 8000 × g for three min, and re-suspended with MES solution (consisting of 10 mM MES, 10 mM MgCl2 and 200 mM Acetosyringone) to OD600 value of 0.6-0.8. The suspensions containing the target gene were injected into the cotyledons of 6-7-d-old cucumber seedlings with a 1ml disposable syringe, and cultured in the dark at 20°C for one day, then the follow- up related treatment experiments were conducted. Phylogenetic analysis, conserved motif and gene structure analysis A phylogenetic tree containing 115 RAV protein sequences from ten species and a phylogenetic tree with the full-length amino acid sequences of nine SlRAVs, 13 AtRAVs and eight CsRAVs were constructed using MEGA 7.0 respectively and the neighbour- joining (NJ) method was used with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 Frontiers in Plant Science 02 frontiersin.org Li et al. 10.3389/fpls.2023.1115874 Vector construction, transient transformation of cucumber cotyledons and Arabidopsis transformation The coding sequence of CsRAVs was recombined into pCAMBIA1300 vector with a GFP tag to obtain 35S:CsRAVs-GFP. The 35S:CsRAV1-GFP construct was transformed into Agrobacterium tumefaciens LBA4404, and then transferred into Arabidopsis (Col-0) or 8-d-old cucumber cotyledons (Li et al., 2020). The homozygous T3 transgenic Arabidopsis lines were screened and identiﬁed for subsequent experiments. The primers used are listed in Supplementary Table 5. Frontiers in Plant Science Identiﬁcation and analysis of RAV Genes in cucumber To identify the putative CsRAV family genes in cucumber genome, BlastP was used to search against cucumber genome database based on 13 Arabidopsis RAV proteins and consensus protein sequences of B3 (PF02362) and AP2 (PF00847). A total of eight CsRAVs were identiﬁed in the cucumber genome and the presence of the B3 and/or AP2 domains was also conﬁrmed by Pfam and SMART. The identiﬁed CsRAVs were named CsRAV1 to CsRAV8 according to the similarities of CsRAVs with their characterized counterparts in Arabidopsis. The information of the CsRAVs, including the gene ID, gene name, location, molecular weight, amino acid length and isoelectric points (pI) was listed in Table 1. These eight CsRAV genes were distributed on chromosome 1, 3, 4, 5 and 6 of cucumber, respectively (Supplementary Figure 1). Except for CsRAV7 and CsRAV8, the pI of the other CsRAV proteins were all greater than 7, indicating that these proteins were basic proteins (Table 1). In order to support the phylogenetic results, the gene structure of RAVs from tomato, Arabidopsis and cucumber were analyzed (Figure 2C). The number of exons in SlRAV, AtRAV and CsRAV genes was conserved, ranging from one to three exons. As shown in Figure 2C, the gene structures of RAVs in the same clade were highly conserved in all three species. For example, all RAVs in Clade I contained only one exon and all RAVs in clade II, except AtRAV-like2, contained three exons. In Clade III and Clade IV, the genes had one or two exons (Figure 2C). To further analyze the structural diversity and predict the function of the RAV proteins, the number and composition of conservative motifs in the SlRAVs, AtRAVs and CsRAVs were analyzed by MEME (Figure 2B; Supplementary Figure 2). We analyzed 10 different motifs named Motif1-Motif10 (Supplementary Figure 2). Motifs 1 and 2, which is representative B3 domain (PF02362), were identiﬁed in all RAVs, and motifs 3 and 4, which are representative AP2 domain (PF00847), were identiﬁed in some RAV proteins, including all the RAVs in Clade I and Clade IV except SlRAV5. Some of the speciﬁc motifs were absent in speciﬁc clade (Figure 2B). For example, motif 6 was absent in all the member of the Clade IV subfamily, which further corroborates the accuracy of subfamily division. Motif 9 was only identiﬁed in Clade I (Figure 2B). Identiﬁcation and analysis of RAV Genes in cucumber Therefore, the functions of these motifs in relation to the functions of these proteins need to be investigated further. Subcellular localization of CsRAVs Tobacco leaf epidermal cells were injected with the empty GFP vector and the 35S: CsRAVs-GFP recombinant plasmids, respectively. Then the injected tobaccos were grown under normal conditions for about 48 h, the subcellular localization of CsRAVs were determined by observing the ﬂuorescence signal under a ﬂuorescence microscope. 03 frontiersin.org Li et al. 10.3389/fpls.2023.1115874 Li et al. tree, conserved motifs and gene structures of eight CsRAVs, 13 AtRAVs and nine SlRAVs were further analyzed in detail (Figure 2). As shown in Figure 2A, the phylogenetic tree divided these RAV proteins into four clades, which CsRAV1 and CsRAV2 were classiﬁed into Clade I, CsRAV5 belonged to Clade II, CsRAV6, CsRAV7 and CsRAV8 belonged to Clade III, and CsRAV3 and CsRAV4 were classiﬁed into Clade IV. Each clade contained Arabidopsis, cucumber, and tomato RAV genes, suggesting that proteins in the same clades might have similar functions. For example, CsRAV1 and CsRAV2 were simultaneously classiﬁed into Clade I with RAV1 and RAV2 of Arabidopsis and tomato, implying that CsRAV1 and CsRAV2 might regulate plant growth and improve plant tolerance (Figure 2A). tree, conserved motifs and gene structures of eight CsRAVs, 13 AtRAVs and nine SlRAVs were further analyzed in detail (Figure 2). As shown in Figure 2A, the phylogenetic tree divided these RAV proteins into four clades, which CsRAV1 and CsRAV2 were classiﬁed into Clade I, CsRAV5 belonged to Clade II, CsRAV6, CsRAV7 and CsRAV8 belonged to Clade III, and CsRAV3 and CsRAV4 were classiﬁed into Clade IV. Each clade contained Arabidopsis, cucumber, and tomato RAV genes, suggesting that proteins in the same clades might have similar functions. For example, CsRAV1 and CsRAV2 were simultaneously classiﬁed into Clade I with RAV1 and RAV2 of Arabidopsis and tomato, implying that CsRAV1 and CsRAV2 might regulate plant growth and improve plant tolerance (Figure 2A). phenotypes of transgenic seedlings and control seedlings were observed at different periods (Li et al., 2020). Frontiers in Plant Science frontiersin.org 04 Phylogenetic analysis, conserved motif and gene structure analysis of RAV gene family in cucumber, tomato and Arabidopsis To explore the homology of RAV genes among different plant species, RAV genes of ten species were selected to construct a phylogenetic tree based on amino acid sequences (Figure 1). As could be seen from Figure 1, the 115 RAV proteins could be roughly divided into 23 groups, among which CsRAV genes were classiﬁed into different groups, such as CsRAV1 was classiﬁed in group 5, CsRAV2 were classiﬁed in group3, CsRAV3 and CsRAV4 were classiﬁed in group 22, CsRAV6 and CsRAV7 were classiﬁed in group 16, and CsRAV8 was classiﬁed in group 19 (Figure 1; Supplementary Table 1). To better assess the phylogenetic relationships of the cucumber RAV proteins, the phylogenetic In general, RAV genes with close evolutionary relationships in the phylogenetic tree contained similar conserved motifs and gene structures, suggesting that each subfamily in the three different species was evolutionarily conserved. TABLE 1 Information of RAV genes in cucumber. Gene ID Gene name Location Molecular weight (kD) Amino acid length (aa) pI CsaV3_5G023010 CsRAV1 Chr5:17061938-17063609 35.6 317 9.30 CsaV3_5G034010 CsRAV2 Chr5:27134879-27137672 38.2 344 9.18 CsaV3_1G042340 CsRAV3 Chr1:27284528-27286805 41.5 356 8.11 CsaV3_1G028810 CsRAV4 Chr1:15745012-15746031 38.8 339 8.93 CsaV3_3G037570 CsRAV5 Chr3:30990269-30994595 32.5 296 8.44 CsaV3_6G008410 CsRAV6 Chr6:6759884-6760601 21.7 184 9.93 CsaV3_5G001410 CsRAV7 Chr5:765460-770215 44.5 399 6.96 CsaV3_4G005920 CsRAV8 Chr4:3907865-3912965 43.9 384 6.40 TABLE 1 Information of RAV genes in cucumber. 04 Li et al. 10.3389/fpls.2023.1115874 FIGURE 1 Phylogenetic relationships of 115 RAV proteins from Cucumis sativus, Arabidopsis thaliana, Solanum lycopersicum, Glycine max, Oryza sativa, Triticum aestivum, Zea mays, Mangifera indica, Pyrus bretschneideri and Nicotiana tabacum. The MEGA 7.0 software was used to construct the phylogenetic tree of 115 RAVs with complete amino acid sequences. The CsRAV proteins were marked with red stars. FIGURE 1 Phylogenetic relationships of 115 RAV proteins from Cucumis sativus, Arabidopsis thaliana, Solanum lycopersicum, Glycine max, Oryza sativa, Triticum aestivum, Zea mays, Mangifera indica, Pyrus bretschneideri and Nicotiana tabacum. The MEGA 7.0 software was used to construct the phylogenetic tree of 115 RAVs with complete amino acid sequences. The CsRAV proteins were marked with red stars. Synteny analysis of RAV genes in cucumber, tomato and Arabidpsis between tomato and Arabidopsis (Supplementary Table 3). These results indicated that these orthologous pairs might already exist before the ancestral divergence. In addition, we found the rest of the CsRAV genes (CsRAV1, CsRAV3, CsRAV4, CsRAV6, CsRAV7 and CsRAV8) were not associated with syntenic gene pairs in Arabidopsis or tomato, suggesting that they might be unique to cucumber during the evolutionary process. Gene duplication is one of the main driving forces for the evolution of genomes and genetic systems (Das et al., 2020). Segmental duplication and tandem duplication are considered to be the two main reasons for the expansion of plant gene families (Cannon et al., 2004). To reveal the duplication of CsRAVs, the syntenic regions were analysed using MCscanX software. In cucumber genome, there were 231 segmental duplication blocks and 1468 tandem duplication gene pairs in all (Supplementary Table 2). The analysis revealed that there was one segmental duplication gene pair (CsRAV7 and CsRAV8) in cucumber RAV gene family, but no tandem duplication gene pairs (Figure 3A). Frontiers in Plant Science Responses of CsRAV genes to different abiotic stresses Abiotic stresses are known to affect many physiological processes. To explore the response of CsRAV genes to different stresses, qRT- PCR was used to analyze the expression patterns of these genes under low temperature (4°C), ABA treatment (100 mΜ ABA) and salt stress (100 mM NaCl), respectively (Figure 5). Under salt stress, CsRAV1 and CsRAV2 genes were signiﬁcantly up-regulated (>15-fold) after 0.5 h. Two genes, CsRAV6 and CsRAV8, were slightly upregulated, while others were obviously down-regulated (Figure 5A). Under ABA treatment, the expression levels of all CsRAV genes were markedly induced (>7-fold). For example, the expression levels of CsRAV5 and CsRAV8 were more than 30 times higher. In particular, the expression patterns of CsRAV1 and CsRAV2 genes showed a similar trend and the expression levels of these two genes were up-regulated 60 times Subcellular localization of cucumber RAVs To conﬁrm the subcellular localization of CsRAV proteins, the GFP fusion protein vectors 35S:CsRAVs-GFP of eight CsRAV genes were constructed, respectively. The 35S:GFP vector was used as empty control. The green ﬂuorescent signal of GFP expression in epidermal cells of tobacco leaves was observed after transformation. Under confocal laser scanning microscope, the different CsRAV fusion proteins were all localized in the nucleus, while 35S:GFP ﬂuorescence signal was distributed throughout the whole cell (Figure 6). The data showed that all CsRAV proteins were nuclear localization proteins, suggesting that these CsRAVs might be involved in transcriptional regulation. FIGURE 2 FIGURE 2 The analysis of phylogenetic relationships, conserved motifs and gene structures in RAV genes from cucumber, Arabidopsis and tomato. (A) A phylogenetic tree containing nine SlRAVs, 13 AtRAVs and eight CsRAVs was constructed by MEGA 7.0 software and divided into four clades named I, II, III and IV, respectively. (B) The conserved motifs in RAVs were identiﬁed using MEME online program. Ten conserved sequences were presented in Supplementary Figure S2. (C) Exon-intron structure of RAV genes. after 0.5 h (Figure 5B). After low temperature treatment, all CsRAV genes were up-regulated except CsRAV4 and CsRAV6, and CsRAV1 and CsRAV2 were up-regulated most signiﬁcantly. CsRAV1 reached maximum values at 1 h, while the expression levels of CsRAV2 reached its maximum at 6 h (Figure 5C). These results suggested that some CsRAV genes were involved in low temperature, salt and ABA responses. involved in regulating plant growth and development were present on promoters of certain CsRAV genes, for example, meristem expression, metabolism regulation and anaerobic induction (Figure 4). Furthermore, the core elements including transcription start site (TSS) and TATA-box of eight CsRAV genes were predicted using TSSPlant online software. The results showed that only four transcription start sites were predicted for CsRAV3 and no TATA- box, while three core elements were predicted for CsRAV5 and CsRAV7 and two core elements were predicted for all the other ﬁve genes (Supplementary Figure 3). In conclusion, the analyses of cis- elements suggested that CsRAVs might play vital functions in abiotic stress responses and multiple plant hormone signaling pathways. Frontiers in Plant Science Cis-element analysis of CsRAVs promoter in cucumber Cis-acting elements on promoters are non-coding DNA sequences that play key roles in gene expression and mutation. To explore the potential function of CsRAV genes in cucumber, the putative cis-elements on the 2-kb promoter regions of CsRAV were analyzed by PlantCARE (Supplementary Table 4). As shown in Figure 4, the cis-elements associated with abiotic stress such as low temperature, drought, wound, defense and stress existed on the promoter of CsRAV genes. Moreover, cis-elements that respond to plant hormones were also found on the promoter regions of CsRAV genes, such as auxin, gibberellin (GA), abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (MeJA). In addition, some cis-elements To further elucidate the phylogenetic mechanism of the cucumber RAV family, a comparison of the syntenic map of cucumber connected with tomato and Arabidopsis, was constructed (Figure 3B; Supplementary Table 3). Interestingly, only two CsRAV genes in cucumber were collinear with RAV genes in tomato and Arabidopsis. As shown in Figure 3, CsRAV2 gene (CsaV3_5G034010) showed syntenic relationship with At1G25560.1 gene in Arabidopsis, Solyc04g007000.1.1 and Solyc05g009790.1.1 genes in tomato. Similarly, CsRAV5 gene also corresponded to syntenic gene pairs 05 frontiersin.org Li et al. 10.3389/fpls.2023.1115874 B C C A B B C C FIGURE 2 The analysis of phylogenetic relationships, conserved motifs and gene structures in RAV genes from cucumber, Arabidopsis and tomato. (A) A phylogenetic tree containing nine SlRAVs, 13 AtRAVs and eight CsRAVs was constructed by MEGA 7.0 software and divided into four clades named I, II, III and IV, respectively. (B) The conserved motifs in RAVs were identiﬁed using MEME online program. Ten conserved sequences were presented in Supplementary Figure S2. (C) Exon-intron structure of RAV genes. C B A Overexpression of CsRAV1 improved cucumber and transgenic Arabidopsis tolerance to NaCl and ABA The expression analysis showed that CsRAV1 was signiﬁcantly induced by NaCl and ABA in cucumber (Figures 5A, B). To Frontiers in Plant Science 06 frontiersin.org Li et al. 10.3389/fpls.2023.1115874 A B FIGURE 3 Gene duplication and synteny analysis of CsRAV genes. (A) Chromosome distribution and interchromosome relationships of CsRAV genes. The red line indicated segmental duplication gene pair. (B) Synteny analysis of RAV genes from cucumber, tomato and Arabidopsis. Gray lines represented the collinear blocks between cucumber and tomato or Arabidopsis; Blue lines represented the collinear blocks of RAV genes between cucumber and tomato or Arabidopsis. A B Gene duplication and synteny analysis of CsRAV genes. (A) Chromosome distribution and interchromosome relationships of CsRAV genes. The red line indicated segmental duplication gene pair. (B) Synteny analysis of RAV genes from cucumber, tomato and Arabidopsis. Gray lines represented the collinear blocks between cucumber and tomato or Arabidopsis; Blue lines represented the collinear blocks of RAV genes between cucumber and tomato or Arabidopsis. determine the function of CsRAV1 in abiotic stress response, ﬁrst, agrobacterium-mediated transient transformation of cucumber cotyledons was used to elucidate the tolerance of CsRAV1 to NaCl and ABA. In the preliminary experiments, the transient transfection of CsRAV1 was successfully veriﬁed by PCR ampliﬁcation, and the expression levels of CsRAV1 in transgenic cucumber cotyledons were observably higher than that in 35S plants by qRT-PCR (Supplementary Figure 4). When cultured in a hydroponic solution containing 100 mM NaCl, the control seedlings (overexpressing 35S empty vector) showed signiﬁcant wilting after 12 h compared with overexpressed CsRAV1 transgenic seedlings, and the difference in wilting degree was more obvious after 24 h (Figure 7A). After 24 h of NaCl treatment, the survival rate of transgenic seedlings was 44%, which was signiﬁcantly higher than that of control, indicating that the overexpression of CsRAV1 produced signiﬁcant salt tolerance (Figure 7C). Similarly, in the presence of exogenous ABA, the growth of both control and 35S:CsRAV1 seedlings was distinctly harmed, but the damage of control was much greater than that of transgenic seedlings. After 24 h of ABA treatment, about 46% of the transgenic seedlings remained green with extended cotyledons, while FIGURE 4 The promoter analysis of the CsRAV genes in cucumber. The potential cis-elements on the 2-kb promoter regions upstream of CsRAV genes were analysed using PlantCARE. Different colored boxes represented the corresponding cis-elements. FIGURE 4 The promoter analysis of the CsRAV genes in cucumber. Overexpression of CsRAV1 improved cucumber and transgenic Arabidopsis tolerance to NaCl and ABA The potential cis-elements on the 2-kb promoter regions upstream of CsRAV genes were analysed using PlantCARE. Different colored boxes represented the corresponding cis-elements. 07 Frontiers in Plant Science frontiersin.org Li et al. 10.3389/fpls.2023.1115874 A B C FIGURE 5 Relative expression of CsRAV genes in cucumber seedlings treated with NaCl, ABA and low temperature. QRT-PCR was used to analyze the expression levels of CsRAVs under NaCl (100 mM) (A), ABA (100 mM) (B) and 4°C (C). Using cucumber b-actin gene as an internal control, three biological replicates were used to analyze gene expression. Error bars were the standard errors (SE). Different lowercase letters represented signiﬁcant differences (P < 0.05). B FIGURE 5 Relative expression of CsRAV genes in cucumber seedlings treated with NaCl, ABA and low temperature. QRT-PCR was used to analyze the expression levels of CsRAVs under NaCl (100 mM) (A), ABA (100 mM) (B) and 4°C (C). Using cucumber b-actin gene as an internal control, three biological replicates were used to analyze gene expression. Error bars were the standard errors (SE). Different lowercase letters represented signiﬁcant differences (P < 0.05). overexpressing CsRAV1 and two highly expressed homozygous transgenic lines (L1 and L2) were selected for further analysis (Supplementary Figure 5). The NaCl and ABA tolerance of CsRAV1 transgenic plants was evaluated. For germination assays, the WT, L1 and L2 on 1/2 MS medium showed similar germination rate (Figure 8A). However, the germination of both WT and 35S: CsRAV1 transgenic seeds was signiﬁcantly inhibited on 1/2 MS medium containing 100 mM NaCl or 2 mM ABA, but the inhibition of the WT was much higher than that of the transgenic seeds. With 100 mM NaCl or 2 mM ABA treatment, nearly 40-50% of CsRAV1 transgenic seeds were able to germinate, while only about 10- 12% of the WT seeds could germinate (Figures 8A, B). The results showed that overexpressing CsRAV1 could remarkably improve seed germination rate under salt and ABA treatment. In addition, we determined the seedling growth of 3-week-old WT and CsRAV1 transgenic lines under 200 mM NaCl and 100mM ABA treatment, respectively. After four days of treatment with 200 mM NaCl or 100mM ABA, the leaves of CsRAV1 transgenic lines were still green, but the leaves of WT were severely yellowed (Figures 8C, D). Frontiers in Plant Science Overexpression of CsRAV1 improved cucumber and transgenic Arabidopsis tolerance to NaCl and ABA After eight days, the CsRAV1 transgenic lines and WT had more obvious differences in NaCl and ABA resistance, indicating that CsRAV1 89% of the control showed obvious injury symptoms, such as wilting and death (Figures 7B, C). To elucidate the underlying regulatory effect of CsRAV1 on tolerance to NaCl and ABA stress, the expression levels of four stress- and ABA- related marker genes were detected by qRT-PCR in 35S and 35S:CsRAV1 cucumber seedlings before and after NaCl and ABA treatments. As shown in Figures 7D–G, only CsCPK11 was slightly higher expressed in 35S:CsRAV1 transgenic plants before NaCl and ABA treatments. However, after NaCl and ABA treatments, all the four genes, CsSOS1 (Salt overly Sensitive 1), CsNHX1 (vacuolar sodium/proton antiporter), CsCPK11 (calcium- dependent protein kinase) and CsABI5 (ABA-insensitive factor 5) were strongly induced in 35S and 35S:CsRAV1 plants. Furthermore, the expression levels of the four genes were signiﬁcantly higher in 35S: CsRAV1 transgenic plants than those in 35S plants after NaCl and ABA stress treatment. The enhanced expression of these genes in 35S: CsRAV1 transgenic plants might conduce to improve plant stress resistance, which also suggested that CsRAV1 may be involved in response to NaCl and ABA stress by inﬂuencing the transcription of multiple stress-related genes. To further study the function of CsRAV1 in plant resistance to abiotic stress, we obtained transgenic Arabidopsis plants 08 frontiersin.org Li et al. 10.3389/fpls.2023.1115874 FIGURE 6 Subcellular localization of the cucumber RAV proteins in tobacco leaf. The green ﬂuorescence signal was observed under confocal microscope 48 h after transformation. A B D E F G C FIGURE 7 Overexpression of CsRAV1 improved NaCl and ABA tolerance in cucumber seedlings. (A, B) Phenotypes of 35S and 35S:CsRAV1 cucumber seedlings treated with NaCl and ABA under hydroponics. (C) Survival rate of 35S and 35S:CsRAV1 plants after 24 h NaCl and ABA treatments. The transcript levels of CsSOS1 (D), CsNHX1 (E), CsCPK11 (F), and CsABI5 (G) genes in 35S and 35S:CsRAV1 cucumber seedlings were analyzed with qRT-PCR under NaCl and ABA treatment for 6 h. The cucumber b-actin gene was used as internal control. Error bars were the standard errors (SE). Different letters indicated FIGURE 6 Subcellular localization of the cucumber RAV proteins in tobacco leaf. The green ﬂuorescence signal was observed under confocal microscope 48 h after transformation. FIGURE 6 Subcellular localization of the cucumber RAV proteins in tobacco leaf. Overexpression of CsRAV1 improved cucumber and transgenic Arabidopsis tolerance to NaCl and ABA The green ﬂuorescence signal was observed under confocal microscope 48 h after transformation. A B D E F G C FIGURE 7 Overexpression of CsRAV1 improved NaCl and ABA tolerance in cucumber seedlings. (A, B) Phenotypes of 35S and 35S:CsRAV1 cucumber seedlings treated with NaCl and ABA under hydroponics. (C) Survival rate of 35S and 35S:CsRAV1 plants after 24 h NaCl and ABA treatments. The transcript levels of CsSOS1 (D), CsNHX1 (E), CsCPK11 (F), and CsABI5 (G) genes in 35S and 35S:CsRAV1 cucumber seedlings were analyzed with qRT-PCR under NaCl and ABA treatment for 6 h. The cucumber b-actin gene was used as internal control. Error bars were the standard errors (SE). Different letters indicated signiﬁcant differences (P < 0.05). A D B FIGURE 7 Overexpression of CsRAV1 improved NaCl and ABA tolerance in cucumber seedlings. (A, B) Phenotypes of 35S and 35S:CsRAV1 cucumber seedlings treated with NaCl and ABA under hydroponics. (C) Survival rate of 35S and 35S:CsRAV1 plants after 24 h NaCl and ABA treatments. The transcript levels of CsSOS1 (D), CsNHX1 (E), CsCPK11 (F), and CsABI5 (G) genes in 35S and 35S:CsRAV1 cucumber seedlings were analyzed with qRT-PCR under NaCl and ABA treatment for 6 h. The cucumber b-actin gene was used as internal control. Error bars were the standard errors (SE). Different letters indicated signiﬁcant differences (P < 0.05). 09 Frontiers in Plant Science frontiersin.org 10.3389/fpls.2023.1115874 Li et al. A B D E F G H C FIGURE 8 CsRAV1 enhanced NaCl and ABA tolerance in transgenic Arabidopsis. (A) Seed germination of WT and 35S:CsRAV1 plants (L1 and L2) after 7 d on 1/2 MS with NaCl and ABA. (B) Seed germination rate in (A). Different letters indicated signiﬁcant differences (P < 0.05). (C, D) Phenotypes of WT and 35S:CsRAV1 transgenic plants treated with NaCl and ABA. After 6 h of NaCl and ABA stress treatment, the expression levels of stress- and ABA- related marker genes AtRD29A (E), AtSOS1 (F), AtNHX1 (G), and AtABI5 (H) were detected in WT and 35S:CsRAV1 plants (L1 and L2). The actin gene was used as internal control, with error bars of three biological replicates. Different letters indicated signiﬁcant differences (P < 0.05). A B B D C C D E F F E G H H G FIGURE 8 CsRAV1 enhanced NaCl and ABA tolerance in transgenic Arabidopsis. Overexpression of CsRAV1 improved cucumber and transgenic Arabidopsis tolerance to NaCl and ABA (A) Seed germination of WT and 35S:CsRAV1 plants (L1 and L2) after 7 d on 1/2 MS with NaCl and ABA. (B) Seed germination rate in (A). Different letters indicated signiﬁcant differences (P < 0.05). (C, D) Phenotypes of WT and 35S:CsRAV1 transgenic plants treated with NaCl and ABA. After 6 h of NaCl and ABA stress treatment, the expression levels of stress- and ABA- related marker genes AtRD29A (E), AtSOS1 (F), AtNHX1 (G), and AtABI5 (H) were detected in WT and 35S:CsRAV1 plants (L1 and L2). The actin gene was used as internal control, with error bars of three biological replicates. Different letters indicated signiﬁcant differences (P < 0.05). transgenic plants were more tolerant to NaCl and ABA stresses than WT. Following NaCl and ABA stress treatment, the expression levels of stress- and ABA- related marker genes were signiﬁcantly up- regulated in transgenic lines (L1 and L2) and WT, but the induction levels of AtRD29A (response to desiccation 29A), AtSOS1, AtNHX1 and AtABI5 genes were markedly higher in L1 and L2 lines than in WT (Figures 8E–H). In conclusion, these results indicated that CsRAV1 might play key roles in ABA signaling and in plant response to high salinity during seed germination and seedling development. transgenic plants were more tolerant to NaCl and ABA stresses than WT. Following NaCl and ABA stress treatment, the expression levels of stress- and ABA- related marker genes were signiﬁcantly up- regulated in transgenic lines (L1 and L2) and WT, but the induction levels of AtRD29A (response to desiccation 29A), AtSOS1, AtNHX1 and AtABI5 genes were markedly higher in L1 and L2 lines than in WT (Figures 8E–H). In conclusion, these results indicated that CsRAV1 might play key roles in ABA signaling and in plant response to high salinity during seed germination and seedling development. Discussion To analyze the molecular evolutionary relationship between cucumber RAV proteins and RAVs among other plant species, a phylogenetic tree of 115 RAV proteins from ten species was constructed, and roughly divided into 23 groups, among which CsRAVs were classiﬁed into different groups (Figure 1). To further study the evolutionary relationship and diversity/conservativeness of RAV genes in tomato, Arabidopsis and cucumber, the phylogenetic tree, conserved motifs and gene structures of eight CsRAVs, 13 AtRAVs and nine SlRAVs were further analyzed in detail (Figure 2). The gene structure pattern and motif composition can give important insights for evolutionary relationships of multi-gene families (Boudet et al., 2001; Babenko et al., 2004). Tomato, Arabidopsis and cucumber are different in anatomy and physiology. Therefore, some branches may have different ways of expansion in the RAV family of tomato, Arabidopsis and cucumber. As shown in Figure 2, the RAVs within the same clade shared similar gene structure and motif composition. Genes with similar gene structures and conserved motifs usually have similar functions. The cucumber RAV proteins were clustered into some Arabidopsis functional clades, which will provide valuable information for studying the function of CsRAV genes. Previous studies have shown that abiotic stress-related marker genes (such as RD29A, ABI5, SOS1, SOS2, NHX3, CPK1, RD22 and APX2) are involved in plant response and defense against environmental stress (Li et al., 2015; Zhang et al., 2020). Changes in the expression of these marker genes may contribute to plant resistance to abiotic stress. The transcriptional levels of stress- and ABA- related genes were regulated in transgenic plants under different stresses (Li et al., 2015; Zhang et al., 2020). Under NaCl treatment, the expressions of ABI1, RD29A and RAB18 were obvious enhanced in 35S:GhRAV1 transgenic plants compared with those in WT (Li et al., 2015). Compared with those in WT, the transcriptional levels AtSOS1, AtRD22 and AtRD29A were upregulated in 35S: TaRAV1 transgenic plants after NaCl treatment (Luo et al., 2022). The ABI5 gene is a key positive regulator of ABA signal transduction, and increased ABI5 gene expression enhances susceptibility to ABA during seed germination and early seedling development (Lopez- Molina et al., 2001). AtRAV1 is involved in ABA signaling by directly binding to the promoter of ABI5 and inﬂuencing its expression (Feng et al., 2014). MdRAV1 plays a key role in ABA signaling by directly binding to the promoters of MdABI3 and MdABI4 (Zhao et al., 2019b). Discussion RAV gene family is widely distributed in higher plants and is one of the plant-speciﬁc regulatory gene families. As one of the B3 gene superfamilies in plants, the RAV family genes regulate many aspects of plant growth and development, including regulating ﬂowering time and heading date (Hu et al., 2004; Woo et al., 2010; Duan et al., 2016; Shin and Nam, 2018). At present, RAV genes have been extensively studied in Arabidopsis, rice, soybean, cotton, pepper and other plants (Giraudat et al., 1992; Sohn et al., 2006; Zhao et al., 2008; Li et al., Frontiers in Plant Science 10 frontiersin.org 10.3389/fpls.2023.1115874 Li et al. However, there were different response patterns under different stresses, indicating functional diversity among the genes. 2015; Osnato et al., 2020). However, little is known about the role of the RAVs in cucumber. In this study, we identiﬁed eight cucumber RAV genes through genome-wide analysis. The phylogenetic relationship, gene structure, conserved domains, gene duplication events and cis-acting elements on promoters of RAV family genes in cucumber were systematically analyzed. The functions of RAV genes in Arabidopsis, soybean, cotton, pepper and rice have been widely reported (Hu et al., 2004; Zhao et al., 2017; Chen et al., 2021; Kabir et al., 2021). Overexpression of AtRAV1 and AtRAV2 can improve the drought resistance in cotton (Matı́as- Hernández et al., 2014). Overexpression of soybean GmRAV3 in Arabidopsis could signiﬁcantly increase the resistance of transgenic lines to high salt and drought, and lead to the insensitivity of transgenic plants to exogenous ABA (Lu et al., 2014; Zhao et al., 2017). CsRAV1 was signiﬁcantly induced by salt and ABA (Figures 5A, B), so CsRAV1 might play key roles in resistance to salt or ABA stress. To further determine the roles of CsRAV1 in abiotic stress response, we obtained the 35S:CsRAV1 transgenic Arabidopsis and transient transformed cucumber cotyledons. Under NaCl and ABA treatments, we found 35S:CsRAV1 cucumber seedlings indeed showed enhanced tolerance to salt and ABA compared with 35S plants (Figures 7A–C). Meanwhile, the transgenic Arabidopsis showed higher germination rates than WT, indicating that CsRAV1 played vital roles in seed germination and development (Figures 8A–D). The expression of CsRAV1 was also induced by low temperature (Figure 5C), but we found that there were no signiﬁcant differences in phenotype between 35S:CsRAV1 transgenic seedlings and control treated at 4°C, which required further veriﬁcation and discussion in the future. References Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H., Shinn, P., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. 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A subset of Arabidopsis RAV transcription factors modulates drought and salt stress responses independent of ABA. Plant Cell Physiol. 55 (11), 1892–1904. doi: 10.1093/pcp/pcu118 Cannon, S. B., Mitra, A., Baumgarten, A., Young, N. D., and May, G. (2004). The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 4, 10. doi: 10.1186/1471-2229-4-10 Giraudat, J., Hauge, B. M., Valon, C., Smalle, J., Parcy, F., and Goodman, H. M. (1992). Isolation of the Arabidopsis ABI3 gene by positional cloning. Supplementary material JL, WH and ZW conceived and designed the experiments. JL, CS, HL, SW, LH and YY performed the experiments. JL analyzed the data and wrote the manuscript. WH and ZW revised the manuscript. All authors contributed to the article and approved the submitted version. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1115874/ full#supplementary-material Funding CsSOS1 and CsNHX1 genes to maintain ion homeostasis, thus enhancing salt tolerance. In addition, stress- and ABA- related marker genes (AtRD29A, AtSOS1, AtNHX1 and AtABI5) in transgenic Arabidopsis were signiﬁcantly up-regulated compared with WT, suggesting that CsRAV1 might alleviate NaCl and ABA stress by regulating stress- related genes in Arabidopsis (Figures 8E– H). These results will provide a theoretical basis for further study on the function of RAV transcription factor in cucumber. CsSOS1 and CsNHX1 genes to maintain ion homeostasis, thus enhancing salt tolerance. In addition, stress- and ABA- related marker genes (AtRD29A, AtSOS1, AtNHX1 and AtABI5) in transgenic Arabidopsis were signiﬁcantly up-regulated compared with WT, suggesting that CsRAV1 might alleviate NaCl and ABA stress by regulating stress- related genes in Arabidopsis (Figures 8E– H). These results will provide a theoretical basis for further study on the function of RAV transcription factor in cucumber. This work was supported by fundings from the National Natural Science Foundation of China (32202484, 31872415 and 32102310), the Natural Science Foundation of Shandong Province (ZR2022QC008), Scientiﬁc Research Leaders Studio of Jinan (2019GXRC052) and Doctoral Fund project of Jinan University (XBS2104). The funds played no role in study design, data analysis, and manuscript preparation. Conﬂict of interest In this study, the cucumber RAV family genes were systematically analyzed. The expression patterns of CsRAV genes under different stress treatments were studied, and the potential functions of CsRAV1 were also analyzed using the transgenic method. This work provides a rich insight into the functions and regulatory mechanism of CsRAV genes in abiotic stress resistance of cucumber. 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. Data availability statement 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. The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. Discussion Previous research showed the sodium/proton antiporter1 (NHX1) maintained Na+ homeostasis in plants and plays a vital role in plant salt tolerance (Zhang et al., 2020). In this study, qRT- PCR analysis indicated that the transcriptional levels of four stress- related genes, including CsSOS1, CsNHX1, CsCPK11 and CsABI5, in transgenic cucumber were higher than those in 35S plants after NaCl and ABA treatment (Figures 7D–G). These results indicated that CsRAV1 might directly regulate the expression of CsABI5, thereby participating in ABA signal pathway and enhancing ABA tolerance in cucumber. Furthermore, CsRAV1 might regulate the transcription of Previous studies have investigated the role of RAV transcription factors in regulating plant responses to biotic and abiotic stresses such as plant hormones and pathogens (Chen et al., 2021). In our study, it was shown that the promoters of CsRAV genes contain cis-elements that respond to abiotic stresses and hormones such as drought, wound, defense and stress, low temperature, auxin, salicylic acid, abscisic acid, gibberellin and jasmonic acid (Figure 4). In addition, TATA box is one of the elements that constitute the eukaryotic promoters, and its sequence is TATA (A/T) A (A/T). It is generally about -30bp (-25~-32bp) upstream of the transcription start site of most eukaryotic genes. The TATA box is the selection that determines the initiation of gene transcription and is one of the binding sites for RNA polymerase, which can only be transcribed after the RNA polymerase is ﬁrmly bound to the TATA box (Pedersen et al., 1999). The transcription start site (TSS) is the location where transcription starts at the 5’-end of a gene sequence (Peng et al., 2006). Our data showed that all CsRAV gene promoters except CsRAV3 contained TSS and TATA-box (Supplementary Figure 3). The speciﬁc TSS and TATA-box on each CsRAV gene promoter need to be determined by robust analysis of 5’-transcript ends (5’-RATE) experiment. Previous studies indicated that AtRAV1, AtRAV2, BnaRAV-1, GhRAV1 and CARAV1 genes could be signiﬁcantly induced after stress treatment (Sohn et al., 2006; Zhuang et al., 2011; Fu et al., 2014; Li et al., 2015). In this study, CsRAV genes were mainly up-regulated by salt, low temperature and ABA, and some of them were signiﬁcantly affected in different degrees (Figure 5). These results suggested that the cucumber RAV genes, like the genes of other plants, played key roles in stress resistance. Frontiers in Plant Science 11 frontiersin.org Li et al. 10.3389/fpls.2023.1115874 10.3389/fpls.2023.1115874 10.3389/fpls.2023.1115874 References PLoS One 10 (2), e0118056. doi: 10.1371/journal.pone.0118056 Wei, Y., Chang, Y., Zeng, H., Liu, G., He, C., and Shi, H. (2018). 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https://openalex.org/W2971355953	https://dialnet.unirioja.es/descarga/articulo/7076488.pdf	Portuguese	null	Memória rastro em poemas de Conceição Evaristo	Anuário de Literatura	2,019	cc-by	4,146	http://dx.doi.org/10.5007/2175-7917.2019v24n1p13 http://dx.doi.org/10.5007/2175-7917.2019v24n1p13 MEMÓRIA RASTRO EM POEMAS DE CONCEIÇÃO EVARISTO Memory as trace in Conceição Evaristo’s poems Rosana Arruda de Souza https://orcid.org/0000-0002-1378-070X Universidade Federal do Mato Grosso, Programa de Pós-Graduação em Estudos de Linguagem, Cuiabá, MT, Brasil. 78060-900 – secretariappgelufmt@gmail.com 1 Entrevista concedida ao projeto “Mulheres que escrevem”. Disponível em: https://medium.com/mulheres-que- escrevem/mulheres-que-escrevem-entrevista-conceicao-evaristo-fa243ff84284. Acesso: 14/03/2019. Rosana Arruda de Souza https://orcid.org/0000-0002-1378-070X Universidade Federal do Mato Grosso, Programa de Pós-Graduação em Estudos de Linguagem, Cuiabá, MT, Brasil. 78060-900 – secretariappgelufmt@gmail.com Resumo: A poesia de Conceição Evaristo é conhecida por desvelar aquilo que há muito permaneceu encoberto: a voz negra feminina, uma voz que, paradoxalmente, sempre existiu, mas era ecoada em ouvidos ensurdecidos. Agora, os tantos gritos perdidos da diferença podem ser ouvidos e os ouvidos são colocados à prova, pois reconhecer e vislumbrar a ausência trazida pelos signos – quando antes acreditávamos que eles traziam a presença – não é tarefa fácil. Assim, pois, entendemos e nos propomos a analisar os poemas da autora: sua poesia encerra o rastro – a ausência, o outro, que carrega em si uma potencialidade significativa justamente enquanto é sombra, pois as vozes negras estão em cena, mas, ainda, não estão à luz, entretanto estar à sombra se faz potência. Para esta discussão, evocaremos, portanto, o conceito derridiano de rastro num diálogo com o conceito de memória, por considerarmos que é através da memória daquelas vozes que se constroem os rastros na poesia de Conceição Evaristo. Palavras-chave: Rastro. Memória. Poesia. Abstract: The poetry of Conceição Evaristo is known for revealing what has long remained hidden: the female black voice, a voice that, paradoxically, always existed, but was echoed in deaf ears. Now the many lost screams of difference can be heard and the ears are put to the test, for recognizing and glimpsing the absence brought by the signs – when we first believed they brought the presence – is no easy task. Thus, we understand and propose to analyze the poems of the author: his poetry promotes the trace – the absence, the other, which carries within itself a significant potential precisely while it is a shadow, because the black voices are on the scene, but still, are not under the light, however to be in the shade becomes potency. For this discussion, we will therefore evoke the Derrida concept of trace in a dialogue with the concept of memory, because we believe that it is through the memory of those voices that the traces are constructed in the poetry of Conceição Evaristo. Keywords: Trace Memory Poetry Keywords: Trace. Memory. Poetry. Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 Esta obra está licenciada sob uma Creative Commons - Atribuição 4.0 Internacional.. Esta obra está licenciada sob uma Creative Commons - Atribuição 4.0 13 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 Introdução Em entrevista1, Conceição Evaristo declara: “há povos que conseguem falar pelos orifícios da máscara. E falam com tanta veemência que são capazes de estilhaçar a máscara”. Podemos, facilmente, atrelar a tal declaração um caráter autobiográfico, visto que a história de Conceição se entrelaça à desses povos, tendo conseguindo alcançar um espaço, ainda reduzido para as mulheres negras, de onde emanam os vários estilhaços de quando se rompem máscaras e barreiras. A escritora declara também que sua escrita se abre às leitoras num espaço onde elas possam se sentir em casa, reconhecidas de fato, em contraponto à lacuna existente na “literatura que nós conhecemos, essa literatura canônica, ela não nos representa e quando nos representa é sempre de uma maneira limitada, de uma maneira estereotipada” (EVARISTO, 2017, p. 01). Propomos, aqui, que, nos poemas da escritora, a eu lírica persegue o entrelaçar de ausência e presença da mulher negra. A escrita insinua um caráter de conhecimento e autorreconhecimento identitário, mas a parcela histórica conferida a todo ato poético engajado traz à luz a ausência daquelas mulheres, cujo rastro (de lágrimas, de luta, de recordações) não se permite chegar ao objeto criador. A força dessas mulheres está exatamente em seu caráter de rastro que não se deixa apreender, na ausência que tem sido potência graças a uma memória, memória que se tenta recuperar por meio da poesia. Queremos pensar como tal embate ocorre dentro da própria língua, especificamente na escrita de Conceição Evaristo, de maneira a oferecer uma análise de como são articulados os elementos que mantêm a instância do rastro. Averiguamos que o rastro pode ser visto como a lógica da diferença e abertura ao outro, pois reitera aquilo que é o próprio resto, potencializado justamente por seu caráter negativo, daquilo que é deixado para trás – um conjunto de memórias jogadas (dentro)fora. 14 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 O dentro e o fora: a identidade negra Segundo Homi Bhabha (1998, p. 321), a escrita “encena o direito de significar dos escravos, não simplesmente por negar ao imperialista o ‘direito de tudo a ser um nome’, mas por questionar a subjetividade masculinista, autoritária, produzida no processo colonizador”. Desde aí, podemos antever a escrita como um processo em que se busca um lugar de reencontro com o outro e com a nossa própria história, pois escrever, como foi dito pelo autor, emana o direito de significar, mas também suscita uma trama de embates. O próprio processo de escrita se constrói através de uma teia de inclusão e exclusão de signos; é como se ela própria sustentasse o direito de nomear de que fala Bhabha. Quem pode nomear carrega o poder da escrita, daquele que pode ser e fazer ser; quem é nomeado fica obscurecido sob nomeações por vezes avulsas, que não integram e que não corroboram as suas memórias. 14 1 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 Escrever-se imprime um ato de emancipação própria do sujeito e também um ato de deixar-se livre da própria escrita, deixar-se livre da palavra e do nome: deixar-se livre do ser e ser, de fato, livre. Por isso que o rastro configura a exterioridade de tudo, um vir a ser. Conforme Jacques Derrida (2009, p. 91), “escrever é retirar-se. Não para a sua tenda para escrever, mas da sua própria escritura. Cair longe da linguagem, emancipá-la ou desampará-la, deixá-la caminhar sozinha e desmunida. Abandonar a palavra”. Derrida se destacou por ter formulado a lógica da desconstrução, a qual se conduz por um caminho outro às teorias metafísicas do ocidente. Com ela se questiona qualquer articulação que pretende a totalização ou a centralização das coisas. Desse modo, o filósofo traz a lume conceitos como o de rastro, em que se conjuga justamente aquilo que não está no centro das coisas. “O rastro não é somente a desaparição da origem, ele quer dizer que a origem não desapareceu sequer, que ela jamais foi retroconstruída, a não ser por uma não-origem, o rastro, que se torna, assim, origem da origem” (DERRIDA, 1973, p. 75). O dentro e o fora: a identidade negra O rastro nos permite ver as relações sociais por meio de um prisma linguístico (e vice-versa), pois se contrapõe, sem anular, o pressuposto da relação intrínseca significante/significado, ao apontar aquilo que fica às margens e não atende a tal lógica de significação, pois o rastro (o traço) não ganha um significado e também está desprendido de uma origem, pois ele próprio já é significante de outro significante e, assim, sucessivamente, numa cadeia eterna. A ótica de Derrida chega a ser entendida como uma ótica engajada, visto que suscita o questionamento das limitações propagadas pelo ato da palavra, mais especificamente, pelos processos de significação entre os sujeitos. Na ideia antiga de signo, temos o significado como se fosse o objetivo final das coisas, quando, no entanto, sob um olhar derridiano, o significado limita a potencialidade das coisas, pois, achando-se o significado de tudo, morre a potência da linguagem imprimida justamente pelo momento em que se não é, em que se está num estado de vir a ser. Trazendo isso às relações humanas, significar o outro – nomeá-lo – conduz um ato de delimitação do mesmo. raremos tal discussão para a análise de poemas de Conceição Evaristo. Começaremos Traremos tal discussão para a análise de poemas de Conceição Evaristo. Começaremos por: por: 15 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 enquanto o outro, o cisco cerceia e da visão que me resta vazo o invisível e vejo as inesquecíveis sombras dos que já se foram. Da língua cortada, digo tudo, amasso o silêncio e no farfalhar do meio som solto o grito do grito do grito e encontro a fala anterior, aquela que emudecida, conservou a voz e os sentidos nos labirintos da lembrança. (EVARISTO, 2008, p. 50) Da língua cortada, digo tudo, amasso o silêncio e no farfalhar do meio som solto o grito do grito do grito e encontro a fala anterior, aquela que emudecida, conservou a voz e os sentidos nos labirintos da lembrança. (EVARISTO, 2008, p. 50) No poema em questão, notamos o trabalho com a relação de metades (“meia lágrima”, “meia gota”, “meio tom”) que percorre um caminho metonímico inverso – da parte ao todo. 2O espectro é alguma coisa que, precisamente, não se conhece, e não se sabe, precisamente, se ele é, se existe, se responde a um nome e se corresponde a uma essência. Não se conhece: não por ignorância, mas porque esse não- objeto, esse presente não-presente, esse estar-lá de alguém ausente ou que partiu já não pertence ao controle do conhecimento. Pelo menos não pertence mais àquilo que pensamos entender pelo nome de conhecimento. É uma coisa que não pode ser nomeada [...] (DERRIDA, 1994, p. 6-7). O dentro e o fora: a identidade negra Então, depois da lágrima vamos ao olho, “um só olho”; e depois do tom do soluço, vamos à língua, que também vem pela metade, “língua cortada”, até chegarmos ao grito, já em seu estado de abjeto, rastro, sendo apenas “o grito do grito do grito”. Podemos aplicar ao encadeamento de metades o postulado derridiano, no sentido de termos um encadeamento de significantes que permanecem em um vir a ser. Acolhe-se, no poema, o sentido da exterioridade do signo quando dá lume àquilo que ficou de fora, a não presença. Percebemos, na última estrofe, que a eu lírica se enuncia por meio da expressão “digo tudo”, mas depois, queda sua ausência, pois o que se ouve é o próprio silêncio, por meio de um grito que já virou eco do próprio eco, mas é este eco que potencializa a “fala anterior e emudecida” e conserva a voz “nos labirintos da lembrança”. Derrida considera por metafísica da presença aquela que tinha por totalidade do logos a relação significante/significado. Para ele, “a época do logos, rebaixa a escritura, pensada como medição e queda na exterioridade do sentido” (DERRIDA, 1973, p. 15). No poema de Conceição Evaristo, observamos que o conhecimento vem a lume justamente através daquilo que demarca a ausência: o grito do grito do grito. Ademais, os trechos “amasso o silêncio” e o “farfalhar do meio som” conduzem uma maneira bem sutil para falarmos daquilo que não pode ser significado. São menções que remetem àquilo que fica no meio caminho, entre a presença e o logos, como um espectro2, como “as inesquecíveis sombras que já se foram” trazidas no poema. Avalia-se também o movimento semântico que se faz na terceira estrofe através daquilo que se pode olhar e daquilo que é invisível. Resta à eu lírica um olho só, pois o outro está 16 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 cerceado por cisco. No entanto, o olho que sobrou dá vazão ao invisível composto pelas inesquecíveis sombras dos que já se foram. Mas este olho que resta ainda vê, “sei ainda ver com um olho só”, o invisível é o material sabido e de sabedoria, pois ao olho que restou, enxergar o resto, “a sombra”, é o que importa. Podemos conferir a este olho a pontencialidade de ele articular muito além de imagens, pois ele articula memórias, é como se ele enxergasse para dentro. O dentro e o fora: a identidade negra Nessa mesma esteira, falamos da necessidade de se falar a potência da escrita feminina negra e de como nela se articulam elementos emancipatórios, seja da própria mulher que escreve, seja da própria escrita levantada como o lugar das memórias, o lugar de desprendimento do signo. Este não mais aparece como imagem da coisa, mas como realização ininteligível da potência de significação. A poesia, na escrita de Conceição Evaristo, percorre o legado de memórias deixadas pelas mulheres negras. Nesse sentido, analisamos o poema abaixo: Na voz de minha filha se fará ouvir a ressonância o eco da vida-liberdade (EVARISTO, 2008, p. 10-11) Na voz de minha filha se fará ouvir a ressonância o eco da vida-liberdade (EVARISTO, 2008, p. 10-11) No título do poema, vislumbramos o signo mulher distorcido de seu caráter presença para tornar-se o rastro, mais especificamente, a voz, que se estende a um conjunto de vozes ancestrais: “a voz de minha mãe”, “a voz de minha vó”, “a voz de minha bisavó”. Henri Bergson chama de memória-lembrança aquela que é construída naturalmente sem uma aplicabilidade prática e que vem à tona sem esforço. “Por ela se tornaria possível o reconhecimento inteligente, ou melhor, intelectual, de uma percepção já experimentada” (BERGSON, 1999, p. 88). No caso do poema em análise, verifica-se como a memória toma o caráter de reconhecimento das “vozes mudas” de liberdade (“vida-liberdade”) presente em todas as gerações de mulheres descritas no poema. Para Bergson, a memória, “a bem da verdade, já não nos representa nosso passado, ela o encena; e, se ela merece ainda o nome de memória, já não é porque conserve imagens antigas, mas porque prolonga seu efeito útil até o momento presente” (1999, p. 89). Já para Soares e Carbonieri (2016, p. 02), a literatura surge como um importante instrumento para esse exame [da escrita feminina negra], não porque seja a transcrição de uma realidade (o que, de fato, não é), mas porque está entremeada de discursos a respeito dessa realidade, discursos que muitas vezes são conflitantes entre si. Assim, compreendemos que o que temos no poema de Conceição Evaristo é antes o efeito das vozes veladas ao longo do percurso feminino negro, do que a experiência vivida por essas mulheres, pois sabemos que, sim, tal experiência é intransferível. Ressoa, aqui, o entendimento de Derrida a respeito de sua relutância em abraçar a correspondência entre significante e significado, entre sensível e inteligível. Com isso, avaliamos que o que nós lemos não pode ser nunca a própria experiência lida – o sensível – mas apenas o rastro daquilo que “as vozes mudas caladas/ engasgadas na garganta” tentaram ecoar. Notemos, também no poema em apreço, a similitude sonora (repetição vogal), utilizada para descrever a presença do homem colonizador, presente em “os brancos-donos de tudo”. Vozes-mulheres A voz de minha bisavó ecoou criança nos porões do navio. ecoou lamentos de uma infância perdida. A voz de minha avó ecoou obediência aos brancos-donos de tudo. A voz de minha mãe ecoou baixinho revolta no fundo das cozinhas alheias debaixo das trouxas roupagens sujas dos brancos pelo caminho empoeirado rumo à favela. A voz de minha mãe ecoou baixinho revolta no fundo das cozinhas alheias debaixo das trouxas roupagens sujas dos brancos pelo caminho empoeirado rumo à favela. A minha voz ainda ecoa versos perplexos com rimas de sangue e fome. A voz de minha filha recolhe em si a fala e o ato. O ontem – o hoje – o agora. 17 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 mas os fundos oceanos não me amedrontam e nem me imobilizam. mas os fundos oceanos não me amedrontam e nem me imobilizam. Uma paixão profunda é a boia que me emerge. p p q g Sei que o mistério subsiste além das águas. (EVARISTO, 2008, p. 09) Neste poema, recuperamos o signo da lágrima do poema anteriormente analisado. Dessa vez, no entanto, o signo vem metaforizado através das “águas lembranças” que transbordam dos olhos. A recordação é um momento em que se aparta do mundo e a memória se volta às águas onde ficaram perdidas as lembranças, outrora duras lembranças, mas que agora sobrevêm para reconstrução. Por isso, a eu lírica tem a liberdade para ser “eternamente náufraga/ [pois] os fundos oceanos não [a] amedrontam e nem imobilizam”. Conforme Joël Candau (2012), a memória tem um caráter mútuo com a identidade. Para ele, “toda tentativa de se descrever a memória comum a todos os membros de um grupo a partir de suas lembranças, em um dado momento de suas vidas, é reducionista, pois ela deixa na sombra aquilo que não é compartilhado” (CANDAU, 2012, p. 34). No entanto, o que havia ficado na sombra pode sobrevir uma hora ou outra, pois fica como um patrimônio das vozes mudas de um conjunto de gerações, como acontece no poema Vozes-mulheres. Assim, o olhar para dentro, que mencionamos durante a análise de Meia lágrima, cabe muito bem a este movimento em que as lembranças, antes não compartilhadas, vêm à tona. Voltando o olhar a Recordar é preciso, conferimos que o oceano, no qual tantas vezes os porões negreiros transitaram, agora serve para cotejar as memórias entre passado e presente, e agora a eu lírica pode reconhecer-se, “sou”, reconstrói-se e revigora-se uma identidade. Do fogo que em mim arde Sim, eu trago o fogo, o outro, não aquele que te apraz. Ele queima sim, é chama voraz que derrete o bivo de teu pincel incendiando até às cinzas O desejo-desenho que fazes de mim. Sim, eu trago o fogo, o outro, aquele que me faz, e que molda a dura pena de minha escrita. é este o fogo, o meu, o que me arde e cunha a minha face na letra desenho do autorretrato meu. (EVARISTO, 2008, p. 19) Segundo Candau (2012), a memória é a identidade em ação, mas ela pode, ao contrário, ameaçar, perturbar e mesmo arruinar o sentimento de identidade tal como mostram os trabalhos 19 19 Anu. Lit., Florianópolis, v. Na voz de minha filha se fará ouvir a ressonância o eco da vida-liberdade (EVARISTO, 2008, p. 10-11) Além disso, observamos a ascendência espacial em que habita a mulher negra que vai dos “porões do navio”, passa pelas “cozinhas alheias”, até chegar à favela e, por fim, ao espaço da filha, que, aliás, não é espaço, mas sim “a fala e o ato”. As delimitações dos ecos afrouxaram- se, não se alimentam mais das paredes entorno, agora há liberdade. Passemos à análise de outro poema: Recordar é preciso O movimento vaivém nas águas-lembranças dos meus marejados olhos transborda-me a vida, salgando-me o rosto e o gosto. Sou eternamente náufraga, 18 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 mas os fundos oceanos não me amedrontam e nem me imobilizam. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 sobre as lembranças de traumas e tragédias. Neste poema, embora o caráter das memórias não esteja diretamente evocado, temos um momento de efervescência memorialística. Corpo e imagem são evocados: o corpo remetido pelo “desejo”, pelo “fogo”, pelo “voraz”; e a imagem é evocada no desenho feito pelo pincel e também pela “letra desenho” do “autorretrato”. Conceição Evaristo declara: se há uma literatura que nos inviabiliza ou nos ficciona a partir de estereótipos vários, há um outro discurso literário que pretende rasurar modos consagrados de representação da mulher negra na literatura. Assenhorando-se “da pena”, objeto representativo do poder falocêntrico branco, as escritoras negras buscam inscrever no corpus literário brasileiro imagens de autorrepresentação. Criam, então, uma literatura em que o corpomulher-negra deixa de ser o corpo do “outro” como objeto a ser descrito, para se impor como sujeito- mulhernegra que se descreve, a partir de uma subjetividade própria experimentada como mulher negra na sociedade brasileira. Pode-se dizer que o fazer literário das mulheres negras, para além de um sentido estético, busca semantizar um outro movimento que abriga todas as nossas lutas. Toma-se o lugar da escrita, como direito, assim como se torna o lugar da vida. (EVARISTO, 2005, p. 54). No poema Do fogo que em mim arde, haveria uma voz inflamada que reclama a própria potência falocêntrica do pincel e da pena. Na segunda estrofe, agora é a eu lírica autora de sua imagem, que não é mais apenas o desenho da primeira estrofe, é “letra desenho”, portanto, agora ela tem o poder das palavras nas mãos e pode escrever-se, fazer um autorretrato, ressignificar-se muito além de seu desenho desejo pintado por mãos alheias. Outro ponto a ser analisado é a entrada deste poema e de Meia lágrima realizada por meio dos advérbios “sim” e “não”, respectivamente. Este tipo de retórica imprime, no caso, não só o eco da informação positiva e negativa que se vai passar, mas também uma exortação ao interlocutor que podemos supor nos dois poemas como sendo o homem colonizador. Exortar alguém ao diálogo confere maior ousadia ao ato de liberdade: falamos, mas queremos quem nos ouça e quem retorne. Conclusão Nos poemas analisados, vimos um trabalho com a memória e com o rastro. A memória é o rastro. E pô-los em evidência exige um olhar para dentro e um olhar para fora, isto porque a própria luta emancipatória se coloca dos dois lados da arena. Conquistar um lugar na escrita não exige e não quer dizer afastar o homem branco, mas negociar a partilha do uso dos instrumentos – o pincel e a pena – e promover uma rasura sobre o signo colonizador. Afinal, colere (do latim) aparece como uma das raízes do signo e quer dizer habitar, cultivar, respeitar, guardar. Deveríamos deixar o inteligível de lado e fazer o sensível ecoar. BERGSON, Henri. Matéria e memória: ensaio sobre a relação do corpo com o espírito. Tradução Paulo Neves. 2. ed. São Paulo: Martins Fontes, 1999. Referências BERGSON, Henri. Matéria e memória: ensaio sobre a relação do corpo com o espírito. Tradução Paulo Neves. 2. ed. São Paulo: Martins Fontes, 1999. 20 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 BHABHA, Homi. O local da Cultura. Trad. de Mvriam Ávila, Eliana Lourenço de Lima Reis e Gláucia Renate Gonçalves. Belo Horizonte: Editora UFMG, 1998. CANDAU, Joël. Memória e identidade. Tradução de Maria Letícia Ferreira. São Paulo: Contexto, 2012. DERRIDA, Jacques. Gramatologia. Trad. de Miriam Chnaiderman e Renato Janine Ribeiro. São Paulo: Perspectiva: USP, 1973. DERRIDA, Jacques. Espectros de Marx: o estado da dívida, o trabalho do luto e a nova Internacional. Trad. de Anamaria Skinner. Rio de Janeiro: Relume-Dumará, 1994. DERRIDA, Jaques. A escritura e a Diferença. 2. ed. São Paulo: Perspectiva, 2009. EVARISTO, Conceição. Gênero e etnia: uma escre(vivência) de dupla face. In: MOREIRA, Nadilza Martins de Barros; SCHNEIDER, Liane (Orgs.). Mulheres no mundo. Etnia, marginalidade e diáspora. João Pessoa: Ideia, 2005, p. 201-212. EVARISTO, Conceição. Poemas da recordação e outros movimentos. Belo Horizonte: Nandyala, 2008. EVARISTO, Conceição. In: Mulheres que escrevem entrevista: Conceição Evaristo, 2017. Disponível em: https://medium.com/mulheres-que-escrevem/mulheres-que-escrevem- entrevista-conceicao-evaristo-fa243ff84284. Acesso em: 14/03/2019. SOARES, Cláudia Regina; CARBONIERI, Divanize. Nervous conditions: a literatura africana escrita por mulheres e o tema da educação formal, Afluente, Bacabal, v. 1, n. 3, p. 133-156, out./dez. 2016. Disponível em: http://www.periodicoseletronicos.ufma.br/index.php/afluente/index. Acesso em: 14/03/2019. p //www.periodicoseletronicos.ufma.br/index.php/afluente/index. Acesso em: 14/03/2019 Rosana Arruda de Souza (rosanaarrudasouza@hotmail.com) é doutoranda em estudos de linguagem, com concentração na área de estudos literários, pelo Programa de Pós-Graduação em Estudos de Linguagem, da Universidade Federal de Mato Grosso. Mestre em estudos de linguagem, com concentração na área de estudos literários, pelo mesmo programa e pela mesma universidade (2017). Graduada em Letras/Literatura pela Universidade Federal de Mato Grosso (2014). Atuou como professora substituta no Departamento de Letras da UFMT, no período de 2016-2018. ç Não se aplica. Não se aplica. Não se aplica. NOTAS DE AUTORIA Como citar esse artigo de acordo com as normas da revista SOUZA, Rosana Arruda de. Memória rastro em poemas de Conceição Evaristo. Anuário de Literatura, Florianópolis, v. 24, n. 1, p. 13-22, 2019. Consentimento de uso de imagem Não se aplica. Não se aplica. Aprovação de comitê de ética em pesquisa Não se aplica. Não se aplica. Financiamento 21 21 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917 O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001; Universidade Federal de Mato Grosso; Fundação de Amparo à Pesquisa do Estado de Mato Grosso. Licença de uso ç Este artigo está licenciado sob a Licença Creative Commons CC-BY. Com essa licença você pode compartilhar, adaptar, criar para qualquer fim, desde que atribua a autoria da obra. Histórico Histórico Recebido em: 18/03/2019 Aprovado em: 20/05/2019 22 Anu. Lit., Florianópolis, v. 24, n. 1, p. 13-22, 2019. ISSNe 2175-7917
https://openalex.org/W3038693402	https://europepmc.org/articles/pmc7337531?pdf=render	English	null	Different acupuncture therapies for spastic paralysis after stroke	Medicine	2,020	cc-by	3,443	Abstract Abstract Background: Stroke is emerging as a signiﬁcant health issue that threatens human health worldwide and as a common sequela of stroke spastic paralysis after stroke (SPAS) has received wide attention. Currently, several systematic reviews have suggested that the commonly used acupuncture therapy (electroacupuncture, ﬁre acupuncture, warm acupuncture, and ﬁliform needle acupuncture) has achieved signiﬁcant efﬁcacy in the treatment of SPAS. In this study, network meta-analysis will be used to analyze the results of different clinical trials and evaluate the differences in the efﬁcacy of different acupuncture treatments for SPAS. Methods: Only randomized controlled trials will be included and all patients were diagnosed as spastic paralysis after stroke. A computer-based retrieval will be conducted at CNKI, WanFang databases, VIP, Sinoed, Pubmed, Embase, Web of Science, and the Cochrane library. The search period limit is from the time the date of database establishment to April 17, 2020. To avoid omissions, we will manually retrieve relevant references and conference papers. The risk of bias in the ﬁnal included studies will be evaluated based on the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions. All data analysis will be conducted by Revman5.3, WinBUGS 1.4.3, and Stata14.2. Results: This study quantiﬁed the effectiveness of each intervention for different outcome indicators. The primary outcomes include the Fugl-Meyer Assessment score, the modiﬁed Ashworth scale for the assessment of spasticity, and Barthel Index. The secondary outcomes include clinical effectiveness and adverse reactions. Conclusion: It will provide evidence-based medical evidence for clinicians to choose more effective acupuncture therapy for SPAS Abbreviations: EA = electroacupuncture, FA = ﬁre acupuncture, RCTs = randomized controlled trials, SPAS = spastic paralysis after stroke, WA = warm acupuncture. Conclusion: It will provide evidence-based medical evidence for clinicians to choose more effective acupuncture therapy for SPAS Abbreviations: EA = electroacupuncture, FA = ﬁre acupuncture, RCTs = randomized controlled trials, SPAS = spastic paralysis after stroke, WA = warm acupuncture. This study is supported by the Construction Project of Double First-Class discipline (traditional Chinese Medicine) in Jiangxi Province (No. JXSYLXK- ZHYI009); National Administration of Traditional Chinese Medicine, Construction Project of Inheritance Studio of National Famous Traditional Chinese Medicine Experts (Grant Number: [2016] No. 42). The authors have no conﬂicts of interest to disclose. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. a Jiangxi University of Traditional Chinese Medicine, b Afﬁliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi Province, China. ∗Correspondence: Chunhua Huang, Department of Neurology, Afﬁliated Hospital of Jiangxi University of Traditional Chinese Medicine, No. 445 Ba-yi Road, Nanchang, 330006, Jiangxi Province, China (e-mail: vich622@163.com). Copyright © 2020 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. How to cite this article: Wang R, Xie R, Hu J, Wu Q, Rao W, Huang C. Different acupuncture therapies for spastic paralysis after stroke: a protocol for systematic review and network meta-analysis. Medicine 2020;99:27(e20974). Received: 26 May 2020 / Accepted: 29 May 2020 http://dx.doi.org/10.1097/MD.0000000000020974 Abstract Keywords: acupuncture, network meta-analysis, protocol, spastic paralysis after stroke sequelae and a major obstacle for patients to recover, the incidence rate of spastic paralysis after stroke (SPAS)is arising in about 30% of patients.[3,4] However, the rate of spasms is highly variable and can occur in the short, medium, or long term Post- stroke period.[5] Relevant articles revealed the possible etiologies that mainly injured the upper motor neurons, resulting in a signiﬁcant increase in muscle tension.[6,7] If failure to take active treatment may cause Joint deformation, muscular atrophy, and pain, which may affect the rehabilitation of the limbs and the quality of life.[8] Medicine ® OPEN Study Protocol Systematic Review OPEN Copyright © 2020 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. How to cite this article: Wang R, Xie R, Hu J, Wu Q, Rao W, Huang C. Different acupuncture therapies for spastic paralysis after stroke: a protocol for systematic review and network meta-analysis. Medicine 2020;99:27(e20974). Medicine ® Medicine ® ∗Correspondence: Chunhua Huang, Department of Neurology, Afﬁliated Hospital of Jiangxi University of Traditional Chinese Medicine, No. 445 Ba-yi Road, Nanchang, 330006, Jiangxi Province, China (e-mail: vich622@163.com). This study is supported by the Construction Project of Double First-Class discipline (traditional Chinese Medicine) in Jiangxi Province (No. JXSYLXK- ZHYI009); National Administration of Traditional Chinese Medicine, Construction Project of Inheritance Studio of National Famous Traditional Chinese Medicine Experts (Grant Number: [2016] No. 42). This study is supported by the Construction Project of Double First-Class discipline (traditional Chinese Medicine) in Jiangxi Province (No. JXSYLXK- ZHYI009); National Administration of Traditional Chinese Medicine, Construction Project of Inheritance Studio of National Famous Traditional Chinese Medicine Experts (Grant Number: [2016] No. 42). The authors have no conﬂicts of interest to disclose. 3.2. Data sources and search strategies interventions, and cannot compare multiple acupuncture thera- pies. At present, there are various types of acupuncture, the 4 acupuncture therapies (electroacupuncture [EA], ﬁre acupunc- ture (FA), warm acupuncture (WA), ﬁliform needle acupuncture) analyzed in this study are commonly used methods. Meanwhile the advantages of various acupuncture therapies are not the same, so the choice of which acupuncture therapy can bring greater curative effect has brought troubles to clinical operators. In this study, the effectiveness of acupuncture therapy commonly used in clinical will be ranked by network meta-analysis, to provide scientiﬁc evidence-based medicine basis for the clinical selection of the best. interventions, and cannot compare multiple acupuncture thera- pies. At present, there are various types of acupuncture, the 4 acupuncture therapies (electroacupuncture [EA], ﬁre acupunc- ture (FA), warm acupuncture (WA), ﬁliform needle acupuncture) analyzed in this study are commonly used methods. Meanwhile the advantages of various acupuncture therapies are not the same, so the choice of which acupuncture therapy can bring greater curative effect has brought troubles to clinical operators. In this study, the effectiveness of acupuncture therapy commonly used in clinical will be ranked by network meta-analysis, to provide scientiﬁc evidence-based medicine basis for the clinical selection of the best. A computer-based retrieval will be conducted at CNKI, WanFang databases, VIP, SinoMed, Pubmed, Embase, Web of Science, and the Cochrane library. The search period limit is from the time the date of database establishment to April 17, 2020. To ensure the comprehensiveness of the search, relevant references and conference literature are also included. A search strategy is as follows:(((((((([stroke [MeSH Terms]) OR (cerebral infarction [MeSH Terms])) OR (cerebral hemorrhage[MeSH Terms])) OR (sequelae)) OR (spasm)) OR (spastic paralysis)) OR (spastic hemiplegia)) AND ((((((((acupuncture) OR (EA)) OR (FA)) OR (WA)) OR (ﬁliform needle acupuncture)) OR (warm needle)) OR (ﬁre-needle)) OR (needle acupuncture))) AND ((((“Randomized Controlled Trial” [Publication Type]) OR “RCT” [Publication Type]) OR “clinical trials” [Publication Type])). 3.4. Risk assessment of bias The risk of bias in the ﬁnal included studies will be evaluated based on the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions. The evaluation criteria include 7 items: selections bias, performance bias, detect bias, attrition bias, reporting bias, and other bias. Each item will be graded into 3 levels: “high risk”, “low risk”, and “not clear”.[24,25] This work will also be done independently by 2 reviewers. 3.1.2. Participants. All adult patients are diagnosed as SPAS. There will also be no restrictions based on gender, race, and the course of the disease. However, the following patients will be excluded: (1) Patients with mental illness who cannot cooperate with treatment. (1) Patients with mental illness who cannot cooperate with treatment. (2) Patients with complications or vital organ failure. (2) Patients with complications or vital organ failure. (3) Pregnant or lactating women. (4) Patients with adverse reactions or pain that cannot withstand acupuncture. (4) Patients with adverse reactions or pain that cannot withstand acupuncture. 3.3. Selection of studies and data extraction Two evaluators (RQW and RFX) will independently screen all relevant literature according to the inclusion and exclusion criteria. Then import them into Endnote for centralized management. We then go through the title abstract and the full text, in turn, to determine which study ultimately meets the criteria. During this process, if there is any dispute, the decision will be made by the third evaluator (JWH). Also, we will establish a document information extraction table in EXCEL, the extracted data includes the following: title, author, publication time, abstract, sample size, number of cases in each group, age, gender, course of the disease, distribution method, intervention, treatment course, outcome indicators, and so on. 3.1. Inclusion criteria 3 1 1 S d O 3.1.1. Study type. Only randomized controlled trials (RCTs) will be included and no restrictions on language. Non-RCTs such as system reviews, meeting abstracts will be excluded. Inter- ventions incompatible with the inclusion criteria and animal experiments will be excluded. Duplicate publications are preferred to the 1 with the most recent and comprehensive data. 2. Protocol registration This systematic review protocol will be reported strictly adherence to the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P).[20] The protocol of the systematic review has been registered in the INPLASY website(registration number is INPLASY202050058). If there are any adjustments during the entire study period, we will ﬁx and update the detailed information in the ﬁnal report in time. 1. Introduction Stroke is emerging as a signiﬁcant health issue that threatens human health worldwide. Besides a previous study shows stroke is a leading cause of death in China.[1,2] As 1 of the most common Nowadays, the common treatment methods for SPAS are medications, surgical interventions, and physical therapy.[9,10] However, concerns have been raised about the side effects of the drugs, the invasive nature of the surgery, and the high cost of treatment. Due to limitations of current SPAS treatment, it is particularly important to explore alternative treatment options with better efﬁcacy, fewer side effects, and low price. As a part of traditional Chinese medicine treatment, acupuncture has been used to treat stroke patients for many years in China and become more popular in Western countries in recent years.[11] Modern research shows that acupuncture can enhance immunity, regulate blood circulation, relieve the pain, help muscles relax and exercise more passively, thereby increasing recovery.[12–14] More and more systematic reviews suggest that acupuncture plays an important role in patients with SPAS.[15–19] But traditional systematic reviews can only directly compare 2 1 1 Medicine Wang et al. Medicine (2020) 99:27 3.2. Data sources and search strategies Author contributions Because of the large number of interventions involved in this study, the Loop inconsistency test of studies with direct and indirect evidence is needed in the evidence network for each outcome indicator. Calculate the inconsistency factor (IF), and judge whether there is inconsistency according to the size of IF value and P-value.[30] At the same time, the node split model will be used to judge whether each node has local inconsistency.[31] If P>.05, there is no signiﬁcant inconsistency, and the consistency model is used. As for the results obtained from the consistency model analysis, the stability of the results can be tested by the inconsistent model .[32,33] Conceptualization: Ruiqi Wang, Chunhua Huang, Rongfang Xie Data curation: Ruiqi Wang, Chunhua Huang, Jinwen Hu Formal analysis: Ruiqi Wang, Chunhua Huang, Rongfang Xie Project administration: Ruiqi Wang, Chunhua Huang, Qingz- hong Wu Supervision: Ruiqi Wang, Chunhua Huang, Wangfu Rao Writing – review & editing: Ruiqi Wang, Chunhua Huang, Rongfang Xie 3.8. Assessment of publication bias If the outcome indicators include in study ≥10, funnel plots will be used to assess the publication bias of the included trials.[36] If there is a difference in symmetry or distribution, there will be a publication bias or a small sample effect. [10] Shi L, Guo L, Zhang H, et al. Acupuncture for poststroke spasticity: a protocol of a systematic review and meta-analysis. Medicine 2019;98: e17124. [11] Lee JD, Chon JS, Jeong HK, et al. The cerebrovascular response to traditional acupuncture after stroke. Neuroradiology 2003;45:780–4. [12] Shin B, Lim H, Lee MS. Effectiveness of combined acupuncture therapy and conventional treatment on shoulder range of motion and motor power in stroke patients with hemiplegic shoulder subluxation: a pilot study. Int J Neurosci 2007;117:519–23. 3.7. Heterogeneity, subgroup analysis, and sensitivity analysis [2] Wu S, Wu B, Liu M, et al. Stroke in China: advances and challenges in epidemiology, prevention, and management. Lancet Neurol 2019; 18:394–405. The heterogeneity between trials is quantiﬁed with the I2 and P values.[34] For the test results with obvious heterogeneity, the source of heterogeneity should be analyzed. Subgroup analysis can be conducted according to the different sources of heterogeneity, such as the following aspects: treatment duration, disease course, underlying disease, race, gender, age, and so on. If no clear source of heterogeneity can be found, only descriptive analysis can be conducted. [3] Nair KPS, Marsden J. The management of spasticity in adults. BMJ 2014;349:g4737. [4] Miller EL, Murray L, Richards L, et al. Comprehensive overview of nursing and interdisciplinary rehabilitation care of the stroke patient: a scientiﬁc statement from the American Heart Association. Stroke 2010;41:2402–48. [5] Wissel J, Manack A, Brainin M. Toward an epidemiology of poststroke spasticity. Neurology 2013;80(3 Supplement 2):S13–9. [6] Lance JW. The control of muscle tone, reﬂexes, and movement: Robert Wartenbeg Lecture. Neurology 1980;30:1303. The purpose of sensitivity analysis is to eliminate low-quality studies and different statistical models.[35] Then, the strength, reliability, and stability of the results will be analyzed by observing the heterogeneity of different tests and whether the combined results changed after various treatments. [7] Jin Y, Jin X, Chen Y, et al. Acupuncture and constraint-induced movement therapy for a patient with chronic stroke: One-year follow-up case report. Medicine 2017;96:e8737. [8] Baricich A, Picelli A, Molteni F, et al.Post-stroke spasticity asa condition: a new perspective on patient evaluation. Funct Neurol 2016;31:179. [9] Cai Y, Zhang CS, Liu S, et al. Add-on effects of Chinese herbal medicine for post-stroke spasticity: a systematic review and meta-analysis. Front Pharmacol 2019;10:734. 3.5. Statistical analysis Revman 5.3 software is used for bias evaluation and traditional Meta-analysis. For continuous variables (FMA, MAS, BI), the results will be reported as mean difference (MD) with 95% conﬁdence interval (CI); Count data (clinical effectiveness and adverse events) will be calculated with the odds ratio (OR) and 95% CI. WinBUGS 1.4.3 and Stata 14.2 are used for network meta-analysis.[26,27] In the WinBUGS software, Bayesian net- work meta-analysis is performed by the Markov Chain Monte Carlo (MCMC) method, which is simulated by 4 chains, the number of iterations is set to 50,000, and the step size is set to 10.[28] At the same time, the potential scale reduction factor (PSRF) is used to evaluate the convergence of the results. When 1.00PRSF1.05, it indicates that the results converge well and the results obtained are highly reliable.[29] 3.1.3. Interventions. The experimental group only use EA, FA, WA or milli-acupuncture, and the control group uses rehabili- tation or a comparison of the above 4 acupuncture methods. Patients in both groups could receive conventional medical treatment and treatment duration and frequency are not limited. 3.1.4. Outcome indicators. The primary outcomes include the Fugl-Meyer Assessment score (FMA), the modiﬁed Ashworth scale (MAS) for the assessment of spasticity, and Barthel Index (BI). These indexes can evaluate the recovery of limb spasm.[21–23] The secondary outcomes include clinical effectiveness and adverse reactions. Clinical effectiveness including 4 grades of recovery, obvious effect, effective and ineffective. Effective rate = ([recovery + obvious effect + effective]/total number of cases) 100%. We can calculate the surface under the cumulative ranking curve (SUCRA) to estimate the possible ranking order of various interventions.[30] The SUCRA value ranges from 0 to 100. The 2 Wang et al. Medicine (2020) 99:27 www.md-journal.com larger the value, the intervention is considered to have better efﬁcacy. larger the value, the intervention is considered to have better efﬁcacy. acupuncture manipulations, and quantiﬁed their effectiveness according to different outcome indicators. 3.9. Ethics and dissemination Due to this is a protocol for systematic review and network meta- analysis, all data of this study are from published studies and do not involve patients, so ethical approval will not be necessary. The ﬁndings of this study will be disseminated to a peer-reviewed journal and presented at a relevant conference. [13] Chen B, Zhao M, Chen B, et al. Effectiveness and safety of acupuncture in post-stroke depression (PSD): protocol for a Bayesian analysis. Medicine 2020;99:e18969. [14] Rabinstein AA, Shulman LM. Acupuncture in clinical neurology. Neurologist 2003;9:137–48. [15] Yang L, Tan J, Ma H, et al. Warm-needle moxibustion for spasticity after stroke: a systematic review of randomized controlled trials. Int J Nurs Stud 2018;82:129–38. References [1] Jones AT, O’Connell NK, David AS. Epidemiology of functional stroke mimic patients: a systematic review and meta-analysis. Eur J Neurol 2020;27:18–26. 4. Discussion [16] Cai Y, Zhang CS, Liu S, et al. Electroacupuncture for poststroke spasticity: a systematic review and meta-analysis. Arch Phys Med Rehab 2017;98:2578–89. With the continuous development and improvement of acupunc- ture technology, there are a variety of acupuncture treatments that are considered to be effective in the treatment of SPAS. However, the therapeutic advantages of different acupuncture therapies are different from each other, which confuses clinical choices. Network meta-analysis overcomes the shortcomings of traditional systematic reviews and can integrate direct and indirect evidence. Besides, it provides an intuitive comparison of the efﬁcacy and safety of available technologies. So the results can provide clinicians and patients with the ability to determine the best treatment options. We will use the network meta-analysis method to assess the effectiveness of 4 commonly used clinical [17] Fan W, Kuang X, Hu J, et al. 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https://openalex.org/W2070907553	https://europepmc.org/articles/pmc4053419?pdf=render	English	null	Correction: Deagrarianisation and Forest Revegetation in a Biodiversity Hotspot on the Wild Coast, South Africa	PloS one	2,014	cc-by	282	File S1. Originally published, uncorrected article. File S1. Originally published, uncorrected article. File S1. Originally published, uncorrected article. File S2. Republished corrected article. File S2. Republished corrected article. Notice of Republication This article was republished on May 22, 2014, to correct errors in figure legends that occurred during the typesetting process. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference. Correction Correction: Deagrarianisation and Forest Revegetation in a Biodiversity Hotspot on the Wild Coast, South Africa The PLOS ONE Staff Correction Correction: Deagrarianisation and Forest Revegetation in a Biodiversity Hotspot on the Wild Coast, South Africa The PLOS ONE Staff Correction Correction: Deagrarianisation and Forest Revegetation in a Biodiversity Hotspot on the Wild Coast, South Africa Correction: Deagrarianisation and Forest Revegetation in a Biodiversity Hotspot on the Wild Coast, South Africa The PLOS ONE Staff Reference 1. Shackleton R, Shackleton C, Shackleton S, Gambiza J (2013) Deagrarianisation and Forest Revegetation in a Biodiversity Hotspot on the Wild Coast, South Africa. PLoS ONE 8(10): e76939. doi:10.1371/journal.pone.0076939 Citation: The PLOS ONE Staff (2014) Correction: Deagrarianisation and Forest Revegetation in a Biodiversity Hotspot on the Wild Coast, South Africa. PLoS ONE 9(6): e100463. doi:10.1371/journal.pone.0100463 Published June 11, 2014 Copyright: 2014 The PLOS ONE Staff. 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. PLOS ONE \| www.plosone.org June 2014 \| Volume 9 \| Issue 6 \| e100463 June 2014 \| Volume 9 \| Issue 6 \| e100463 1
https://openalex.org/W2096698467	https://ccsenet.org/journal/index.php/elt/article/download/31107/18199	English	null	The Effect of Large Classes on English Teaching and Learning in Saudi Secondary Schools	English language teaching	2,013	cc-by	8,602	Abstract The effect of class size on teaching and learning English as a foreign language (EFL) has been through a contentious debate among researchers for a long time. Before the 1950's the concern about the effect of class size and the learning outcomes of students in such classes waned for some time. Yet, researchers have reconsidered the case once again and up to now it is a field of investigation. Therefore, through reviewing the existing literature on this area and examples culled from the teachers' experience, the aim of the present study are twofold: (1) to ascertain the impact of large classes on the teaching and learning outcomes, and (2) to suggest appropriate strategies Saudi secondary teachers can utilize in their large classes to facilitate English teaching and learning. To reach the aim of this study, six Saudi English teachers from two public schools were interviewed to express their perceptions about teaching in large classes. Research results present that all participants find it daunting to teach large classes. Teachers also comment that despite their efforts to elevate the level of language learning in such context, the outcome of their students is considered to be unsatisfactory. They believe that this can be contributed to the fact that there are big numbers of students in one class and wish for the number to be reduced. The study findings shed the light on some useful and effective methods to be applied in large classes which may facilitate the language teaching and learning. Keywords: EFL, large classes, learning outcomes, secondary schools, Saudi Arabia Received: August 12, 2013 Accepted: September 10, 2013 Online Published: October 10, 2013 doi:10.5539/elt.v6n11p49 URL: http://dx.doi.org/10.5539/elt.v6n11p49 Received: August 12, 2013 Accepted: September 10, 2013 Online Published: October 10, 2013 doi:10.5539/elt.v6n11p49 URL: http://dx.doi.org/10.5539/elt.v6n11p49 Received: August 12, 2013 Accepted: September 10, 2013 Online Published: October 10, 2013 doi:10.5539/elt.v6n11p49 URL: http://dx.doi.org/10.5539/elt.v6n11p49 English Language Teaching; Vol. 6, No. 11; 2013 ISSN 1916-4742 E-ISSN 1916-4750 Published by Canadian Center of Science and Education English Language Teaching; Vol. 6, No. 11; 2013 ISSN 1916-4742 E-ISSN 1916-4750 Published by Canadian Center of Science and Education The Effect of Large Classes on English Teaching and Learning in Saudi Secondary Schools Dalal A. Bahanshal1 1 English Language Institute, King Abdulaziz University, Saudi Arabia Correspondence: Dalal A. Bahanshal, English Language Institute, King Abdulaziz University, P.O. Box 80203, Jeddah, 21589, Saudi Arabia. Tel: 96-650-568-4068. E-mail: d.bahanshal@gmail.com 1.2 Problems Perceived Due to Large Classes There are considerable studies concerning the influence of class size on students' improvement but a shortage of consensus on the definition of large and small classes. Thus, this section and the one that follows intend to probe into the causes to plateau of English learning and teaching in large classes. It is the language teachers who hold a negative view against large classes as they believe having many students in one class can cause some problems that affect them and their students. Some commonly comments heard by teachers are: there is no communication, the class is out of control, lack of individual attention and difficult to set effective group activities. Other teachers always emphasize that it is impossible to concentrate on all students and therefore not enough reinforcement will be made to encourage all to participate in different activities. Participants in Al- Jarf's (2006) study commented that "due to the size of the class they do not have enough time to pay attention to each and give every student a chance to speak and participate" (p. 24). In words, such context makes some students, especially the shy or weak, to be neglected and left behind. Kennedy & Kennedy (1996) shared similar view as they believed it is difficult to overcome all of what occur in class when the number of students exceeds a certain limit. On the contrary, teachers in small classes are able to pay great attention to their students and the benefit from the presented activities is considered to be high. From that we can say that it is abundantly evident that English teachers encounter great challenges when teaching large classes as they encounter difficulties in knowing all students in the class, having time for all individuals or presenting effective activities and therefore many students, especially the weak ones, tend to lose concentration. Another problem which is borne out in the words of the teachers and hinders the learning process in large classes is identifying and controlling students who tend to distract their classmates from concentrating on the lesson. Lazear (2003) assumed that if a student misbehaves and begins disturbing the class, the teacher has to attend the disturbance and control the noise. Such action from one student or more in a large class will block the learning of that moment and demolish the capability of others to learn. 1. Introduction It seems that English teachers have no doubt that large size of classes is a prime impediment for the efficient English teaching and learning. With more students enrolling in schools, large classes in a country like Saudi Arabia and other developing countries like Egypt, India and Pakistan tend to be obligatory rather than exceptional. Therefore, the problem seems to expand widely. As a matter of fact, large classes are not in and of themselves an issue to look forward to with dread. Admittedly, they require a great intention and careful preparation from teachers before the teaching process takes place. Heppner (2007) views large classes as a small business which should be set up and handled carefully. Generally speaking, experienced English teachers in general and teachers beginning teaching in particular find it onerous to manage a class with a big number of students with various levels and different personalities. Therefore, it is helpful for teachers to divert a lot of class responsibility to students and back up a little and that will give students space to teach each other. No one is absolutely certain whether small classes are inherently better than the large once in regard to the roles both classes play and their effectiveness on the process of English teaching and learning. Therefore, finding a definition to an idealistic class size which could be applicable in almost all teaching and learning environment tends to be difficult. Interestingly enough, teachers in general and English teachers in particular always favor small classes and believe that students in small classes learn better than their counterparts in large classes as more practice and activities are employed. In fact, their beliefs are not supported by lots of research in this field. To throw light on this phenomenon, an empirical study was conducted where six Saudi secondary English teachers were interviewed. The general purpose of investigating teachers' experience in teaching large classes was to realize their perceptions towards teaching large classes and the strategies to cope with the existing adverse situations in the classroom. Such understanding is expected to be beneficial in possible improvement of some fruitful and appropriate methods to yield the maximum learning output. However, review on the related literature provided deep insights about the effect of large classes and practical methods English teachers could utilize in 49 English Language Teaching Vol. 6, No. 11; 2013 www.ccsenet.org/elt such contexts. 1.2 Problems Perceived Due to Large Classes Pedder confirms: In larger classes, more time is needed for non-academic activities related to administrative and organizational procedures and to the management and control of discipline. Reductions in the quantity of learning opportunities constrain teachers from achieving the necessary pace, depth and breadth of curriculum coverage as class size increases (2006: 224). Noise level of some students is also considered to be a problematic issue as it will produce disturbance and prevent their classmates from learning. AL-Jarf (2006) recorded that "large class sizes inhibit small group activities and individualized instruction, because of the noise level and lack of space in the classroom" (p. 24). Admittedly, some researchers have found that classroom noise is a result of students' boredom, vague instructions to activities presented in class and teachers' low voice (Harmer, 2000; Lacastro, 2001 and Zhang, 2008). 1.1 Significance of the Study Considering teaching English in large classes as a great problem and a real world issue which is worrying Saudi English teachers of all levels in general and secondary teachers in particular, this study aims to investigate the effect of large English classes on the teaching and learning process and suggest some useful strategies to cope with this dilemma as to prompt the language level. To do this, six Saudi secondary English teachers were interviewed to express their point of views towards this phenomenon and to discuss the strategies they have adopted to promote effective teaching and learning in their overcrowded classes. It is the researcher's belief that the findings of the study will help all stakeholders to have a clearness view of the case which may raise the awareness of the problem and add to the body of knowledge about teaching and learning in large classes. 1. Introduction It is anticipated that large English classes do not offer ideal setting where effective teaching and learning could take place. For example, student-centered teaching/learning is almost impossible in large classes. Nevertheless, a strong willingness to investigate the effect of large classes on English teaching according to teachers' perceptions made the researcher undertake this study where the main theme of this study is to list the problems Saudi secondary teachers/students encounter when learning English in large classes. Therefore, it is the researcher intention to draw on a selection of studies from different research to, firstly, examine the study main goal which is the influence of class size on English teaching and learning, secondly, provide personal examples culled from actual experience, thirdly, recognize the issues of concern to English teachers facing these large classes and finally, furnish some guidance to facilitate the teaching and learning process. 2. Literature Review Although, there is vast literature on issues regarding teaching and learning English in large classes yet the issue 50 English Language Teaching Vol. 6, No. 11; 2013 www.ccsenet.org/elt still invites many different areas which ought to be explored. This study concentrates on the challenges teachers and students encounter daily in large classes. Through reading the literature, the following studies have been found to quiet closely related to the present study. still invites many different areas which ought to be explored. This study concentrates on the challenges teachers and students encounter daily in large classes. Through reading the literature, the following studies have been found to quiet closely related to the present study. Many researchers believe that large classes offer few opportunities to teachers to employ quality teaching and learning environment for learners (Blatchford et al., 2002; Hattie, 2005; Pedder, 2006). Zhang (2002) examined the effect of large classes among college students and proposed that problems are one of three or all. First, discipline problems. Second, students' and teachers' weary. Third, efficient learning. Similar finding was also allocated by Yu (2004) who conducted a study on college students as well studying in large classes. The study showed that individuals' variant is neglected and anxiety is a class predominant. She also pointed that limited practice for the target language due to the large number of students hindered students from improving their level of oral English. Such a conclusion also appeared in a study conducted by Harmer (2000) confirming that large classes bring difficulties to the teaching and learning process in general and to teachers and students in particular as with the big number, teachers find it difficult to organize effective and creative activities, pay attention or contact with all individuals especially those who tend to hide by sitting at the back. Another interesting conclusion found in Harmer's study is that large classes are troubling for experienced teachers but extremely daunting for the new teachers and that emphasize the need of good training and sufficient technical strategies to be employed in large English classes. The literature review also reveals that the number of studies conducted in the developing countries about class size and large classes are not enough compared with those addressing the issues in the developed world. Therefore, more studies are needed to tackle this field in countries like Saudi Arabia, Oman, Egypt and Jordan. 2. Literature Review Kumaravadevelu (2006) believed that there is a need for a pedagogical method to investigate the features of large classes in the developing countries and that will provide teachers of those contexts with helpful strategies suitable for their students. Locastro (1989) conducted a study in three urban locations in Japan, Tokyo, Nagoya and Kyoto to investigate the attitudes and expectations of English teachers teaching large classes where the Ministry of Education has no policy on class size. The participants were university teachers as well as teachers and students from different secondary public schools. Classes in schools ranged from 40 – 50 students while in university they ranged from 20 – 100 students. The researcher was able to collect 96 responses from teachers and lecturers and 94 from students. 33 out of 47 teachers and lecturers were familiar to teaching large classes where they had 41 to 60 students per class. They believed that the average class size is 38 as reaching 39 and above tends to create a serious problem for them and their students. Among the 94 students, 35 had experienced being in classes of 40 to 49 students and only 12 had great experience to be in classes with 200 to 209 classmates and only 8 students studied in classes which had 300 to 309 students. The study showed that 51 students from those who experienced large classes prefer to be in classes not fewer than 11 and not more than 20. Interestingly enough, 4 students rated a class of 41 students to 51 as ideal. Some students considered a class of 21 students and up to 30 too large while others thought a class of 10 students or fewer is too small. Therefore, Locastro suggested that for effective language teaching to take place in large classes, appropriate methodologies and practices should be applied. The findings of this study were also allocated in different studies carried by other researchers on primary and secondary school students (Su, 2005; Tan, 2009; Zhang, 2010). Other studies have also found that students perform better in large classes than in small ones as Pong and Pallas (2001) hypothesized that this could be because teachers with more experience are assigned large classes as they are able to control them but new teachers or less experienced are given small classes which have small numbers of weak students. 2.2 Small or Large Classes: Which Are Better? As mentioned at the outset of this paper, there are some scholars who favor small classes as they believe that small classes provide ample opportunities for teachers and their students to accomplish the highest level of language achievement. One of these is Miller-Whitehead (2003) who suggested that small classes assist raising teachers’ spirit and decrease most of the discipline issues. She elaborates by stating that small classes allow teachers to identify problems quickly and then apply immediate remedial solution and that will eliminate or at least minimize any future disruption. On the other hand, Finn et al. (2003) revealed that class size has great affects on students' social and academic involvement in the class and on the teacher personality as well. That is to say that students in small classes, on contrary to their peers in large ones, are always under pressure to participate in class activities as "they are on the frying line, by being more visible to the teacher and may be called upon at any time to answer questions or to participate in a class activity" (p. 346). On a similar view, Resnick (2003) claimed that smaller classes elevate students' achievement as teachers in such context pay greater attention to each and every student leaving students with no time to either be destructive or distracted by any means. Consequently, students in small classes encounter continuous pressure to engage in various activities and become active class members and that will make the attention to learning goes up and off-task actions as well as any disruptive behaviors go down. On a similar view, Normore and Ilon (2006) conclude that classes of a small size positively influence the teaching process as they encourage students and teacher engagement, allow students to be more cognitively engaged, offer ample time for teachers to cover the whole materials and provide safe school environment with fewer misbehaved students. Finn et al. (2003) conducted a study to examine how class size affects the social behavior of students and sought to focus on antisocial attitudes such as disruptiveness and misbehavior. From the result of their study, they found that small classes encounter less misbehavior actions than large classes as students in small classes are engaged in more social interactions with their teachers which give them less space to misbehave or fool around. Shamim et al. 2.2 Small or Large Classes: Which Are Better? (2007) reinforced this idea when they revealed that teachers in large classes have problems establishing discipline in their classrooms and dealing with the increasing noise level and that according to Al-Jarf (2006) creates difficulty for students to hear the teacher and concentrate because they are distracted by the actions of their peers. From what have been mentioned in this part, it is to say that large classes have negative influence on teachers and students which reflects on the teaching and learning outcomes. 2.1 The Concept of Large Classes The effectiveness of class size on students’ achievement and motivation, and its synchronous relation to teaching process and teachers' workload, attitudes and motivation, is probably the most written about, however least explored topic in the educational field. Yet, there is no consensus definition in literature to what constitutes a large class as material developers, teachers and students in different parts of world have various perceptions of what frames large, small or ideal classes. Their ideations, therefore, are affected by the educational philosophies, theories and experience of all parties involved in the teaching and learning process. According to Hayes (1997) there is no numerical determination of what shape a large class as teachers’ perceptions of large classes differs from one context to another. Hess (2001) assumed that a class is considered large if it has 30 students or more. As regard to the Saudi context, a class of 45 students is considered to be large while in some Far East countries such as Japan, China, Pakistan and India a class of 80 students and more is deemed to be large. 51 English Language Teaching Vol. 6, No. 11; 2013 www.ccsenet.org/elt Ur (1996) believed that regardless to the number of students in a class, it is teachers' perceptions towards the class size in a certain context with particular tools and facilities that are provided that make classes either small or large. Hence, we can say that large classes are those with a specific number of students that teachers cannot handle and resources are not enough to facilitate the teaching and learning process and that pose insurmountable problems for both teachers and students. Mulryan-Kyne (2010) also shares that view and points to a large class as "a class that is too large for effective teaching to occur" (p. 176). Brown (2001), on the other hand, believed that an ideal class should not exceed 12 students. In another word, a class should be big enough to offer variation and allow interaction and small enough to provide students with opportunities for participating and receiving individual attention. 3.1 Research Site This study was conducted in two public secondary schools in the city of Jeddah. Classes in government schools vary in terms of size as the average number of students is from 30 to 45. Such number of students is found in schools in the south, west and east parts of the city where schools in the north side of Jeddah usually have fewer students. This variance is due to the number of private schools in the north where most of the upper class families live and those are the ones who could afford the expenses of private schools. All schools in Saudi Arabia, private or government are single sex, where female teachers teach only in girls' schools and male teachers teach boys only. 3. Methodology The main goal of this study is to explore the Saudi secondary teachers' perception towards teaching English in large classes and to determine the practical methodology to teach English in such context. Therefore, it is necessary to obtain the insider's view of the case by studying the participants' perceptions and the effect of class size on the teaching and learning process. In a case as such, a qualitative approach seems best convenient to classify these issues as it is interested on people as the tool of inquiry. It also consists of data that focuses on observation, interviews and surveys. In words, it is needed to provide deep understanding of the factors effecting teaching and learning in large classes. For collecting the data, this exploratory qualitative research employs personal interviews of six Saudi secondary English teachers. The conducted interview is a semi structured one (see Appendixes A) where the "interviewer has a set of predetermined questions, but the interviewer is free to follow up a question with additional questions that probe further" (Perry, 2005: 119). Interviews let the researchers comprehend the phenomenon of learning desire through the interviewees' points of view not as the researchers might perceive it. In this interview, the researcher concentrated on class size and asked each participant to describe the size of her class using a four point scale as follow:1) very large, 2) large, 3) average, 4) small. Based on their answers, more open- end questions were asked regarding their perceptions of the appropriate number of students in each class and the reasons of their preference. 2.3 Improving Learning in Large Classes In words, engaging students in challenging activities where individuals or groups compete with one another would bring positive effects of large number in classes. Moreover, creating interesting activities that make students effectively occupied at the same time will increase students' on-task behavior, minimize students' boredom and bring loafing to its lowest level and that of course will promote the language learning. On a study conducted by Adrian (2010) on college students, he finds that active learning involves "students in doing things and thinking about the things they are doing" (p. 2). Another beneficial remedy to overcome any problem tends to occur in large classes is to allow students to play an active role in class and involve them in all decision making about the classroom life as controlling the noise level or handling any disciplinary actions by setting rules and punishment among groups or individuals. Renaud, Tannenbaum & Stantial (2007) believe that students are willing to abide by rules they set and not only that but they will make sure that these rules are effectively applied. In words, such action is expected to be useful in possible growth of some fruitful and compatible strategies in order to minimize the level of disturbance in class and maximize the level of learning. Other scholars have also emphasized the importance of students' evaluation as a way of promoting the learning level (Blatchford et al., 2003; Al-Jarf, 2006 and Herington & Weaven, 2008). Shepard (2000) reiterates this idea by stressing that assessing and evaluating students is a significant tool to the teaching and learning process, that: To the right is an emergent, constructivist paradigm in which teachers' close assessment of students' understandings, feedback from peers, and student self- assessments would be a central part of the social processes that mediate the development of intellectual abilities, construction of knowledge, and formation of students' identities (p. 4). 2.3 Improving Learning in Large Classes With the great number of students enrolling each year in schools and the limited facilities, reducing class size in some teaching context in the developing countries like Saudi Arabia seems to be impossible. Therefore, many scholars have proposed shifting from concentrating on class size to seeking more realistic and effective ways of coping with the situation and exploring the kind of teaching, either in large or small classes, that can produce a difference (Blatchford & Mortimore, 1994; Blatchford, 2003 and O'Sullivan, 2006). In order to reach this goal, some of the useful advices that have been mentioned in the literature for effective large class teaching will be considered. It is believed that there is no one way to teach large classes. Therefore, a substantial body of research suggests that for effective teaching and learning in large classes, teachers have to consider the followings: the course objectives; the characteristics of their students; and their teaching styles. Some studies have emphasized that it is not the class size that affects the learning process yet it is the teacher's quality and methodology that greatly counts (Maged, 1997; Nakabugo, 2003 and O'Sullivan, 2006). Even though, teachers believe it is extremely important to decrease the number of students in classes, they confirm the significance of finding alternative teaching methodologies that are beneficial for elevating language learning in large classes. One way that is considered to be significant is to set good rapport between teachers and students and among students themselves which will help to minimize the anxiety where all in the class will feel as part in the same learning context. In spite of the different constraints large classes have on the teaching and 52 English Language Teaching Vol. 6, No. 11; 2013 www.ccsenet.org/elt learning outcomes, many teachers attempt to improve their level of teaching by employing multiple techniques such as team teaching, group work, reduce the workload and utilize whole class discussion. Chen (2005); Yazedjian et al. (2007) and Herington & Weaven (2008) confirm that forming small group of students help to reduce the noise, save the time of performing a certain activity and allow teachers to concentrate on small numbers of groups instead on focusing on many individuals. It is also a useful technique to bring the distractions to its lowest limit by involving every student in the learning process. 3.2 Participants Most public secondary schools in Saudi Arabia have about four or five English teachers due to the large number of classes in this level. Two of these schools have been chosen to be the site of this research. Upon the researcher 53 English Language Teaching Vol. 6, No. 11; 2013 www.ccsenet.org/elt visit to both schools and after presenting the research plan to all English teachers, as the intention was to ask four volunteers from each school to join the research but only three teachers from each school were interested to take part. Before discussing my research in the introductory meeting, the researcher had to express her sincere gratitude to the volunteers who willingly joined even though they have a very busy schedule with not only teaching but also correcting note books and work books, cover the class of any absent teachers and take part, at least 2 periods weekly, in a school general activity. visit to both schools and after presenting the research plan to all English teachers, as the intention was to ask four volunteers from each school to join the research but only three teachers from each school were interested to take part. Before discussing my research in the introductory meeting, the researcher had to express her sincere gratitude to the volunteers who willingly joined even though they have a very busy schedule with not only teaching but also correcting note books and work books, cover the class of any absent teachers and take part, at least 2 periods weekly, in a school general activity. Six Saudi 2nd and 3rd grade English teachers at two secondary schools in Jeddah had joined this study. All participants have taught classes which they consider large but have no experience of teaching smaller ones. All participants have long experience, 15 years and more, in teaching English to large classes. Such long experience seems to be a significant factor since the teachers' perceptions about large classes and the way they deal with them will be beneficial sources of data for this study. In the last ten years of their teaching, all participants have experienced teaching large classes ranging from 35 to 48 students per class with limited space and insufficient technical materials as tapes, tape recorder or data show. Yet, students' books, workbooks and teachers' book are always provided to all at the beginning of each academic year. 3.2 Participants The aim of the study was explained to participants as to gain some understanding of their attitudes and beliefs towards teaching in large class which will help to expand the knowledge of this significant phenomenon. All participants were interviewed once where the questions concentrated on their experience in teaching large classes in secondary level, the challenges they encounter and the strategies they employ to overcome the problem. The transcripts were coded and analyzed according to teachers' responses to the research questions. Table 1 below displays interviewees' information. Table 1. Demographic information of interviewees Participants Education level Age Years of teaching experience No. of classes per day Average No. of students per class P. 1 BA/College of Arts & Humanities/English Language Dept 38 10 4 35–45 P. 2 BA/College of Arts & Humanities/English Language Dept 40 13 5 30–48 P. 3 BA/College of Arts & Humanities/English Language Dept 40 11 4 30–40 P. 4 BA/College of Arts & Humanities/English Language Dept 43 18 4 35–50 P. 5 MA/College of Arts & Humanities/English Language Dept 45 15 3 32–48 P. 6 BA/College of Arts & Humanities/English Language Dept 45 10 5 35–45 Table 1. Demographic information of interviewees 4.2 Managing Large Classes One of the most problems related to large classes is classroom management. Therefore, it is certainly much of the art that large classes must involve assertiveness, leadership and active teacher who is able to control most of the class actions. To this, participants acknowledged that clear and strict rules must be declared at the beginning of the class and it is the teachers' role to effectively apply those rules when needed and may become a bit lenient when actions turn out to be under control. Discussing questions that are related to classroom management, participant (4) professes that "when meeting students for the first time, setting classroom' rules is one of my priorities. She carries on "I find it very useful to discuss all my rules, write them in Arabic and English and then pass them around for all students to read and sign. Not only have that, I even encouraged my students to set forth a punishment for those who may not abide by the class rules. She also raises another point by saying "I don't mind teaching large number of students where there is adequate space for me to move around and be able to reach the students at the back to control any noise or distraction". On the other hand, as a respond to the question about the number that makes a class large or small, participant (3) says "for me a class of 35 students is considered to be good where as a class of 25 is ideal but 36 and more is really a big class". 4. Data Analysis and Findings Analyzing the data is a step that a researcher makes to understand and interpret all of what have been said or seen. It is according to Merriam (2009) the process of making meaning. Through analyzing the interviews, the researcher could list a number of reasons that all participants believe to hinder the effectiveness of learning and teaching process and these are: 1) Mob manners of some students force teachers to focus on behaving students instead of using the time on more productive actions. 1) Mob manners of some students force teachers to focus on behaving students instead of using the time on more productive actions. 2) Classes with big number of students don't provide teachers with neither amble chances nor time to correct 2) Classes with big number of students don't provide teachers with neither amble chances nor time to correct 54 Vol. 6, No. 11; 2013 English Language Teaching www.ccsenet.org/elt students either in their oral production or the written form. students either in their oral production or the written form. 3) Due to the number of classes teachers are teaching, 3 to 5 per day, which have big numbers of students up to 50, teachers have less opportunity to assess and evaluate their students' work and achievement, discuss their problems or provide any useful and constructive feedback. 4) Diversity among students confuses and puts too much pressure on their teachers leaving them mentally and physically exhausted. 4) Diversity among students confuses and puts too much pressure on their teachers leaving them mentally and physically exhausted. 5) Students in large classes receive less individual attention than their peers in small classes and that leads to dissatisfaction among students especially weak ones who will feel marginalized. 5) Students in large classes receive less individual attention than their peers in small classes and that leads to dissatisfaction among students especially weak ones who will feel marginalized. 6) Students in large classes seem to be demotivated as they show no interest in either the lesson explained or activities presented by the teacher and that is due to the limited or lack of teachers' support. 6) Students in large classes seem to be demotivated as they show no interest in either the lesson explained or activities presented by the teacher and that is due to the limited or lack of teachers' support. 4. Data Analysis and Findings 7) Big number of students with limited space in class does not allow teachers to move around freely to monitor, observe and assess students during activities. 7) Big number of students with limited space in class does not allow teachers to move around freely to monitor, observe and assess students during activities. 8) With the growth of class size, students have less chance to effectively participate and use the target language in class. As a consequence, loss of language interest will be class dominant. 8) With the growth of class size, students have less chance to effectively participate and use the target language in class. As a consequence, loss of language interest will be class dominant. Despite all the above mentioned problems which are believed to negatively impact the teaching and learning process, through their long experience in teaching and from the useful suggestions presented in workshops and seminars as well as discussion with peers, participants assume that concentrating on the teaching process and implementing various techniques lead to effective English teaching and learning. In the following section, some of the participants' remedial solutions are listed and elaborated. 4.1 Challenges of Large Classes Even though all participants believe that teaching large classes is a huge burden as they end up feeling exhausted, they also find lots of opportunities and challenges while teaching big number of students. One of the participants (P.3) asserts that "teaching large classes provides me with many ways to improve my teaching". When asked about her preference for teaching small or large classes, participant (6) concedes that "even though I prefer to teach small classes, I find my current class (45 students) enjoyable, rewarding and challenging as we (me and my students) always dig for ways that help us to sail safely through the extreme situation". Similarly, participant (1) strongly admits that large classes provide her with many chances to develop her organizational talent and use all managerial proficiency she acquired during her teaching. By applying that, she manages to creatively organize the large classroom into a convenient welcoming learning setting. Agreeing to that, participant (5) confirms that "large classes are interesting and stimulating because they challenge me to try different methods and always come up with applicable solutions to make the class conducive to learning". 4.4 Small Group not the Whole Class Regarding the big number of students in class, all participants strongly express the difficulty to monitor, observe or correct individuals. Therefore, in certain tasks, participant (1) tends to apply the technique of dividing her class into small group which makes it easier and less hectic to monitor 8 groups instead of going around 45 students. She mentions that "one of the biggest problems I encounter in my large class is observing the class while performing activities. Therefore, dividing my students into small groups facilitates my job and saves my time". Participant (6), who managed to find remedial solutions, declares "games, role plays and interviews are my favorite methods. By dividing the class into small group and involving each member in a certain task which is relevant to her needs, I manage to create a highly competitive setting with less or almost no disturbance. In this regard, Cooper and Robinson (2000) found that small-group work can "contribute to effective teaching and learning by promoting cognitive elaboration, enhancing critical thinking, providing feedback, promoting social and emotional development, appreciating diversity and reducing student attrition" (p. 181). From all that mentioned above, we can say that it is possible for English to be taught and improved in large classes in Saudi secondary schools by producing an interactive model of teaching/learning process where there is a great interaction between teachers and students and among classmates in the shape of pair and group work. It is also recommended that even though large classes are daunting and lead to huge burden, English teachers should not be distracted by any chaotic situation and carefully plan to control the class as well as to create challenging activities, employ appropriate materials and valid methodology which may result on great impact on English teaching. It should emphasized that teachers have to be flexible and adventurous by using various teaching strategies, detecting problems created by large classes, identifying their students' needs and then gear teaching to overcome the problem. 4.3 Students-Centered rather than Teacher-Students Approach Generally speaking, teaching all subjects in Saudi Arabia, where English is no exception, involves the traditional passive view of teaching where teachers are spoon feeding the information to students and presenting the materials to them through a lesson-based format. Recently, many seminars and workshops for all subjects are held to concentrate on a more modern view of leaning to shift from the old method of delivering information to students to more active and engaged students. From the finding of their study, De Capraiis, Barman & Magee (2001) suggest that lesson directs students to remember facts while group discussion produces positive outcomes 55 English Language Teaching Vol. 6, No. 11; 2013 www.ccsenet.org/elt and leads to higher level of thinking, recognition and elaborating. Participants, in this study, strongly affirmed that encouraging students to be active members in class by dominating class discussion leads to efficacious language learning. They also emphasize the importance of collaborative tasks as group work, pair work and peer editing. As a respond to Q. 1 about her teaching method, participant 4, points out that "in large classes, I always try different methods that facilitate the learning process". She explains "if method X is not good enough, I switch to another one. Saying more "I started using the teacher-centered approach then realized it is not working effectively so I turned to more effective method which is students-centered and only then I found that my students are becoming more responsible and much involved in the learning process which make them better recipients rather than passive learners". Interestingly enough, through her long experience of teaching, participant (5) finds it difficult to assess all individuals in the class and that encourages her to search for a leading method which offers the best for all in her class. Thus, implementing group work helps her to generate more interactive settings and embrace affective teaching environment which elevate her students' autonomy and arise their responsibility towards learning. On the other hand, participant (2) concludes that peer editing encourages her students to take part in different writing tasks starting with easy task to high level of editing and that boosts students' awareness of the writing task and elevates their self-confident through correcting others' work. 5. Limitations of the Study The general purpose of the present study is to determine what problems are presented in teaching and learning English in large classes and to find remedial solutions to elevate the teaching/learning process. However, it must be admitted that this study is a personal effort that may have shortcomings that can be tackled in other studies. From those, first, the small number of participants in this study (6 teachers) constitutes a clear limitation as the represented data generated from their interviews may not be enough to generalize the result to other contexts either inside or outside Saudi Arabia. Further, the data is collected from English teachers teaching large classes in secondary schools in Saudi Arabia where the strategies proposed by the review may not be suitable for the above mentioned context as it has been proven that teaching success or failure is affected by context. Moreover, as mentioned earlier, the methodology used for data collection is interviews only with no classes' observation while including observational method would allow the researcher to actually see a real practice in large classes and that would enrich the data. In a word, the problem of teaching and learning English in large classes is a large domain with many aspects that can not be tackled in one study. Yet, it seems fair to say that the result of this study is a path to others that leads to understand this dilemma where ample chances are provided for further investigation to find remedial solution and that will bring a new dynamism to the body of literature about teaching English in large classes in the developing countries. It will also offer teachers with deep understanding 56 English Language Teaching Vol. 6, No. 11; 2013 www.ccsenet.org/elt about the realities of large classes and encourage them to share ideas and exchange experiences with others in similar contexts. about the realities of large classes and encourage them to share ideas and exchange experiences with others in similar contexts. 6. Conclusion Synthesizing the earlier views, we can say that large classes are not firmly a pedagogical dilemma as the complications found in large classes raise more demands and actions from language teachers in large classes compared with their counterparts teaching smaller ones. Taken together, the literature review and the study findings we can say that class size has a significant role in the teaching and learning process. Therefore, it is important to explore various methods and apply effective strategies that minimize the effects of large classes and elevate the teaching and learning level to its highest standard. Hence, the effectiveness of any techniques may vary from one context to another as it relies heavily on different aspects such as students, teachers and facilities available in a certain school. References Adrian, L. M. (2010). Active learning in large classes: Can small interventions produce greater results than are statistically predictable? Active learning in large classes. The Journal of General Education, 59(4), 223-237. Al-Jarf, R. (2006). Large student enrollments in EFL programs: Challenges and consequences. Asian EFL Journal Quarterly, 8(4), 8-34. Blatchford, P. (2003). A systematic observational study of teachers' and pupils' behavior in large and small classes. Learning and Instruction, 13, 569-595. Blatchford, P., & Mortimore, P. (1994). The issue of class size for young children in schools: What can we learn from research? Oxford Review of Education, 24, 411-428. Blatchford, P., Moriarty, V., Edmonds, S., & Martin, C. (2002). Relationships between class size and teaching: A multimethod analysis of English infant school. American Educational Research Journal, 39(1), 101-132. Brown, H. D. (2001). Teaching by principles. White Plains: Addison Wesley Longman, Inc. Chen, P. (2005). Effectively implementing a collaborative task-based syllabus (CTBA) in EFL large-size Business classes. English for Specific Purpose World, 1-14. Cooper J. L., & Robinson, P. (2000). The argument for making large classes seem small. New Directions for Teaching and Learning, 81, 5-16. De Caprariis, P., Barman, C., & Magee, P. (2001). 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B. (2009). Qualitative research: A guide to design and implementation. San Francisco, CA: Jossey-Bass. Miller-Whitehead, M. (2003). Compilation of class size findings: Grade level, school and district. Paper presented at the Annual Meeting of the Mid-south Educational Research Association. Mulryan-Kyne, C. (2010). Teaching large classes at college and university level: Challenges and opportunities. Teaching in Higher Education, 15(2), 175-185. Nakabugo, M. G. (2003). Closing the gap? Continuous assessment in primary education in Uganda. Thesis presented for the Degree of Doctor of Philosophy, Graduate School in Humanities, University of Cape Town. Normore, A., & Ilon, L. (2006). Cost-effective school inputs: Is class size reduction the best educational expenditure for Florida? Educational Policy, 20, 429-454. O’Sullivan, M. C. (2006). Teaching large classes: The international evidence and a discussion of some good practices in Ugandan primary schools. 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Zhang, L. (2010). Problems of teaching English in primary large classes in rural areas and application of group study. Journal of Guangxi Normal University for Nationalities, 27(1), 129-131. Appendix 11) What are the roles your students have in large classes? 12) In your opinion what is the ideal students' number that facilitates the learning process? 13) Throughout your years of teaching, what factors have influenced you the most? 14) What instructional techniques do you use to maximize the level of outcome in large class? 15) Do you use any sort of technology? If yes, how? If no, why? 16) Do you feel stratified about what you have offered to your students? If yes, why? If no, why? Appendix Teachers' Interview Teachers' Interview 1. Personal details: 1) Name 2) Qualifications 3) Teaching experience 58 Vol. 6, No. 11; 2013 English Language Teaching www.ccsenet.org/elt 4) School 5) Grade 6) No. of classes per term 7) No. of students in each class 2. Teaching methodology 1) What kind of teaching method you employ in class? 2) As a teacher list some of your strengths points and weaknesses (if any). 3) Do you perceive your class as very large, large, average or small? Justify! 4) What is the students' number that makes the class large? 5) Do you prefer teaching large/small classes? Why? 6) What are the advantages of large/small classes? 7) In your opinion what are the disadvantages of large/small classes? 8) In your opinion what is the most difficult task for your students to perform in Large classes? 9) In your opinion how do large classes constrain teachers, affect students and hinder the learning process? 10) What are your best methods to control any misbehaved actions? 11) What are the roles your students have in large classes? 12) In your opinion what is the ideal students' number that facilitates the learning process? 13) Throughout your years of teaching, what factors have influenced you the most? 14) What instructional techniques do you use to maximize the level of outcome in large class? 15) Do you use any sort of technology? If yes, how? If no, why? 16) Do you feel stratified about what you have offered to your students? If yes, why? If no, why? 4) School 5) Grade 6) No. of classes per term 7) No. of students in each class 2. Teaching methodology 1) What kind of teaching method you employ in class? 2) As a teacher list some of your strengths points and weaknesses (if any). 3) Do you perceive your class as very large, large, average or small? Justify! 4) What is the students' number that makes the class large? 5) Do you prefer teaching large/small classes? Why? 6) What are the advantages of large/small classes? 7) In your opinion what are the disadvantages of large/small classes? 8) In your opinion what is the most difficult task for your students to perform in Large classes? 9) In your opinion how do large classes constrain teachers, affect students and hinder the learning process? 10) What are your best methods to control any misbehaved actions? Copyrights yright for this article is retained by the author(s), with first publication rights granted to the journal. Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 59
https://openalex.org/W4210368908	https://jamsi.jurnal-id.com/index.php/jamsi/article/download/178/96	Indonesian	null	Memperdalam Kemampuan Berbahasa Inggris Anak Panti Asuhan (Studi Pada Anak Panti Asuhan Taruna Melati Pematangsiantar)	Jurnal Abdi Masyarakat Indonesia	2,022	cc-by	1,940	Jurnal Abdi Masyarakat Indonesia (JAMSI) https://jamsi.jurnal-id.com Jurnal Abdi Masyarakat Indonesia (JAMSI) https://jamsi.jurnal-id.com Vol. 2, No. 1 Januari 2022, Hal. 133-136 DOI: https://doi.org/10.54082/jamsi.178 Abstrak Permasalahan yang dihadapi oleh anak-anak di Panti Asuhan pada umumnya terkait dengan pembelajaran di masa pandemic seperti sekarang ini. Anak-anak panti yang berkisar dari SD sampai SMA memiliki kesulitan dalam belajar, termasuk belajar ber Bahasa Inggris. Selain karena Bahasa Inggris bukan bahasa sehari-hari yang membuat mereka asing menggunakannya, Bahasa Inggris juga menurut mereka adalah bahasa yang sulit. Hal ini banyak terjadi di banyak panti asuhan, seperti anak-anak di Panti Asuhan Taruna Melati Muhammadiyah Pematangsiantar. Mereka ingin bersaing dengan Bahasa Inggris karena Bahasa Inggris merupakan Bahasa Internasional. Solusi yang ditawarkan dalam kegiatan praktikum untuk Kelompok (Groupwork) ini adalah pendidikan secara komprehensif tentang pentingnya paham mengenai Bahasa Inggris, kemudian dilanjutkan menjawab pertanyaan berbahasa Inggirs. Program Intervensi yang digunakan dalam kegiatan Praktikum ini adalah Intervensi Mezzo oleh Zastrow yang terdiri dari; Persiapan (dengan Pendekatan Non-Direktif), Intake / Engangement, Assesment, Perencanaan, Implementasi dan Rencana Aksi, Evaluasi dan Terminasi. Hasil dari kegiatan ini adalah bertujuan agar anak-anak di Panti Asuhan ini mampu bersaing dengan teman-temannya yang lain, tidak ketinggalan, serta agar masa depan mereka lebih cerah dan cemerlang. Agus Suriadi1, Syifa Salsabila Damanik2 1,2Jurusan Kesejahteraan Sosial, Fakultas Ilmu Sosial dan Ilmu Politik, Universitas Sumatera Utara, Indonesia e-mail: agusur@gmail.com1, syifaadmk@gmail.com2 P-ISSN 2807-6605 \| E-ISSN 2807-6567 Keywords: Ability, English, Orphanages Abstract The problems faced by children in orphanages are generally related to learning during a pandemic like today. The orphanage children ranging from elementary to high school have difficulties in learning, including learning English. Apart from the fact that English is not their everyday language that makes them foreign to use it, English is also a difficult language for them. This happens a lot in many orphanages, such as the children at the Taruna Melati Muhammadiyah Pematangsiantar Orphanage. They want to compete with English because English is an International language. The solution offered in this group work is a comprehensive education on the importance of understanding English, followed by answering questions in English. The Intervention Program used in this practicum is the Mezzo Intervention by Zastrow which consists of; Preparation (with a Non-Directive Approach), Intake / Engagement, Assessment, Planning, Implementation and Action Plan, Evaluation and Termination. The results of this activity are aimed at making the children at the orphanage able to compete with other friends, not to be left behind, and so that their future is brighter. Keywords: Ability, English, Orphanages Jurnal Abdi Masyarakat Indonesia (JAMSI) https://jamsi.jurnal-id.com Vol. 2, No. 1 Januari 2022, Hal. 133-136 DOI: https://doi.org/10.54082/jamsi.178 Anak Panti Asuhan juga memerlukan perhatian dan kasih sayang. Apalagi di usia mereka yang menginjak remaja sangat lah memerlukan kehadiran orang tua yang berguna untuk memberikan semangat dan kasih sayang dari kedua orang tua. Anak yang berada di Panti Asuhan dituntut untuk menjadi orang yang mandiri, dan menjadi anak yang tidak bisa bersandar kepada orang tua, dan dituntut untuk menjadi dewasa sebelum waktunya. Peran keluarga apalagi orang tua sangatlah penting bagi keberhasilan tumbuh kembang dari sang anak. Hal ini di karenakan oleh orangtua bertanggungjawab untuk menyediakan lingkungan yang aman, memantau aktivitas anak, membantu mengembangkan emosi sosial dan kognitif, serta menyediakan arahan dan panduan dalam kehidupan sehari-hari. Dengan menyediakan lingkungan rumah yang aman dan kondusif, anak akan senang bermain, mengeksplorasi hingga menemukan berbagai hal baru yang dapat meningkatkan level perkembangan kognitif, sosial, dan emosional. Harapannya kelak dapat menjadi pribadi yang bertanggungjawab dan produktif. Kegiatan ini berlangsung selama kurang lebih 2 sampai 3 bulan di Panti Asuhan Taruna Melati Pematangsiantar. Dalam pembelajaran Bahasa Inggris ini, banyak sekali manfaatnya. Salah satunya adalah kesempatan mendapat pekerjaan lebih besar, dapat mengikuti perkembangan zaman, paham teknologi, dan meningkatkan pengetahuan. Di Indonesia Bahasa Inggris adalah bahasa asing, namun menempati posisi yang penting dalam keseharian masyarakat kita. Hal ini terlihat jelas dalam dunia pendidikan di Indonesia. Bahasa Inggris adalah salah satu pelajaran yang diajarkan kepada peserta didik mulai dari tingkat dasar sampai dengan perguruan tinggi. Selain daripada itu, Bahasa Inggris telah menjadi satu kata kunci yang sanggup menggenggam segala aspek, baik bisnis, politik, sosial, maupun budaya. Bahwa dalam belajar, tidak ada kata terlambat jika ada kemauan. Pendidikan bahasa asing untuk anak-anak di Amerika dan Eropa sudah dimulai sejak tahun lima puluhan dan menjadi sangat populer pada tahun enam puluhan, namun agak menurun pada tahun tujuh puluhan. Pendidikan dan pembelajaran bahasa Inggris didasari suatu pemikiran bahwa belajar bahasa asing atau bahasa kedua akan lebih baik bila dimulai lebih awal (Hammerby, 1982: 265). Banyak asumsi tentang usia dan pembelajaran bahasa antara lain adalah anak-anak belajar bahasa lebih baik dari pebelajar dewasa, pembelajaran bahasa asing disekolah sebaiknya dimulai seawal mungkin, lebih mudah menarik perhatian dan minat anak-anak daripada orang dewasa seperti diungkapkan oleh Ur (1996: 296). 1. PENDAHULUAN Setiono (2009:24) keluarga adalah sekelompok orang yang ada hubungan darah atau perkawinan. Seorang laki-laki dan seorang perempuan dari keluarga yang berbeda lalu disatukan dalam ikatan perkawinan atau pernikahan maka dapat dikatakan sebagai keluarga. Sepasang suami istri yang dikaruniai seorang anak maka antara orang tua dan anak dapat dikatakan sebuah keluarga karena disatukan oleh ikatan darah. Keluarga idealnya terdiri dari ayah, ibu, dan anak. Ayah dan Ibu merupakan orang terpenting bagi kehidupan dan keberlangsungan hidup anaknya. Anak yang kehilangan orangtua tentu akan memberikan pengaruh dalam tumbuh kembang anak tersebut. Anak menjadi kehilangan arah dalam kehidupan yang akan datang. Kehilangan satu atau dua orangtua dapat mengakibatkan masalah tertentu yang akan berpengaruh besar terhadap kehidupan anak dimasa yang datang dan masa depan sang anak. P-ISSN 2807-6605 \| E-ISSN 2807-6567 133 Jurnal Abdi Masyarakat Indonesia (JAMSI) https://jamsi.jurnal-id.com 3.1. Survei Pendahuluan dan Persiapan Materi Kegiatan Survei Pendahuluan ini pada awalnya dilaksanakan pada tanggal 19-Agustus-2021 di Panti Asuhan Taruna Melati Kota Pematangsiantar, yang bertempat di Jln. Sumbawa, Bantan, Kec Siantar Barat, Pematangsiantar, Sumatera Utara. Kegiatan ini bertujuan untuk menanyakan ketersediaan pengelola Panti Asuhan terhadap kegiatan pengabdian masyarakat. Setelah itu, diperoleh lah kesepakatan bahwa akan diadakannya kegiatan pengabdian masyarakat sekitar 2- 3 bulan lamanya. Setelah memperoleh kesepakatan tersebut, maka penulis juga menjelaskan kegiatan penjadwalan kegiatan, dan menjelaskan beberapa topik yang akan dilakukan di Panti Asuhan ini, salah satunya sosialisasi, dan belajar Bahasa Inggris. 2. METODE Metode yang dilakukan kepada klien pada kegiatan ini yaitu melalui metode Groupwork oleh Zastrow secara General, dan tahapannya adalah sebagai berikut: a. Intake dan Contract: berisi tentang pengenalan Klien dan perjanjian berapa lama proses intervensi akan dilakukan. b. Assesment: mendengarkan para klien memaparkan permasalahan individu, inti dan juga pernyataan masalah. b. Assesment: mendengarkan para klien memaparkan permasalahan individu, inti dan juga pernyataan masalah. c. Planning / Perencanaan: Pada tahap ini penulis merancang strategi berupa keberlanjutan atau pengaruh langsung dan juga merupakan teknik pertolongan yang mana dalam bimbingan sosial perorangan ini dilaksanakan setelah pekerja sosial memahami situasi klien dan mempunyai pengertian yang dalam masalahnya mengenai prosedur yang tertentu. Mungkin diantaranya adalah dengan memberikan dorongan (supportive relationship) juga menjelaskan persoalan (clarification of the problem). d. Intervensi dan Formulasi Program: Tentunya disini ada proses yang dilakukan oleh penulis. Penulis melakukan pendampingan untuk menghasilkan perubahan berencana dalam diri para klien. Pemberian edukasi tentang konsep pentingnya belajar Bahasa Inggris dan mengajarkan para kliennya untuk bisa suka dan paham tentang Bahasa Inggris. e. Evaluasi: tahap ini dilakukan penulis dengan memberikan pertanyaan-pertanyaan yang akan dijawab oleh klien. Pada tahap ini, Syifa mengetahui yang mana klien yang memang sudah paham dengan Bahasa Inggris dasar, dan yang mana yang belum. e. Evaluasi: tahap ini dilakukan penulis dengan memberikan pertanyaan-pertanyaan yang akan dijawab oleh klien. Pada tahap ini, Syifa mengetahui yang mana klien yang memang sudah paham dengan Bahasa Inggris dasar, dan yang mana yang belum. P-ISSN 2807-6605 \| E-ISSN 2807-6567 134 Jurnal Abdi Masyarakat Indonesia (JAMSI) https://jamsi.jurnal-id.com Vol. 2, No. 1 Januari 2022, Hal. 133-136 Vol. 2, No. 1 Januari 2022, Hal. 133-136 DOI: https://doi.org/10.54082/jamsi.178 f. Terminasi: pada tahap ini penulis menutup kontrak yang sudah disepakati diawal, dengan memberikan buku Bahasa Inggris agar klien mampu mendalami Bahasa Inggris dengan baik. f. Terminasi: pada tahap ini penulis menutup kontrak yang sudah disepakati diawal, dengan memberikan buku Bahasa Inggris agar klien mampu mendalami Bahasa Inggris dengan baik. P-ISSN 2807-6605 \| E-ISSN 2807-6567 4. KESIMPULAN Dalam pelaksanaan kegiatan dalam di Panti Asuhan Taruna Melati yang telah dilakukan, membawa pengaruh yang baik terhadap seluruh anak panti asuhan. Anak panti asuhan lebih rajin dan lebih minat untuk belajar Bahasa Inggris. Hal ini diapresiasi oleh bapak Pengelola Panti Asuhan karena membawa dampak yang positif dan baik pada anak-anak yang berada disitu. Saran untuk anak-anak yang ada di panti asuhan adalah agar lebih semangat lagi dalam belajar, termasuk belajar Bahasa Inggris agar masa depan lebih cemerlang. Jurnal Abdi Masyarakat Indonesia (JAMSI) https://jamsi.jurnal-id.com Jurnal Abdi Masyarakat Indonesia (JAMSI) https://jamsi.jurnal-id.com Vol. 2, No. 1 Januari 2022, Hal. 133-136 Vol. 2, No. 1 Januari 2022, Hal. 133-136 DOI: https://doi.org/10.54082/jamsi.178 Tidak hanya memberikan sosialisasi dan kegiatan tentang Bahasa Inggris, penulis juga memberikan edukasi mengenai bagaimana cara memakai masker yang benar, dan kesalahan apa saja yang sering dilakukan dalam menggunakan masker. Kegiatan ini juga dilakukan dengan menempelkan poster di dalam Panti Asuhan Taruna Melati mengenai topik tersebut. Gambar 3. Pemempelan poster. Gambar 3. Pemempelan poster. Hasil dari kegiatan ini adalah warga Panti Asuhan Taruna Melati Pematangsiantar sudah mulai merasakan bahwa Bahasa Inggris adalah Bahasa yang penting dan asik untuk dipelajari. Perbedaan dari sebelum belajar Bahasa Inggris sampia sudah belajar Bahasa Inggris adalah anak-anak di Panti Asuhan Taruna Melati Pematangsiantar lebih percaya diri, mampu berbicara kepada teman-temannya secara baik dengan menggunakan Bahasa Inggris dan mampu menjawab soal-soal Bahasa Inggris yang sederhana dengan mudah. UCAPAN TERIMA KASIH Ucapan terima kasih disampaikan kepada Bapak Pengelola Panti Asuhan Taruna Melati, Bapak Supriadi dan Istri yang telah banyak membantu penulis dalam kegiatan ini, serta menerima penulis layaknya seperti saudara sendiri. Serta anak-anak yang berada di Panti Asuhan Taruna Melati yang menyambut dengan ramah dan hangat sehingga kegiatan ini dapat berjalan dengan lancar. 3.2. Sosialisasi dan Kegiatan Pelatihan Kegiatan sosialisasi dan pelatihan mengenai Bahasa Inggris yang dilakukan di Panti Asuhan Taruna Melati Kota Pematangsiantar dilakukan selama seminggu dua kali terhitung dari tanggal 19 Agustus 2021. Pelatihan kegiatan mengenai Bahasa Inggris dihadiri oleh 10-12 orang warga panti asuhan tersebut, dan disambut dengan ramah dan hangat. g p g g Kegiatan awalnya bermula dari sosialisasi mengenai pentingnya mengetahui Bahasa Inggris, dan menjelaskan alasan-alasan yang menarik untuk belajar Bahasa Inggris yang bertujuan untuk menarik antusias warga panti asuhan tersebut mengenai Bahasa Inggris. Kemudian, dilanjutkan dengan membuat permainan-permainan dari Bahasa Inggris agar warga panti asuhan tersebut tertarik untuk belajar Bahasa Inggris. Warga panti asuhan tersebut juga diajak berbicara dengan menggunakan Bahasa Inggris secara mudah. Misalnya untuk menanyakan kabar, alamat, dan nama. Gambar 1. Proses Pembelajaran Bahasa Inggris. Gambar 1. Proses Pembelajaran Bahasa Inggris. Pada kegiatan pelatihan mengenai Bahasa Inggris ini, warga panti asuhan tersebut diberikan kesempatan yang sebesar-besarnya untuk bertanya apapun yang mereka tidak paham mengenai Bahasa Inggris. Warga panti asuhan tersebut juga menanyakan mengenai pekerjaan rumah mereka mengenai Bahasa Inggris. Beberapa warga panti asuhan tersebut juga sudah mulai paham mengenai Bahasa Inggris dasar. Gambar 2. Bermain permainan mengenai Bahasa Inggris. Gambar 2. Bermain permainan mengenai Bahasa Inggris. 135 P-ISSN 2807-6605 \| E-ISSN 2807-6567 Jurnal Abdi Masyarakat Indonesia (JAMSI) https://jamsi.jurnal-id.com Tamura, Elena Taralunga. (2006). Concepts on the methodology of Teaching English. The Economic Journal of Takasaki City University of Economics vol. 48 No. 3 pp. 169-188. DAFTAR PUSTAKA Hammerly, Hector (1982). Synthesis in Second Language Teaching. Blane: Second Language. Maduwu, Byslina. 2016. Pentingnya Pembelajaran Bahasa Inggris di Sekolah. Jurnal Warta Edisi: 50. Setiono, Kusdwiratri. 2011. Psikologi Keluarga. Bandung: PT. Alumni. Setiono, Kusdwiratri. 2011. Psikologi Keluarga. Bandung: PT. Alumni. Tamura, Elena Taralunga. (2006). Concepts on the methodology of Teaching English. The Economic Journal of Takasaki City University of Economics vol. 48 No. 3 pp. 169-188. Ur, Penny. (1996). A Course in Language Teaching. Cambridge: Cambridge University Press. P-ISSN 2807-6605 \| E-ISSN 2807-6567 136
https://openalex.org/W3109763027	https://iris.uniroma1.it/bitstream/11573/1473090/1/Durastanti_Environmental-impacts-cement_2020.pdf	English	null	Environmental Impacts of Cement Production: A Statistical Analysis	Applied sciences	2,020	cc-by	11,934	Claudio Durastanti 1 and Laura Moretti 2,* 1 Department of Basic and Applied Sciences for Engineering, Sapienza University of Rome, Via Antonio Scarpa 16, 00161 Rome, Italy; claudio.durastanti@uniroma1.it 2 D f Ci il C i l d E i l E i i S i U i i f R 1 Department of Basic and Applied Sciences for Engineering, Sapienza University of Rome, Via Antonio Scarpa 16, 00161 Rome, Italy; claudio.durastanti@uniroma1.it 2 Department of Civil, Constructional and Environmental Engineering, Sapienza University of Rome, Via Eudossiana 18, 00184 Rome, Italy * Correspondence: laura.moretti@uniroma1.it; Tel.: +39-06-44-58-51-14 * Correspondence: laura.moretti@uniroma1.it; Tel.: +39-06-44-58-51-14 Received: 6 November 2020; Accepted: 17 November 2020; Published: 19 November 2020 Abstract: The attention to environmental impacts of cement production has grown fast in recent decades. The cement industry is a signiﬁcant greenhouse gases emitter mainly due to the calcinations of raw materials and the combustions of fuels. This paper investigates on the environmental performances of cement production and on the identiﬁcation of factors driving emissions. For this purpose, a sample of 193 diﬀerent recipes of gray cement produced in Italy from 2014 to 2019 according to the European standard EN 197-1. This paper identiﬁes the consumption impact categories (e.g., fossil fuels, renewable and non-renewable secondary fuels) that explain the assessment of the Global Warming Potential, one of the most crucial impacts of cement production. Having regard to the overall examined dataset and each cement type, a set of predictive models is implemented and evaluated. A similar approach has been adopted to produce accurate predictive models for further environmental impact categories that quantify emissions to air. The obtained results provide important information that can support cement producers to develop low-impacting cement recipes. Keywords: life cycle analysis; cement production; EN 15804; regression analysis; AIC criterion applied sciences Article Environmental Impacts of Cement Production: A Statistical Analysis Claudio Durastanti 1 and Laura Moretti 2,* 1 Department of Basic and Applied Sciences for Engineering, Sapienza University of Rome, Via Anto Scarpa 16, 00161 Rome, Italy; claudio.durastanti@uniroma1.it 2 Department of Civil, Constructional and Environmental Engineering, Sapienza University of Rome Via Eudossiana 18, 00184 Rome, Italy * Correspondence: laura.moretti@uniroma1.it; Tel.: +39-06-44-58-51-14 Received: 6 November 2020; Accepted: 17 November 2020; Published: 19 November 2020 Abstract: The attention to environmental impacts of cement production has grown fast in decades. The cement industry is a signiﬁcant greenhouse gases emitter mainly due to the calcin of raw materials and the combustions of fuels. This paper investigates on the environm performances of cement production and on the identiﬁcation of factors driving emissions. F purpose, a sample of 193 diﬀerent recipes of gray cement produced in Italy from 2014 to 2019 acc to the European standard EN 197-1. This paper identiﬁes the consumption impact categorie fossil fuels, renewable and non-renewable secondary fuels) that explain the assessment of the Warming Potential, one of the most crucial impacts of cement production. Having regard to the examined dataset and each cement type, a set of predictive models is implemented and eva A similar approach has been adopted to produce accurate predictive models for further environ impact categories that quantify emissions to air. The obtained results provide important inform that can support cement producers to develop low-impacting cement recipes. applied sciences Article Environmental Impacts of Cement Production: A Statistical Analysis Claudio Durastanti 1 and Laura Moretti 2,* 1 Department of Basic and Applied Sciences for Engineering, Sapienza University of Rome, Via Anto Scarpa 16, 00161 Rome, Italy; claudio.durastanti@uniroma1.it 2 Department of Civil, Constructional and Environmental Engineering, Sapienza University of Rome Via Eudossiana 18, 00184 Rome, Italy * Correspondence: laura.moretti@uniroma1.it; Tel.: +39-06-44-58-51-14 Received: 6 November 2020; Accepted: 17 November 2020; Published: 19 November 2020 Abstract: The attention to environmental impacts of cement production has grown fast in decades. The cement industry is a signiﬁcant greenhouse gases emitter mainly due to the calcin of raw materials and the combustions of fuels. This paper investigates on the environm performances of cement production and on the identiﬁcation of factors driving emissions. F purpose, a sample of 193 diﬀerent recipes of gray cement produced in Italy from 2014 to 2019 acc to the European standard EN 197-1. This paper identiﬁes the consumption impact categorie fossil fuels, renewable and non-renewable secondary fuels) that explain the assessment of the Warming Potential, one of the most crucial impacts of cement production. Having regard to the examined dataset and each cement type, a set of predictive models is implemented and eva A similar approach has been adopted to produce accurate predictive models for further environ impact categories that quantify emissions to air. The obtained results provide important inform that can support cement producers to develop low-impacting cement recipes. applied sciences Environmental Impacts of Cement Production: A Statistical Analysis Claudio Durastanti 1 and Laura Moretti 2,* 1. Introduction In recent years, the interest for environmental protection has grown faster, becoming an important criterion for public policy in social and political contexts [1,2]. The most pursued objective is to reduce emissions of greenhouse gases (e.g., carbon dioxide, nitrous oxide and methane), which are responsible for the greenhouse eﬀect [3]. In particular, the cement industry contributes about 5% of global anthropogenic CO2 emissions excluding land-use change [4,5], as the production of the binder is a highly energy-intensive and emitting process. Calcination of raw materials for the cement production (e.g., limestone, clay, calcareous marl and other clay-like materials) and burning (fossil) fuels to maintain high temperature in the kiln are the processes with highest environmental impact. The former is a chemical emission, the latter a physical emission. Indeed, raw materials are heated inside large rotating furnaces at 1400 ◦C to form a solid substance called clinker [6]. During this process, chemical emissions mainly come from calcium carbonate (CaCO3) and magnesium carbonate (MgCO3) calcination according to Equations (1) and (2) [7,8]: CaCO3(s) + heat →CaO(s) + CO2 (g) (1) MgCO3(s) + heat →MgO(s) + CO2 (g) (2) (1) (2) Clinker is then ground or milled with gypsum and other constituents (e.g., products, raw materials, additives, recycled waste) to produce cement [9]. According to [9], 5 main types of cement (CEM I to Appl. Sci. 2020, 10, 8212; doi:10.3390/app10228212 Appl. Sci. 2020, 10, 8212 2 of 25 CEM V) and 27 products in the family of common cements are deﬁned. They diﬀer for composition (proportion by mass) of the main constituents, but all contain clinker. Therefore, its production aﬀects the environmental performances of the ﬁnal product and cannot be overlooked in this study. The chemical process described in Equations (1) and (2) implies more than 60% of total CO2 emissions due to clinker production, as conﬁrmed by the mass balance published in the last Environmental Product Declaration (EPD) of the Italian cement production [10]. Chemical emissions are not reducible, but physical emissions resulting from fuel combustion for kiln ﬁring can be managed and reduced using alternative/waste fuels [11] and/or by adopting energy-saving technologies [12]. However, these changes in production processes have to turn out in agreement with the required quality of the obtained products, related to construction or industrial uses [13], or with some special performances to obtain [14–16]. 1. Introduction Moreover, in order to reduce the impacts of cement production, in the last decade the content of clinker in cement has decreased [17] using supplementary cementitious materials (e.g., gypsum, ground limestone, coal ﬂy ash or blast furnace slag) [18–20]. Therefore, the cement industry is making eﬀorts to reduce its environmental impacts in terms of greenhouse gases that reﬂect on other environmental performances. The standard EN 15804 “Sustainability of construction works, environmental product declarations, core rules for the product category of construction products” [21] deﬁnes the core rules for the product category of construction Products in order to assess the life cycle impact and develop a Type III environmental declaration (i.e., EPD) for any construction product and construction service [22]. According to [21], diﬀerent parameters describe the environmental performances of a product, such as environmental impacts, resource use, waste categories, and output ﬂows [23–27]. In the literature, several studies assessed the impacts of cement production considering its upstream processes (i.e., production of commodities, raw materials, transport to the factory plant, production process) [28]. The obtained results allow the cement industry to identify the best strategies to reduce its environmental impact. More in detail, the authors have Life Cycle Analysis (LCA) results of the Italian cement production from 2014 to 2019; this data refers to the environmental performances of one of the most important cement industries in Europe with over 19 million of Mg produced in 2019 [29]. The impacts of more than 190 cement powders produced according to [9] have been assessed with a “from cradle to gate” boundary approach using the database Ecoinvent 2.2 (Version 2.2, Ecoinvent, Zurich, Switzerland, 2007) and the software package SimaPro 8.0.5.13 (Version 8.0.5.13, Pré Consultants: Amersfoort, The Netherlands, 2016) [30]. 15 diﬀerent impact categories were assessed to describe the characteristics of each cement powder recipe. p p The goal of this work is to identify among these features the most relevant variables to predict the behavior of Global Warming Potential (GWP). To obtain these results, diﬀerent models have been implemented, by means of linear regression and variable selection procedures, more speciﬁcally, the Akaike Information Criterion. Analogous models are developed also for the other impact categories that quantify emissions to air. A comparative analysis shows that the most important impact category to control GWP and other emissions is represented by the Abiotic depletion-fossil fuels (ADPf). 2. Data and Methods After a quick introduction of the available dataset, this section provides the reader with the key concepts to investigate the behavior of the Global Warming Potential (GWP) and its connections with the production parameters, as well as a short summary of the statistical methods here employed. The examined impact categories (ICs) comply with the standard EN 15804 [21]; Table 1 lists their name and units of measure. 3 of 25 Appl. Sci. 2020, 10, 8212 Table 1. Assessed impact categories. Table 1. Assessed impact categories. p g Impact Category IC Unit Global Warming Potential GWP kg CO2 eq Ozone layer Depletion Potential ODP kg CFC-11 eq Acidiﬁcation Potential AP kg SO2 eq Eutrophication Potential EP kg PO4 eq Photochemical oxidation Potential POCP kg C2H4 eq Abiotic depletion-elements ADPe kg Sb eq Abiotic depletion-fossil fuels ADPf MJ Total Use of Renewable Primary Energy TRPE MJ Total Use of non-Renewable Primary energy TNRPE MJ Use of secondary raw materials SRM kg Non-renewable secondary fuels nonRSF MJ Renewable secondary fuels RSF MJ Water depletion Water m3 Electricity El kWh Particulates P kg PTS ICs in Table 1 are available for a sample of 193 diﬀerent recipes to produce 1 kg of gray cement, which can be classiﬁed into four types of cement (from CEM I to CEM IV according to [9]): • CEM I (i.e., Portland cement). It is the most impacting type because it contains at least 95% by mass of clinker and gypsum as minor additional constituent to control the “setting of cement”. • CEM I (i.e., Portland cement). It is the most impacting type because it contains at least 95% by mass of clinker and gypsum as minor additional constituent to control the “setting of cement”. • CEM II (i.e., Portland composite cement). It is composed of clinker with diﬀerent proportion by mass (65–94%), main constituents (e.g., bastfurnace slag, silica fume, pozzolana, ﬂy ash, burnt shale or limestone), and gypsum as minor additional constituent. In the examined panel, CEM II A-LL and CEM II-BLL (i.e., portland limestone cements) are taken into account. The former has 80–94% clinker, 6–20% limestone and 0–5% gypsum by mass, the latter has 65–79% clinker, 21–35% limestone and 0–5% gypsum by mass: • CEM III (i.e., blastfurnace cement) is composed of 35–64% clinker, 36–65% blastfurnace slag, 0–5% gypsum by mass; and • CEM IV (i.e., pozzolanic cement). Emission Consumption GWP, AP, EP, POCP, P ADPe, ADPf, TRPE, SRM, nonRSF, RSF, Water, El 2. Data and Methods The answer to the ﬁrst question is given by ﬁtting and evaluating a linear regression model linking GWP to the Consumption ICs, (Equation (3)): Are the relevant variables to predict GWP also useful to predict other Emission ICs? The answer to the first question is given by fitting and evaluating a linear regression model GWP = βADPe ADPe + βADPf ADPf + βTRPE TRPE + βSRM SRM + βnonRSF nonRSF + βRSF RSF + βWater Water + βEl El + ε (3) del (3) inking GWP to the Consumption ICs, (Equation (3)): GWP = βADPe ADPe + βADPf ADPf + βTRPE TRPE + βSRM SRM + βnonRSF nonRSF + βRSF RSF + βWater Water + βEl El + ε (3) where ε is the so-called noise, a vector of size n = 193 of independent and identically distributed random variables, while (βADPe, βADPf, βTRPE, βSRM, βnonRSF, βRSF, βWater, βEl) is the vector of parameters to be estimated [31]. In statistics, linear regression is a widely used approach to establish the relationship between the so-called response (in our case GWP for Sections 3.2 and 3.3, the other Emission ICs in Section 3.4) and a set of explanatory variables (here the Consumption ICs) [32]. The relationship between the response and the explanatory variables is modeled by means of a linear predictor unction whose unknown model parameters are estimated from the data. In this work, all the linear d l fit b f th ll d l t h hi h i i i th f th where ε is the so-called noise, a vector of size n = 193 of independent and identically distributed random variables, while (βADPe, βADPf, βTRPE, βSRM, βnonRSF, βRSF, βWater, βEl) is the vector of parameters to be estimated [31]. In statistics, linear regression is a widely used approach to establish the relationship between the so-called response (in our case GWP for Sections 3.2 and 3.3, the other Emission ICs in Section 3.4) and a set of explanatory variables (here the Consumption ICs) [32]. The relationship between the response and the explanatory variables is modeled by means of a linear predictor function whose unknown model parameters are estimated from the data. 2. Data and Methods It is composed of clinker and pozzolanic constituents (i.e., bastfurnace slag, silica fume, pozzolana, and ﬂy ash). Two types of CEM IV are in the panel: CEM IV/A (65–89% clinker, 11–35% pozzolanic materials, 0–5% gypsum) and CEM IV/B (45–64% clinker, 11–35% pozzolanic materials, 0–5% gypsum). A preliminary analysis on the correlation matrix, performed to study relations between variables, is given in Figure 1. Positive and negative correlations are displayed in blue and red color, respectively, while the intensity of the color of each circle and its size is proportional to the absolute value of the corresponding correlation coeﬃcient. Figure 1 shows that ODP, ADPf and TNRPE are pairwise perfectly correlated, that is, the corresponding correlation coeﬃcient is equal to 1. This implies that these variables match deterministically, up to a multiplicative factor. For this reason, ODP and TNRPE are discarded in the further analysis. The remaining ICs have been used as independent variables and split into two groups, “Emissions” and “Consumption” (Table 2). 4 of 25 Appl. Sci. 2020, 10, 8212 Figure 1. Correlation matrix of ICs. Figure 1. Correlation matrix of ICs. Figure 1. Correlation matrix of ICs. Figure 1. Correlation matrix of ICs. Table 2. Classes of ICs. This work will focus on the following questions: Table 2. Classes of I This work will focus on the following questions: Emission Consumption GWP, AP, EP, POCP, P ADPe, ADPf, TRPE, SRM, nonRSF, RSF, Water, El 1. Which ones are the most relevant variables among the Consumption ICs in order to explain the behavior of GWP? Thi k ill f th f ll i ti 2. Are diﬀerent variables important to predict GWP for the four types of cement? This work will focus on the following questions: 3. Are the relevant variables to predict GWP also useful to predict other Emission ICs? 1. Which ones are the most relevant variables among the Consumption ICs in order to explain the behavior of GWP? 2 Are different variables important to predict GWP for the four types of cement? The answer to the ﬁrst question is given by ﬁtting and evaluating a linear regression model linking GWP to the Consumption ICs, (Equation (3)): 1. Which ones are the most relevant variables among the Consumption ICs in order to explain the behavior of GWP? 2 Are different variables important to predict GWP for the four types of cement? 2. Data and Methods In this work, all the linear models are ﬁt by means of the so-called least squares approach, which minimizes the sum of the squared residuals (the diﬀerences between the observed value and the one predicted by the model. In our case, solving the regression problem produces a vector of estimators ( ˆβADPe, ˆβADP f , ˆβTRPE, ˆβSRM, ˆβnSRM, ˆβSRM, ˆβWater, ˆβEl). The most relevant estimators can be then selected by means of the so-called Akaike Information 5 of 25 Appl. Sci. 2020, 10, 8212 Criterion (AIC) [32]. This procedure results in a model where only statistically signiﬁcant variables in terms of the highest variances are selected, while the others are iteratively discarded. A model validation is then performed by means of a 10-fold cross validation procedure [31] to assess and compare the accuracy of the two models. Cross validation evaluates the accuracy of a predictive model, estimating its ability to predict new data. In the k-fold cross validation, the original dataset is randomly partitioned into k subsamples of equal size. Then, one subsample (the validation data) is used to test the model obtained by using the remaining k-1 subsamples (the so-called training data). This procedure is then repeated k times and averaged, so that all the observations are used for both validation and testing. The measure of the accuracy of each method is provided by the root mean square error, the risk function which measures the square root of the average squared diﬀerence between observations and the estimated values [31]. As far as the second question is concerned, it is now investigated if diﬀerent types of cement inﬂuence GWP in diﬀerent ways and, consequently, if the statistical model’s accuracy can be improved by ﬁtting a separate regression model for each class. These regression models can be expressed as Equation (4): GWP = βADPe;i ADPei + βADPf;i ADPfi + βTRPE;i TRPEi + βSRM;i SRMi + βnonRSF;i nonRSFi + βRSF;i RSFi + βWater;i Wateri + βEl;i Eli + εi (4 (4) re i = I, II, III, IV and solving the regression model produces estimates for the set of parameters where i = I, II, III, IV and solving the regression model produces estimates for the set of parameters (βADPe;i, βADP f;i, βTRPE;i, βSRM;i, βnonRSF;i, βWater;i, βEl;i), i = I, . . . , IV. 2. Data and Methods It is also relevant to check if some (βADPe;i, βADP f;i, βTRPE;i, βSRM;i, βnonRSF;i, βWater;i, βEl;i), i = I, . . . , IV. It is also relevant to check if some types of cement behave diﬀerently in terms of the regression models and relevant variables. Again, 10-fold cross validation procedures [31] are used to compare the results and to verify how accurate each predictive model is. Regarding the third question, a multiple linear regression using the full set of Consumption ICs is performed and used to evaluate two alternative models. The ﬁrst alternative model uses as input variables only the ones selected for GWP. The other alternative model develops diﬀerent variables for each Emission IC, by means of the AIC criterion. If suﬃciently accurate, the ﬁrst model would allow the producer to focus on the same subset of variables to control jointly all the emissions. If it is not the case, the second model establishes which Consumption ICs are relevant to predict other emissions than GWP. Also in this case, a 10-fold cross validation procedure has been applied to compare the accuracy of the models. The statistical analysis has been performed within the R Cran environment [33] and the support of additional packages [34,35]. 3.1. Exploratory Analysis Table 4 presents the i Fi 4 First, it is investigated if there is some relation linking GWP with the other Emission ICs, and it can be observed that all the Emission variables are positively correlated (i.e., green lines) with GWP, as shown in Table 3 and described in Figure 2. in Figure 4. Table 4. Correlation of GWP with Consumption ICs. IC ADPe ADPf TRPE SRM NRSF RSF Water El First, it is investigated if there is some relation linking GWP with the other Emission ICs, and it can be observed that all the Emission variables are positively correlated (i.e., green lines) with GWP, as shown in Table 3 and described in Figure 2. in Figure 4. Table 4. Correlation of GWP with Consumption ICs. IC ADPe ADPf TRPE SRM NRSF RSF Water El Table 3. Correlation of GWP with Emission ICs. IC AP EP POCP P Correlation coeﬃcients 0.575 0.703 0.381 0.529 IC ADPe ADPf TRPE SRM NRSF RSF Water El Correlation coefficients −0.018 0.706 −0.387 −0.372 −0.213 −0.152 0.221 0.757 Having regard to the correlation coefficients, Figure 2 shows that all Emission ICs are positively correlated. Figure 2. Correlation among Emission ICs. Figure 2. Correlation among Emission ICs. Table 3. Correlation of GWP with Emission ICs. −0.018 0.706 −0.387 −0.372 −0.213 Figure 2. Correlation among Emission ICs. Figure 2. Correlation among Emission ICs. Figure 2. Correlation among Emission ICs. Figure 2. Correlation among Emission ICs. Figure 3 shows the correlation between GWP and the Consumption ICs. GWP is strongly positively correlated to ADPf and El and mildly negatively correlated to TRP and SRM. The results Table 4 presents the correlation coeﬃcients of GWP with the Consumption variables, presented in Figure 4. Pf comply with the physical meaning of these IC onsumed more greenhouse gases are emitted On Table 4. Correlation of GWP with Consumption ICs. bout GWP, El and ADPf comply with the physical meaning of these ICs. Indeed, the more foss uels and electricity are consumed, more greenhouse gases are emitted. On the other hand, the use o econdary fuels (both renewable and non-renewable) reduces the consumption of fossil fuel orrelation between ADPe and water is due to the upstream processes (i.e., quarry extraction) of th ement production. Table 4. Correlation of GWP with Consumption ICs. 3. Results In this section, details concerning the performed data analysis are presented and discussed to answer the questions introduced in Section 2. In particular, Section 3.1 includes some preliminary exploratory analysis. Section 3.2 concentrates on Question 1, by studying and comparing two models to predict GWP, the ﬁrst model obtained by ﬁtting a linear regression, and the second model by selecting the most relevant variables by means of the AIC criterion. Section 3.3 is concerned with Question 2. For each type of cement, a linear regression is ﬁt and then the most important Consumption ICs are selected by the AIC criterion. Analogies and diﬀerences shown by the models here developed and the ones in Section 3.1 are then investigated. Section 3.4 is focused on the other Emission ICs and, then, on Question 3. For each type of Cement and for each Emission, three diﬀerent models are studied and then compared. The ﬁrst model is a linear regression which uses all the available Consumption ICs to predict each emission. The second model is a linear regression where only the relevant variables to GWP established in Section 3.2 are used. The third model selects the relevant variables for each Emission by the AIC criterion. The three models are then examined and compared to establish whether the same Consumption ICs can be used to predict accurately all the Emissions or not. Appl. Sci. 2020, 10, 8212 IC Correlation 6 of 25 3.1. Exploratory Analysis Table 4 presents the i Fi 4 IC ADPe ADPf TRPE SRM NRSF RSF Water El Correlation coeﬃcients −0.018 0.706 −0.387 −0.372 −0.213 −0.152 0.221 0.757 Having regard to the correlation coeﬃcients, Figure 2 shows that all Emission ICs are positively correlated. Having regard to the correlation coeﬃcients, Figure 2 shows that all Emission ICs are positively correlated. Figure 3 shows the correlation between GWP and the Consumption ICs. GWP is strongly positively correlated to ADPf and El and mildly negatively correlated to TRP and SRM. The results about GWP, El and ADPf comply with the physical meaning of these ICs. Indeed, the more fossil fuels and electricity are consumed, more greenhouse gases are emitted. On the other hand, the use of secondary fuels (both renewable and non-renewable) reduces the consumption of fossil fuels. Correlation between ADPe and water is due to the upstream processes (i.e., quarry extraction) of the cement production. 7 of 25 7 of 25 7 of 25 7 of 25 Appl. Sci. 2020, 10, 8212 Appl. Sci. 2020, 10, x; doi Figure 3. Correlation between GWP and the Consumption ICs. Figure 3. Correlation between GWP and the Consumption ICs. Figure 3. Correlation between GWP and the Consumption ICs. Therefore, the most relevant variables among the Consumption ICs that could explain the behavior of GWP are electricity and fossil fuels consumption. This complies with the Italian energy mix, whose main energy consumption is driven by petroleum and other liquids and natural gas [36]. On the other hand, the correlations between GWP and other Emission ICs (Table 3) and GWP and the Consumption ICs (Table 4) justify the international approach to protect the environment reducing greenhouse gas emissions. At this purpose, in 2003 the European Parliament and the Council Therefore, the most relevant variables among the Consumption ICs that could explain the behavior of GWP are electricity and fossil fuels consumption. This complies with the Italian energy mix, whose main energy consumption is driven by petroleum and other liquids and natural gas [36]. On the other hand, the correlations between GWP and other Emission ICs (Table 3) and GWP and the Consumption ICs (Table 4) justify the international approach to protect the environment reducing greenhouse gas emissions. At this purpose, in 2003 the European Parliament and the Council established the Emissions Trading Scheme [37] to limit or reduce greenhouse gas emissions. greenhouse gas emissions. 3.1. Exploratory Analysis Table 4 presents the i Fi 4 At this purpose, in 2003 the European Parliament and the Council established the Emissions Trading Scheme [37] to limit or reduce greenhouse gas emissions. Figure 4 provides the reader with explicit correlation coefficients (in the top-right cells with respect to the main diagonal), an estimation of the density function by a histogram and a kernel density estimation (KDE) (in the main diagonal) and, finally, scatterplots with fitted nonparametric regression lines to stress the relationship between pairs of different variables (in the bottom-left cells with respect to the main diagonal). In the first column, each plot displays values for GWP paired with all the Consumption ICs, while in the first row the correlation coefficients between GWP and the Figure 4 provides the reader with explicit correlation coeﬃcients (in the top-right cells with respect to the main diagonal), an estimation of the density function by a histogram and a kernel density estimation (KDE) (in the main diagonal) and, ﬁnally, scatterplots with ﬁtted nonparametric regression lines to stress the relationship between pairs of diﬀerent variables (in the bottom-left cells with respect to the main diagonal). In the ﬁrst column, each plot displays values for GWP paired with all the Consumption ICs, while in the ﬁrst row the correlation coeﬃcients between GWP and the Consumption ICs are listed. all the Consumption ICs, while in the first row the correlation coefficients between GWP and the Consumption ICs are listed. In Figure 4 both x- and y- axis labels refer to the corresponding iCs listed in the main diagonal; their units comply with those listed in Table 1. Therefore, GWP values obtained in the LCA range between 0.6 and 1.0 kg CO2 eq./1 kg of produced cement; ADPe ranges between 1.0 × 10−7 and 5.0 × In Figure 4 both x- and y- axis labels refer to the corresponding iCs listed in the main diagonal; their units comply with those listed in Table 1. Therefore, GWP values obtained in the LCA range between 0.6 and 1.0 kg CO2 eq./1 kg of produced cement; ADPe ranges between 1.0 × 10−7 and 5.0 × 10−7 kg Sb eq./1 kg of produced cement. 8 of 25 8 of 25 Appl. Sci. 2020, 10, 8212 Appl Sci 2020 10 x; d Figure 4. Correlation matrix analysis (GWP vs. Consumption ICs). “*” (p-value ≤ 0.001), “” (p-value ≤ 0.01), “” (p-value ≤ 0.05), “.” (p-value ≤ 0.1), otherwise. 3.1. Exploratory Analysis Table 4 presents the i Fi 4 This notation is valid throughout the article. Figure 4. Correlation matrix analysis (GWP vs. Consumption ICs). “” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-val and “ ” otherwise. This notation is valid throughout the article. n matrix analysis (GWP vs. Consumption ICs). “” (p-value ≤ 0.001), “” (p-value ≤ 0.01), “” (p-value ≤ 0.05), “.” (p-value ≤ 0.1), and “ “ n matrix analysis (GWP vs. Consumption ICs). “” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), is notation is valid throughout the article Figure 4. Correlation matrix analysis (GWP vs. Consumption ICs). “” (p-value ≤ 0.001), “” (p-value ≤ 0.01), “” (p-value ≤ 0.05), “.” (p-value ≤ 0.1), and “ “ otherwise. This notation is valid throughout the article. Figure 4. Correlation matrix analysis (GWP vs. Consumption ICs). “” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. This notation is valid throughout the article. Appl. Sci. 2020, 10, 8212 9 of 25 3.2. Linear Regression and Variable Selection for GWP 3.2. Linear Regression and Variable Selection for GWP 3.2. Linear Regression and Variable Selection for GWP In this Section two predictive models for GWP are ﬁt. The ﬁrst model is a linear regression where GWP is the scalar response and the Consumption ICs are the input variables. All variables have been preliminarily normalized to simplify the interpretation. The estimated coeﬃcients are listed in Table 5, together with the related standard deviations (St. dev.) and the corresponding signiﬁcance for the p-values associated to the signiﬁcance test of the model. Table 5. Linear regression summary (GWP vs. Consumption ICs). ICs Linear Regression (GWP vs. Consumption ICs) Coeﬃcients St. Dev. p-Value ADPe −0.127 0.038 * ADPf 1.503 0.077 * TRPE −0.082 0.026 SRM 0.532 0.025 NRSF 0.531 0.052 * RSF 0.563 0.039 * Water −0.017 0.041 El −0.006 0.043 “” (p-value ≤0.001), “” (p-value ≤0.01) and “ ” otherwise. Table 5. Linear regression summary (GWP vs. Consumption ICs). To develop the second model, an AIC backward selection procedure is then performed on the linear regression, to ﬁnd the best subset of Consumption ICs to accurately predict GWP, leading to the model described in Table 6. Table 6. Linear regression model summary (after variable selection). Table 6. Linear regression model summary (after variable selection). ICs Linear Regression (GWP vs. Selected Consumption ICs) Coeﬃcients St. Dev. p-Value ADPe −0.140 0.022 ADPf 1.486 0.044 * TRPE −0.085 0.025 * SRM 0.527 0.024 NRSF 0.527 0.040 * RSF 0.558 0.037 * “*” (p-value ≤0.001), “” (p-value ≤0.01). Then, a 10-fold cross-validation procedure is performed to compare the two models. The root mean square errors (RMSE) are computed by Equations (5) and (6): Then, a 10-fold cross-validation procedure is performed to compare the two models. 3.2. Linear Regression and Variable Selection for GWP The root mean square errors (RMSE) are computed by Equations (5) and (6): RMSElin = ( 1 n nP i=1 (GWPi −ˆβlin ADPeADPei −ˆβlin ADP f ADPfi −ˆβlin TRPETRPEi −ˆβlin SRMSRMi −ˆβlin NRSFNRSFi −ˆβlin RSFRSFi −ˆβlin WaterWateri −ˆβlin ELELi) 2) 1/2 (5) RMSElin = ( 1 n nP i=1 (GWPi −ˆβlin ADPeADPei −ˆβlin ADP f ADPfi −ˆβlin TRPETRPEi −ˆβlin SRMSRMi −ˆβlin NRSFNRSFi −ˆβlin RSFRSFi −ˆβlin WaterWateri −ˆβlin ELELi) 2) 1/2 (5) RMSEAIC = ( 1 n nP i=1 (GWPi −ˆβAIC ADPeADPei −ˆβAIC ADP f ADPfi −ˆβAIC TRPETRPEi −ˆβAIC SRMSRMi ˆβAIC NRSF ˆβAICRSF ˆβAIC Water ˆβAICEL ) 2) 1/2 (6) (5) RMSEAIC = ( 1 n nP i=1 (GWPi −ˆβAIC ADPeADPei −ˆβAIC ADP f ADPfi −ˆβAIC TRPETRPEi −ˆβAIC SRMSRMi −ˆβAIC NRSFNRSFi −ˆβAIC RSFRSFi −ˆβAIC WaterWateri −ˆβAIC EL ELi) 2) 1/2 (6) (6) for the linear and AIC model, respectively. The sample size n = 193 is the number of observations, while ˆβlin ADPe, ˆβlin ADP f , ˆβlin TRPE, ˆβlin SRM, ˆβlin NRSF, ˆβlin RSF, ˆβlin Water, ˆβlin E , and ˆβAIC ADPe, ˆβAIC ADP f , ˆβAIC TRPE, ˆβAIC SRM, ˆβAIC NRSF, ˆβAIC RSF are the estimated parameters with the linear model and then selected by the AIC criterion, respectively. Recall that the RMSE is a risk function aimed to measure the discrepancy between the observations 10 of 25 Appl. Sci. 2020, 10, 8212 and the corresponding estimated values. In Table 7 RMSElin is higher than RMSEAIC, thus the variable reduction produces a more accurate model. and the corresponding estimated values. In Table 7 RMSElin is higher than RMSEAIC, thus the variable reduction produces a more accurate model. Table 7. Comparison of RMSE between the two models. OR PEER REVIEW 10 of 25 Table 7. Comparison of RMSE between the two models. Model RMSE Linear 0.313 AIC 0.305 Appl. Sci. 2020, 10, x; doi: FOR PEER REVIEW 10 of 25 Table 7. Comparison of RMSE between the two models. Model RMSE Linear 0.313 AIC 0.305 Model RMSE Linear 0.313 AIC 0.305 arison of RMSE between the two models. Model RMSE Linear 0.313 AIC 0.305 Finally, Figure 5 describes the size of each regression slope coeﬃcient, after the variable selection. The highest contribution to GWP is given by ADPf. This result complies with the release of carbon dioxide into the atmosphere by burning of fossil fuels [38–40]. 3.2. Linear Regression and Variable Selection for GWP Finally, Figure 5 describes the size of each regression slope coefficient, after the variable selection. The highest contribution to GWP is given by ADPf. This result complies with the release of carbon dioxide into the atmosphere by burning of fossil fuels [38–40]. Figure 5. Size of the coefficients (AIC selected components—absolute values). Figure 5. Size of the coeﬃcients (AIC selected components—absolute values). Figure 5. Size of the coefficients (AIC selected components—absolute values). Figure 5. Size of the coeﬃcients (AIC selected components—absolute values). In answer to Question 1, the most relevant variables among the Consumption ICs to predict GWP are ADPf, NRSF and RSF. The energy-intensive industry of cement manufacturing can motivate this variable selection: all these ICs quantify the energy, mainly fossil but also alternative, spent in the process. This result complies with the efforts to implement in the cement sector different management systems, process-integrated techniques and end-of-pipe measures identified as Best Available Techniques (BAT) to have environmental benefits (e.g., thermal energy optimization techniques in the kiln system; reduction of electrical energy use; recovery of excess heat from the process and cogeneration of steam and electrical power) [41]. 3 3 Li R i d V i bl S l ti f E h T f C t In answer to Question 1, the most relevant variables among the Consumption ICs to predict GWP are ADPf, NRSF and RSF. The energy-intensive industry of cement manufacturing can motivate this variable selection: all these ICs quantify the energy, mainly fossil but also alternative, spent in the process. This result complies with the eﬀorts to implement in the cement sector diﬀerent management systems, process-integrated techniques and end-of-pipe measures identiﬁed as Best Available Techniques (BAT) to have environmental beneﬁts (e.g., thermal energy optimization techniques in the kiln system; reduction of electrical energy use; recovery of excess heat from the process and cogeneration of steam and electrical power) [41]. 3.3. Linear Regression and Variable Selection for Each Type of Cement The different types of cement are now studied separately to 3.3. Linear Regression and Variable Selection for Each Type of Cement The different types of cement are now studied separately to evaluate their impact on GWP. Figure 6, which contains the scatterplots related to GWP and the Consumption ICs, shows that the points associated to the class CEM I (in blue) are isolated in the GWP scatterplots with respect to the data belonging to the other types. Moreover, the environmental impacts of CEM I are higher than other investigated cement types: both the qualitative and the quantitative observed trends suggest investigating whether predictive models built separately for each class (type of cement) could achieve more accurate predictions for GWP. Furthermore, it is of extreme interest to check if different variables result to be important for each separate class with respect to the ones selected for the whole dataset. Figure 6 contains a matrix of scatterplots used to visualize the relationship between pairs of variables, all listed in the main diagonal. For each scatterplot, the variables in the x-axis (y-axis, respectively) can be found in the entry belonging to the main diagonal in the same column (row, respectively). The units of each axis label are listed in Table 1. The diﬀerent types of cement are now studied separately to evaluate their impact on GWP. Figure 6, which contains the scatterplots related to GWP and the Consumption ICs, shows that the points associated to the class CEM I (in blue) are isolated in the GWP scatterplots with respect to the data belonging to the other types. Moreover, the environmental impacts of CEM I are higher than other investigated cement types: both the qualitative and the quantitative observed trends suggest investigating whether predictive models built separately for each class (type of cement) could achieve more accurate predictions for GWP. Furthermore, it is of extreme interest to check if diﬀerent variables result to be important for each separate class with respect to the ones selected for the whole dataset. Figure 6 contains a matrix of scatterplots used to visualize the relationship between pairs of variables, all listed in the main diagonal. For each scatterplot, the variables in the x-axis (y-axis, respectively) can be found in the entry belonging to the main diagonal in the same column (row, respectively). The units of each axis label are listed in Table 1. 11 of 25 11 of 25 Appl. Sci. 2020, 10, 8212 Appl. Sci. 2020, 10, x; doi: nd types of cement. 3.3. Linear Regression and Variable Selection for Each Type of Cement The different types of cement are now studied separately to 3.3. Linear Regression and Variable Selection for Each Type of Cement (CEM I = blue, CEM II = red, CEM III = green, CEM IV = yellow). Figure 6. Data and types of cement. (CEM I = blue, CEM II = red, CEM III = green, CEM IV = yellow). ement. (CEM I = blue, CEM II = red, CEM III = green, CEM IV = yellow). Figure 6. Data and types of cement. (CEM I = blue, CEM II = red, CEM III = green, CEM IV = yellow). 12 of 25 Appl. Sci. 2020, 10, 8212 The dimensions of each class are given in Table 8. Table 8. Dimensions of datasets related to each type of cement. Table 8. Dimensions of datasets related to each type of cement. Table 8. Dimensions of datasets related to each type of cement. Table 8. Dimensions of datasets related to each type of cement. Cement Type Dimension of Classes CEM I 44 CEM II 84 CEM III 4 CEM IV 61 Cement Type Dimension of Classes CEM I 44 CEM II 84 CEM III 4 CEM IV 61 Cement Type Dimension of Classes Due to the small number of observations, CEM III is ﬁltered out. Due to the small number of observations, CEM III is ﬁltered out. For each class, a linear regression is ﬁt, where GWP corresponds to the scalar response and the ICs to the explanatory variables. The estimated regression coeﬃcients for CEM I, II, and IV are listed in Tables 9–11, respectively. Table 9. Linear regression summary (GWP vs. Consumption ICs)—CEM I. Table 9. Linear regression summary (GWP vs. Consumption ICs)—CEM I. ICs Linear Regression (GWP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe 0.217 0.115 ADPf 0.701 0.128 * TRPE 0.105 0.115 SRM 0.046 0.111 NRSF 0.090 0.079 RSF 0.181 0.061 Water −0.165 0.092 . El 0.052 0.050 “*” (p-value ≤0.001), “” (p-value ≤0.01), “.” (p-value ≤0.1), and “ ” otherwise. Table 10. Linear regression summary (GWP vs. Consumption ICs)—CEM II. Table 10. Linear regression summary (GWP vs. Consumption ICs)—CEM II. ICs Linear Regression (GWP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe −0.052 0.099 ADPf 1.237 0.156 * TRPE −0.132 0.044 SRM 0.052 0.148 NRSF 0.389 0.104 * RSF 0.414 0.077 * Water −0.029 0.089 El −0.052 0.081 “*” (p-value ≤0.001), “” (p-value ≤0.01) and “ ” otherwise. Table 11. Linear regression summary (GWP vs. Consumption ICs)—CEM IV. Table 11. Linear regression summary (GWP vs. Consumption ICs)—CEM IV. ICs Linear Regression (GWP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe −0.049 0.045 ADPf 1.234 0.157 *** TRPE −0.095 0.037 * SRM −0.007 0.032 NRSF 0.420 0.091 * RSF 0.412 0.082 * Water −0.016 0.062 El −0.038 0.064 “**” (p-value ≤0.001), “” (p-value ≤0.05), and “ ” otherwise. Appl. Sci. 2020, 10, 8212 13 of 25 Then, AIC backward selection procedures yield the models for the classes CEM I, II and IV described in Tables 12–14, respectively. Then, AIC backward selection procedures yield the models for the classes CEM I, II and IV described in Tables 12–14, respectively. Table 8. Dimensions of datasets related to each type of cement. Table 12. AIC selection model (GWP vs. Consumption ICs)—CEM I. Table 12. AIC selection model (GWP vs. Consumption ICs)—CEM I. ICs Linear Regression (GWP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe 0.267 0.103 * ADPf 0.598 0.069 * RSF 0.163 0.053 Water −0.190 0.085 * El 0.094 0.041 * “*” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05). Table 13. AIC selection model (GWP vs. Consumption ICs)—CEM II. Table 13. AIC selection model (GWP vs. Consumption ICs)—CEM II. ICs Linear Regression (GWP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe −0.082 0.043 . ADPf 1.18 0.117 TRPE −0.133 0.042 NRSF 0.364 0.075 * RSF 0.409 0.072 * “*” (p-value ≤0.001), “” (p-value ≤0.01), “.” (p-value ≤0.1). Table 14. AIC selection model (GWP vs. Consumption ICs)—CEM IV. ICs Linear Regression (GWP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe −0.057 0.059 ADPf 1.173 0.035 * TRPE −0.100 0.114 NRSF 0.387 0.073 * RSF 0.394 0.075 * “*” (p-value ≤0.001), “” (p-value ≤0.01), and “ ” otherwise. Table 14. AIC selection model (GWP vs. Consumption ICs)—CEM IV. A 10-fold cross-validation procedure shows that the root mean square error (RMSE) related to the AIC selection model, is lower than the one computed for the full linear regression model (Table 15). Table 15. Comparison of RMSE between the two models. Table 15. Comparison of RMSE between the two models. Table 15. Comparison of RMSE between the two models. Model RMSE linear 0.275 AIC 0.266 Model RMSE linear 0.275 AIC 0.266 Model RMSE linear 0.275 AIC 0.266 To answer to Question 2, also in this case a variable selection procedure leads to a more accurate model. Furthermore, diﬀerent variables are selected as relevant depending on the type of cement. It can be observed that while for the class CEM I, the ICs TRPE and NRSF are discarded, for the classes CEM II and CEM IV the ones rejected by the AIC criterion are Water and El and the same ICs are selected. These ICs are also consistent also with the ones chosen for the whole dataset (except to SRM). The model for the class CEM I is characterized by a diﬀerent set of variables and associated sizes, including Water and El (Figure 7 shows the size of the coeﬃcients for each class and for the aggregate Appl. Sci. Table 8. Dimensions of datasets related to each type of cement. 2020, 10, 8212 A l S i 2020 10 FOR 14 of 25 14 of 25 data, Figure 7a–d, respectively). It also conﬁrms the qualitative interpretation of Figure 6, where data belonging to CEM I was mostly isolated. data, Figure 7a,b–d, respectively). It also confirms the qualitative interpretation of Figure 6, where data belonging to CEM I was mostly isolated. Figure 7. Size of the coefficients (AIC selected components) for each type of cement (absolute values). (a) aggregated data; (b) CEM I; (c) CEM II; (d) CEM IV. Figure 7. Size of the coeﬃcients (AIC selected components) for each type of cement (absolute values). (a) aggregated data; (b) CEM I; (c) CEM II; (d) CEM IV. Figure 7. Size of the coefficients (AIC selected components) for each type of cement (absolute values). (a) aggregated data; (b) CEM I; (c) CEM II; (d) CEM IV. Figure 7. Size of the coeﬃcients (AIC selected components) for each type of cement (absolute values). (a) aggregated data; (b) CEM I; (c) CEM II; (d) CEM IV. Figure 7. Size of the coefficients (AIC selected components) for each type of cement (absolute values). (a) aggregated data; (b) CEM I; (c) CEM II; (d) CEM IV. Figure 7. Size of the coeﬃcients (AIC selected components) for each type of cement (absolute values). (a) aggregated data; (b) CEM I; (c) CEM II; (d) CEM IV. The results of CEM I (Figure 7b) differ from CEM II and CEM IV (Figure 7c,d) due to its composition (i.e., at least 95% by mass of clinker and not more than 5% by mass of gypsum according to [9]), while other cement types contain less clinker and other main constituents. The results of CEM I (Figure 7b) diﬀer from CEM II and CEM IV (Figure 7c,d) due to its composition (i.e., at least 95% by mass of clinker and not more than 5% by mass of gypsum according to [9]), while other cement types contain less clinker and other main constituents. “*” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. 3.4. Other Emission Variables 3.4. Other Emission Variables This section focuses on producing accurate models for the other Emission ICs. The first model here fit is a multiple linear regression model containing as target variables all the emissions. The results are collected in Tables 16–18 for CEM I, II and IV, respectively. Since in Section 3.3, subsampling data with respect to the type of cement has led to a more accurate model, also in this Section each type of cement is examined separately. This section focuses on producing accurate models for the other Emission ICs. The ﬁrst model here ﬁt is a multiple linear regression model containing as target variables all the emissions. The results are collected in Tables 16–18 for CEM I, II and IV, respectively. Since in Section 3.3, subsampling data with respect to the type of cement has led to a more accurate model, also in this Section each type of cement is examined separately. 15 of 25 Appl. Sci. 2020, 10, 8212 Table 16. Linear regression summary—CEM I. ICs Linear Regression (AP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe 0.199 0.314 ADPf 0.184 0.349 TRPE 0.424 0.313 SRM 0.424 0.302 NRSF −0.148 0.217 RSF −0.057 0.166 Water 0.380 0.251 El −0.070 0.135 ICs Linear Regression (EP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe 0.744 0.409 ADPf 1.571 0.454 ** TRPE 0.085 0.408 SRM 0.732 0.394 . NRSF 0.187 0.283 RSF 0.355 0.216 Water −0.615 0.327 . El −0.273 0.176 ICs Linear Regression (POCP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe −0.034 0.427 ADPf −0.567 0.474 TRPE 0.364 0.425 SRM 0.798 0.410 . NRSF −0.440 0.295 RSF −0.314 0.226 Water 0.786 0.341 * El −0.105 0.183 ICs Linear Regression (P vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe −0.341 0.471 ADPf −1.325 0.523 * TRPE −0.514 0.469 SRM −0.504 0.453 NRSF −0.873 0.325 * RSF −0.590 0.249 * Water 0.514 0.376 El 0.520 0.203 * “*” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. C Linear Regression (AP vs. Consumption ICs) CEM I 16 of 25 Appl. Sci. 2020, 10, 8212 Table 17. Linear regression summary—CEM II. Table 17. Linear regression summary—CEM II. ICs Linear Regression (AP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. “*” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. 3.4. Other Emission Variables 3.4. Other Emission Variables p-Value ADPe 0.067 0.180 ADPf 0.638 0.284 TRPE −0.003 0.079 SRM 0.175 0.269 NRSF 0.116 0.189 RSF 0.017 0.140 Water 0.605 0.160 * El −0.160 0.147 ICs Linear Regression (EP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe 0.084 0.187 ADPf 1.709 0.296 * TRPE 0.145 0.083 . SRM 0.627 0.281 * NRSF 0.411 0.197 * RSF 0.427 0.146 ** Water −0.025 0.168 El −0.394 0.153 * ICs Linear Regression (POCP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe −0.178 0.199 ADPf −0.289 0.314 TRPE −0.033 0.088 SRM 0.495 0.298 . NRSF 0.094 0.209 RSF −0.065 0.155 Water 0.950 0.178 * El −0.296 0.162 . ICs Linear Regression (P vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe −0.010 0.249 ADPf −0.045 0.393 TRPE −0.084 0.110 SRM −0.022 0.372 NRSF 0.022 0.261 RSF −0.287 0.193 Water 0.141 0.222 El −0.028 0.203 “” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. Linear Regression (AP vs. Consumption ICs) CEM II 17 of 25 Appl. Sci. 2020, 10, 8212 Table 18. Linear regression summary—CEM IV Table 18. Linear regression summary—CEM IV ICs Linear Regression (AP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.199 0.314 * ADPf 0.185 0.349 TRPE 0.424 0.313 SRM 0.424 0.302 NRSF −0.148 0.217 RSF −0.057 0.166 Water 0.380 0.251 El −0.070 0.135 ICs Linear Regression (EP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.744 0.409 . ADPf 1.571 0.454 ** TRPE 0.084 0.408 SRM 0.732 0.394 . NRSF 0.187 0.283 RSF 0.355 0.216 Water −0.615 0.327 . El −0.273 0.176 ICs Linear Regression (POCP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe −0.033 0.427 ADPf −0.568 0.474 TRPE 0.364 0.425 SRM 0.798 0.411 NRSF −0.440 0.295 . RSF −0.314 0.226 Water 0.785 0.341 * El −0.105 0.184 ICs Linear Regression (P vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe −0.340 0.471 ADPf −1.325 0.523 * TRPE −0.514 0.470 SRM −0.505 0.453 NRSF −0.873 0.326 * RSF −0.589 0.249 * Water 0.514 0.376 El 0.520 0.202 * “*” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. Linear Regression (AP vs. Consumption ICs) CEM IV For each class, the second model consists in a linear model, which exploits only the variables established in Section 3.3 as relevant to predict GWP. 3.4. Other Emission Variables 3.4. Other Emission Variables The models are summarized in Tables 19–21 for CEM I, II, and IV, respectively. 18 of 25 Appl. Sci. 2020, 10, 8212 Table 19. AIC selection for GWP regression summary—CEM I. Table 19. AIC selection for GWP regression summary—CEM I. ICs Linear Regression (AP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe 0.340 0.288 ADPf 0.478 0.191 * RSF 0.031 0.148 Water 0.262 0.237 El −0.051 0.115 ICs Linear Regression (EP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe 1.080 0.370 ADPf 1.411 0.247 * RSF 0.302 0.190 Water −0.850 0.306 El −0.170 0.149 ICs Linear Regression (POCP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe 0.158 0.407 ADPf 0.147 0.270 RSF −0.149 0.209 Water 0.588 0.335 . El −0.167 0.163 ICs Linear Regression (P vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe −0.816 0.462 ADPf −0.278 0.307 . RSF −0.365 0.237 Water 0.759 0.381 . El 0.164 0.186 “” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. Table 20. AIC selection for GWP regression summary—CEM II. Table 20. AIC selection for GWP regression summary—CEM II. ICs Linear Regression (AP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe 0.671 0.084 ADPf 0.368 0.231 * TRPE −0.041 0.083 NRSF −0.205 0.147 RSF −0.098 0.142 ICs Linear Regression (EP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe 0.055 0.085 ADPf 1.272 0.235 * TRPE 0.130 0.084 NRSF 0.173 0.150 RSF 0.372 0.145 * 19 of 25 Appl. Sci. 2020, 10, 8212 Table 20. Cont. ICs Linear Regression (AP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe 0.766 0.101 ADPf −0.176 0.278 *** TRPE −0.091 0.100 NRSF −0.429 0.178 * RSF −0.246 0.171 ICs Linear Regression (P vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPe 0.132 0.107 ADPf −0.103 0.293 TRPE −0.094 0.105 NRSF −0.049 0.187 RSF −0.315 0.181 . “**” (p-value ≤0.001), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. Linear Regression (AP vs. Consumption ICs) CEM II Table 21. AIC selection for GWP regression summary—CEM IV. Table 21. AIC selection for GWP regression summary—CEM IV. ICs Linear Regression (AP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.379 0.071 * ADPf 0.828 0.235 * TRPE −0.015 0.063 NRSF 0.094 0.150 RSF 0.135 0.154 ICs Linear Regression (EP vs. 3.4. Other Emission Variables 3.4. Other Emission Variables Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.211 0.059 * ADPf 0.858 0.195 * TRPE −0.037 0.052 NRSF −0.006 0.124 RSF 0.178 0.129 ICs Linear Regression (POCP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.379 0.096 ADPf 0.357 0.314 * TRPE −0.028 0.084 NRSF −0.115 0.200 RSF 0.040 0.207 ICs Linear Regression (P vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.071 0.095 ADPf 1.021 0.314 TRPE −0.172 0.084 * NRSF 0.292 0.200 RSF 0.231 0.206 “*” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), and “ ” otherwise. Appl. Sci. 2020, 10, 8212 20 of 25 A third model is ﬁnally produced applying the AIC criterion separately to each Emission IC for each class. Then, each of this model selects the most important variables for the corresponding IC. The results are summarized in the Tables 22–24 for CEM I, II, and IV, respectively. Table 22. AIC selection regression summary—CEM I. Table 22. AIC selection regression summary—CEM I. ICs Linear Regression (AP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPf 0.310 0.71 SRM 0.505 0.260 . Water 0.530 0.68 * ICs Linear Regression (EP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe 0.821 0.387 * ADPf 1.338 0.243 *** SRM 0.687 0.379 . RSF 0.305 0.185 Water −0.649 0.317 * El −0.203 0.146 ICs Linear Regression (POCP vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPe −0.593 0.344 . SRM 0.766 0.355 * NRSF −0.470 0.225 * RSF −0.292 0.196 Water 0.765 0.114 * ICs Linear Regression (P vs. Consumption ICs) CEM I Coeﬃcients St. Dev. p-Value ADPf −1.465 0.502 SRM −0.667 0.414 NRSF −1.004 0.307 RSF −0.709 0.231 Water 0.255 0.128 . El 0.479 0.199 * “*” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), “.” (p-value ≤0.1), and “ ” otherwise. Table 23. GWP signiﬁcant—AIC selection regression summary—CEM II. Table 23. GWP signiﬁcant—AIC selection regression summary—CEM II. ICs Linear Regression (AP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPf 0.437 0.068 Water 0.660 0.061 * ICs Linear Regression (EP vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPf 1.819 0.251 *** TRPE 0.165 0.077 * SRM 0.611 0.278 * NRSF 0.468 0.166 RSF 0.486 0.120 * El −0.403 0.148 21 of 25 Appl. Sci. 2020, 10, 8212 Table 23. Cont. ICs Linear Regression (AP vs. 3.4. Other Emission Variables 3.4. Other Emission Variables Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value ADPf 0.313 0.083 * SRM 0.486 0.292 Water 0.778 0.070 *** El −0.268 0.118 * ICs Linear Regression (P vs. Consumption ICs) CEM II Coeﬃcients St. Dev. p-Value RSF −0.225 0.078 Water 0.120 0.078 “” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), and “ ” otherwise. Linear Regression (AP vs. Consumption ICs) CEM II Table 24. AIC selection regression summary—CEM IV. Table 24. AIC selection regression summary—CEM IV. ICs Linear Regression (AP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.148 0.075 * ADPf 0.375 0.080 * TRPE −0.179 0.053 SRM −0.162 0.053 Water 0.500 0.97 ICs Linear Regression (EP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.280 0.071 * ADPf 1.009 0.147 *** RSF 0.254 0.122 * Water −0.132 0.090 El −0.122 0.087 ICs Linear Regression (POCP vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value TRPE −0.178 0.065 NRSF −0.233 0.081 Water 0.721 0.092 * ICs Linear Regression (P vs. Consumption ICs) CEM IV Coeﬃcients St. Dev. p-Value ADPe 0.314 0.107 ADPf 1.504 0.272 *** NRSF 0..411 0.172 * RSF 0.458 0.180 * Water −0.557 0.134 * “” (p-value ≤0.001), “” (p-value ≤0.01), “” (p-value ≤0.05), and “ ” otherwise. A 10-fold cross-validation procedure shows that the root mean square error (RMSE) computed for AIC selection model built for each emission IC is lower than the one related to the full linear regression model and to the one associated to the variables selected to predict GWP. The two last RMSEs are among them comparable (Table 25). 22 of 25 Appl. Sci. 2020, 10, 8212 Table 25. Comparison of RMSE between the two models. IC Model RMSE AP linear 0.54 AIC (GWP variables) 0.53 AIC 0.44 EP linear 0.59 AIC (GWP variables) 0.61 AIC 0.56 POCP linear 0.61 AIC (GWP variables) 0.67 AIC 0.54 P linear 0.73 AIC (GWP variables) 0.75 AIC 0.71 Table 25. Comparison of RMSE between the two models. In answer to Question 3, the results in Table 25 highlight that, while in general using the same subset of Consumption IC relevant to predict GWP does not improve the accuracy of the linear model, performing separately for each Emission IC a variable selection procedure leads to a meaningful enhancement in terms of predictive assessment of the model. 4. Discussion Due to a dependence on fossil fuels and the calcination of raw materials, the cement industry generates about 5% of global greenhouse gas emissions. Within this framework, several eﬀorts are on-going to protect the environment and increase energy eﬃciency using renewable resources or alternative fuels. In order to analyze comparable environmental performances, cement companies are conducting life cycle assessment of their “from cradle to gate” processes in order to identify the best strategies to meet the need for sustainable development. In this study, having regard to the European approach compliant with the standard EN 15804, the environmental impacts of 193 diﬀerent recipes of gray cement produced in Italy from 2014 to 2019 have been assessed. Fifteen diﬀerent impact categories have been considered and split into two classes, “Emissions” and “Consumption”. One of the main results of this work concerns the identiﬁcation of the signiﬁcant Consumption ICs to predict the behavior of Emission ICs, In particular, the target of this paper consists in answering to the following questions: 1. Which ones are the most relevant variables among the Consumption ICs in order to explain the behavior of GWP? 1. Which ones are the most relevant variables among the Consumption ICs in order to explain the behavior of GWP? 2. Are diﬀerent variables important to predict GWP for the four types of cement? 3. Are the relevant variables to predict GWP also useful to predict other Emission ICs? As far as Question 1 is concerned, it is shown that the most important variable to predict the behavior of GWP is ADPf (Figure 8), while NRSF and RSF are the two other most relevant consumption variables. To answer Question 2, a more accurate model is produced by ﬁtting a linear regression and applying the AIC criterion for diﬀerent types of cement (i.e., CEM I, CEM II, CEM IV) separately. Also in this case, ADPf is proved to be the most important Consumption IC. However, scatterplots related to GWP and the Consumption ICs show that the environmental performances of CEM I diﬀer from those of the other types, and their values are higher. Predictive models built separately for each type of cement revealed more accurate predictions for GWP. Finally, concerning Question 3, the authors investigated if the relevant variables to predict GWP could predict other Emission ICs. 3.4. Other Emission Variables 3.4. Other Emission Variables In Figure 8, all the important variables related to each Emission IC listed in Tables 19–21 are represented proportionally to their size. Appl. Sci. 2020, 10, x FOR PEER REVIEW 22 of 25 Figure 8. Size of the coefficients (AIC selected components) for each class and for all the remaining Emission ICs. (absolute values). Figure 8. Size of the coeﬃcients (AIC selected components) for each class and for all the remaining Emission ICs. (absolute values). Figure 8. Size of the coefficients (AIC selected components) for each class and for all the remaining Emission ICs. (absolute values). Figure 8. Size of the coeﬃcients (AIC selected components) for each class and for all the remaining Emission ICs. (absolute values). Figure 8 confirms that ADPf plays a pivotal role for the majority of IC emissions, that is, AP, EP, POCP and P (CEM I and CEM IV). CEM II differs from CEM I and CEM IV due to its limestone-based composition; particularly, POCP CEM II has its highest correlation with the Water consumption IC. Figure 8 conﬁrms that ADPf plays a pivotal role for the majority of IC emissions, that is, AP, EP, POCP and P (CEM I and CEM IV). CEM II diﬀers from CEM I and CEM IV due to its limestone-based Appl. Sci. 2020, 10, 8212 23 of 25 composition; particularly, POCP CEM II has its highest correlation with the Water consumption IC. It is conﬁrmed by the upstream processes necessary to quarry natural raw materials. composition; particularly, POCP CEM II has its highest correlation with the Water consumption IC. It is conﬁrmed by the upstream processes necessary to quarry natural raw materials. 4. Discussion In this case, it is shown that ﬁtting separately regression models and selecting the most important variables leads to more accurate predictions for all the other Emissions ICs (Table 25) in comparison to the standard linear model or the one which uses the same Consumption ICs for GWP. Also in this case, ADPf is conﬁrmed to be a strong predictor in the models related to the emission variables AP, EP, POCP (for CEM I and CEM II), and P (for CEM I and CEM IV). Therefore, the obtained results underline the need for policies and strategies that could reduce consumption of energy, both fossil and secondary fuels, and justify the European policies about Emission trading and Best Available Techniques to be implemented in the cement industry. Author Contributions: Conceptualization, C.D. and L.M.; methodology, C.D.; software, C.D. and L.M.; validation, L.M. and C.D.; formal analysis, C.D.; data curation, L.M.; writing—original draft preparation, C.D. and L.M.; review and editing, C.D. and L.M. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Miccoli, S.; Finucci, F.; Murro, R. Assessing project quality: A multidimensional approach. Adv. Mater. Res. 2014, 1030–1032, 2519–2522. [CrossRef] 1. Miccoli, S.; Finucci, F.; Murro, R. Assessing project quality: A multidimensional approach. Adv. Mater. Res. 2014, 1030–1032, 2519–2522. [CrossRef] 2. Miccoli, S.; Finucci, F.; Murro, R. 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Best Available Techniques (BAT) Reference Document for the Production of Cement, Lime and Magnesium Oxide: Industrial Emissions Directive 2010/75/EU (Integrated Pollution Prevention and Control.); European Commission: Brussels, Belgium, 2013. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. References 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/).
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https://openalex.org/W2767824561	https://hal.archives-ouvertes.fr/hal-03453561/file/2017_Nat_Commun_Emergent%20patterns%20of%20collective%20cell%20migration%20under%20tubular%20confinement.pdf	English	null	Emergent patterns of collective cell migration under tubular confinement	Nature communications	2,017	cc-by	17,910	ARTICLE Emergent patterns of collective cell migration under tubular conﬁnement Wang Xi1,2, Surabhi Sonam1,3,4, Thuan Beng Saw 1,5, Benoit Ladoux 1,4 & Chwee Teck Lim1,2,3,5,6 Wang Xi1,2, Surabhi Sonam1,3,4, Thuan Beng Saw 1,5, Benoit Ladoux 1,4 & Chwe Collective epithelial behaviors are essential for the development of lumens in organs. How- ever, conventional assays of planar systems fail to replicate cell cohorts of tubular structures that advance in concerted ways on out-of-plane curved and conﬁned surfaces, such as ductal elongation in vivo. Here, we mimic such coordinated tissue migration by forming lumens of epithelial cell sheets inside microtubes of 1–10 cell lengths in diameter. We show that these cell tubes reproduce the physiological apical–basal polarity, and have actin alignment, cell orientation, tissue organization, and migration modes that depend on the extent of tubular conﬁnement and/or curvature. In contrast to ﬂat constraint, the cell sheets in a highly constricted smaller microtube demonstrate slow motion with periodic relaxation, but fast overall movement in large microtubes. Altogether, our ﬁndings provide insights into the emerging migratory modes for epithelial migration and growth under tubular conﬁnement, which are reminiscent of the in vivo scenario. 1 Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411, Singapore. 2 Centre for Advanced 2D Materials and Graphene Research Centre, National University of Singapore, 6 Science Drive 2, Singapore 117546, Singapore. 3 Department of Biomedical Engineering and Department of Mechanical Engineering, National University of Singapore, Singapore 117575, Singapore. 4 Institut Jacques Monod, Université Paris Diderot & CNRS UMR 7592, 75205 Paris cedex 13, France. 5 NUS Graduate School of Integrative Sciences and Engineering, National University of Singapore, Singapore 117456, Singapore. 6 Biomedical Institute for Global Health Research and Technology, National University of Singapore, #14-01, MD6, 14 Medical Drive, Singapore 117599, Singapore. Wang Xi, Surabhi Sonam and Thuan Beng Saw contributed equally to this work. Correspondence and requests for materials should be addressed to B.L. (email: benoit.ladoux@ijm.fr) or to C.T.L. (email: ctlim@nus.edu.sg) 1 NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x M M any human internal organs contain epithelial lumens such as cysts and tubules, which are composed of curved epithelial monolayers enclosing a central cavity. The organization and development of these various epithelial luminal architectures aid in the essential functioning of the organs and are essential in organogenesis1. One common form of morphogenetic process that promotes epithelial tubulogenesis is the collective migration of cell cohorts while maintaining epi- thelial integrity2–5. Emergent patterns of collective cell migration under tubular conﬁnement For example, in mammalian mammary morphogenesis, ductal elongation is accomplished by the move- ment of a group of interconnected cells at the ductal tip6. Simi- larly, coordinated migration of epithelial cells contributes to the positioning of the zebraﬁsh pronephric nephron segment boundaries and to the convolution of the proximal tubule4. Importantly, anomalies in these epithelial motilities have con- sequences for a series of diseases such as cancers6–8. Thus, understanding the key cellular processes in collective cell migra- tion can provide signiﬁcant insights into epithelial morphogenesis as well as contribute toward disease therapies. and the dynamics of multicellular assemblies on planar surfaces as well as in conﬁned environments11–15. Spatial constraint has been highlighted to induce epithelial migration modes that differ from unrestricted ﬂat microenvironments16,17. For instance, epithelial cell monolayers show diffusion-like motion in rectan- gular microchannels18 but undergo epithelial–mesenchymal transition (EMT) when exposed to scattering periodic micropillar restriction19. In addition, the degree and geometry of conﬁne- ments pose another regulation on patterns of collective cell migration. While cell monolayers demonstrate caterpillar-like migratory motion in narrow rectangular strips12, they exhibit coordinated rotating motion under circular boundary restric- tions20,21. Furthermore, the importance of in-plane curvature cues in modulating the polarization22, proliferation23, wound healing processes24, and organization25 of expanding epithelial sheets has been conﬁrmed recently. It is also noteworthy that most of the prior studies investigating the role of physical cues on tissue migration have mainly employed two-dimensional (2D) ﬂat cell culture systems, whereas morphogenetic movements26 or tumor progression27 are facing out-of-plane spatial constrictions and signals. Also, the 2D approaches mainly study planar epithelial sheets whose topo- graphy is fundamentally different from that of lumens. On the other hand, conventional in vitro approaches for epithelial lumen The movement of interconnected cells during tubule formation commonly happens in complex physiological environments consisting of a plethora of physical features such as conﬁned spaces with out-of-plane curvatures2,9,10. External physical cues are known to have profound impacts on epithelial architectures Nuclei Nuclei E-cadherin Actin d e Small Microtube diameters Big Microtubes embedded in a PDMS block Cell reservoir a b c rmation of epithelial lumens inside PDMS microtubes. a Schematic illustration of the experimental setup. MDCK cells (blue, nuclei and yellow, cell re seeded on a ﬁbronectin reservoir in front of a PDMS block containing cylindrical microtubes of different sizes. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x b The cell density at different locations away from the TCS front edges in various microtubes (n = 5 from three independent experiments). c Average cell height of TCSs in different microtubes and ﬂat surface (n = 21 from six independent experiments). t-test has been performed between each microtube diameter and ﬂat condition, *P < 0.001. NS non-signiﬁcant. Data are presented as mean ± s.e.m. formation involve making use of gels analogous to collagen matrices that encompass cells. Although such methods allow epithelial cells to reproduce tissue-like organization28 and to mimic tubular branching morphogenesis in the presence of growth factors28,29, the direction of epithelium advancement and lumen formation in gel-based systems is non-controllable, and thus renders the systematic study of epithelial dynamics in 3D environments very challenging. To this end, recent studies25,30 grew cell sheets on the outer surfaces of cylindrical templates with varying diameter to investigate the collective cell behaviors in a more controllable manner. However, these systems resulted in epithelial tubules having inverted polarity that is incomparable with physiological situations25 and no in-depth study on the dynamics was provided. While fabricating circular microchannels with conventional photolithography technique remains challen- ging, in some successful cases, cell monolayers that were cultured inside such channels under perfusion mainly investigated endothelialisation31–33. Up to now, most of the studies trying to reproduce epithelial cavity networks have aimed at understanding the molecular mechanisms responsible for lumen development, and very few have tried to unravel the dynamical aspects of coordinated epithelial behaviors across space and time. MCF-10A cells demonstrated coordinated migration under these tubular conﬁnements. Further, they form lumens enclosed by cell sheets and exhibit similar apicobasal polarity as seen in vivo, even for the tubes with the most restrictive conditions. Interestingly, the progression of these hollow, tubular epithelial cell sheets (TCSs) in microtubes smaller than three cell lengths (≤50 μm) are led by clusters of interconnected cells plugging the tubes, remi- niscent of sprouting and branching morphogenesis6. We show that cells adopt different migratory patterns, which strongly depend on the extent of tubular constraint such as overall forward cell motion in large microtubes and onward and backward movements in small tubes. Our analysis reveal that these modes are distinct from that observed on ﬂat tracks12. Additionally, the effect of tubular conﬁnement on the epithelial architecture is reﬂected in actin stress ﬁber organization and cellular orienta- tions. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x 3D-reconstructed image 3D-reconstructed image Cross-sectional view Radial Longitudinal Cross-sectional view Radial Φ = 25 μm Φ = 100 μm Longitudinal 10 8 6 4 2 0 25 50 75 100 150 250 Flat Tube diameter (μm) NS NS * * * * Cell height (μm) 6 5 4 3 2 1 0 50 100 150 200 250 Distance from cell front edge (μm) 25 μm 100 μm 250 μm Cell count per 1000 μm2 Nuclei Gp135 Actin a b c Fig. 2 Epithelial TCS organization in microtubes of varying diameter. a 3D reconstructed ﬂuorescent images of anti-Gp135- and phalloidin-stained MDCK cells (nuclei in blue, DAPI) in microtubes of different diameters (25 μm (left) and 100 μm (right)). Lower panels show radial and longitudinal cross-sections of the representative TCSs. Cyan arrows indicate the direction of the collective migration. White arrow indicates the blocked tube front and orange arrow, the formed epithelial lumen in a 25 μm microtube. Scale bars: 40 μm. b The cell density at different locations away from the TCS front edges in various microtubes (n = 5 from three independent experiments). c Average cell height of TCSs in different microtubes and ﬂat surface (n = 21 from six independent experiments). t-test has been performed between each microtube diameter and ﬂat condition, P < 0.001. NS non-signiﬁcant. Data are presented as mean ± s.e.m. a 3D-reconstructed image Cross-sectional view Radial Φ = 25 μm Longitudinal Nuclei Gp135 Actin a 3D-reconstructed image Cross-sectional view Radial Longitudinal Φ = 100 μm 10 8 6 4 2 0 25 50 75 100 150 250 Flat Tube diameter (μm) NS NS * * * Cell height (μm) c 6 5 4 3 2 1 0 50 100 150 200 250 Distance from cell front edge (μm) 25 μm 100 μm 250 μm Cell count per 1000 μm2 b b c Distance from cell front edge (μm) Fig. 2 Epithelial TCS organization in microtubes of varying diameter. a 3D reconstructed ﬂuorescent images of anti-Gp135- and phalloidin-stained MDCK cells (nuclei in blue, DAPI) in microtubes of different diameters (25 μm (left) and 100 μm (right)). Lower panels show radial and longitudinal cross-sections of the representative TCSs. Cyan arrows indicate the direction of the collective migration. White arrow indicates the blocked tube front and orange arrow, the formed epithelial lumen in a 25 μm microtube. Scale bars: 40 μm. Emergent patterns of collective cell migration under tubular conﬁnement The cells start crawling into the s of the microtubes once they are in full conﬂuence (white arrows indicate the leading edge of the cohort and the cyan arrow, the direction of e migration). b, c Representative SEM image (b) and optical image (c) of the microtubes embedded in the PDMS blocks (the diameters of the be: 25, 50, 75, 100, 150, and 250 μm from left to right). d, e Typical ﬂuorescent z-stack projections showing groups of migrating MDCK cells inside bes: the cells were stained for nuclei (DAPI, blue) and in d, actin (phalloidin, red) or in e, E-cadherin (green). The cross-sectional views of the TCS sections in the right panels demonstrate the formation of hollow lumens inside the microtubes. Scale bars: 250 μm for b and c; 50 μm d e Small Microtube diameters Big Microtubes embedded in a PDMS block Cell reservoir a a Cell reservoir Microtubes embedded in a PDMS block Microtubes embedded in a PDMS block Big c b b c d Nuclei Actin d e Nuclei E-cadherin e Fig. 1 Formation of epithelial lumens inside PDMS microtubes. a Schematic illustration of the experimental setup. MDCK cells (blue, nuclei and yellow, cell body) are seeded on a ﬁbronectin reservoir in front of a PDMS block containing cylindrical microtubes of different sizes. The cells start crawling into the openings of the microtubes once they are in full conﬂuence (white arrows indicate the leading edge of the cohort and the cyan arrow, the direction of collective migration). b, c Representative SEM image (b) and optical image (c) of the microtubes embedded in the PDMS blocks (the diameters of the microtube: 25, 50, 75, 100, 150, and 250 μm from left to right). d, e Typical ﬂuorescent z-stack projections showing groups of migrating MDCK cells inside microtubes: the cells were stained for nuclei (DAPI, blue) and in d, actin (phalloidin, red) or in e, E-cadherin (green). The cross-sectional views of the selected TCS sections in the right panels demonstrate the formation of hollow lumens inside the microtubes. Scale bars: 250 μm for b and c; 50 μm for d and e NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Altogether, our ﬁndings provide important clues to understand the pivotal role of external physical constraints in dictating epithelial morphogenesis. formation involve making use of gels analogous to collagen matrices that encompass cells. Although such methods allow epithelial cells to reproduce tissue-like organization28 and to mimic tubular branching morphogenesis in the presence of growth factors28,29, the direction of epithelium advancement and lumen formation in gel-based systems is non-controllable, and thus renders the systematic study of epithelial dynamics in 3D environments very challenging. To this end, recent studies25,30 ll h h f f l d l l h NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x The microtubes offer out-of-surface negative curvatures with tubular conﬁne- ment, which is fundamentally different from the previous experimental setups based on cells cultured on planar sur- faces12,18,37 or borderless cylindrical conﬁnement that provides positive curvatures25. We observed that in all circumstances, once cells wrapped around the whole circumference, they collectively migrated along the microtubes with typical lengths of ~1 mm while maintaining their cell–cell adhesion to form a hollow TCS (Fig. 1e; Supplementary Fig. 3; Supplementary Movie 1). Sur- prisingly, lumens were able to develop even in the smallest tubes with a diameter of only one or two cell lengths (~20–50 μm) (Fig. 1e). The epithelial lumens formed in our microtube platform was then used to stencil the shapes of the metal wires. After cross- linking, the metal wires were removed from the polymerized PDMS, resulting in PDMS blocks embedded with straight circular microtubes (Fig. 1b, c; Supplementary Fig. 2) of exact diameters of the metal wires. This technique can produce transparent and biocompatible microtubes with smooth inner surfaces for long- term cell culture34. We used the microtubes with diameters of 25–250 μm in our experiments because 25 μm represents the diameter of a distal tubule (~30 μm) in kidney nephrons35, while 250 μm is the approximate size of papillary collecting ducts (200–300 μm)36. The inner surfaces of the microtubes were functionalized with ﬁbronectin before placing the PDMS block in front of a cohort of advancing MDCK cells. The narrow gap between the bottom of the microtubes and the substrate enabled MDCKs to crawl into the microtubes (Fig. 1a, d). The microtubes offer out-of-surface negative curvatures with tubular conﬁne- ment, which is fundamentally different from the previous experimental setups based on cells cultured on planar sur- faces12,18,37 or borderless cylindrical conﬁnement that provides positive curvatures25. We observed that in all circumstances, once cells wrapped around the whole circumference, they collectively migrated along the microtubes with typical lengths of ~1 mm while maintaining their cell–cell adhesion to form a hollow TCS (Fig. 1e; Supplementary Fig. 3; Supplementary Movie 1). Sur- prisingly, lumens were able to develop even in the smallest tubes with a diameter of only one or two cell lengths (~20–50 μm) (Fig. 1e). The epithelial lumens formed in our microtube platform Tubular conﬁnement inﬂuences epithelial TCS organization. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x LCs FCs LCs FCs Cells advancing direction Φ = 25 μm Φ = 100 μm a LCs FCs LCs LCs FCs FCs Cells advancing direction Φ = 25 μm Φ = 100 μm Φ = 250 μm 0.8 1.0 0.6 0.4 0.2 Sa=<cos(2)> 0.0 0 Tube diameter (μm) 50 100 150 200 250 40 30 20 10 50 0 Number ofcells 0 0 30 NS 60 90 120 150 180 30 60 90 120 150 180 0 Flat 60 90 120 150 180 0 30 60 90 120 150 180 30 Φ = 25 μm Φ = 250 μm Φ = 100 μm * ** * * * a b c Fig. 3 Epithelial cytoskeleton and cell orientation in different TCSs. a Phalloidin-stained MDCK TCSs exhibiting basal actin stress ﬁber organization in different tube dimensions (25 μm (left), 100 μm (middle), and 250 μm (right)). LC is the representative image of the cells at leading cell front of the tube and FC is the representative image of the following cells. Cyan arrow points in the direction of migration. White arrowheads point to the actin microﬁlaments oriented along the tube long axis. Scale bars: 15 μm. b Order parameter (Sa = < cos(2θ) > ) of the actin ﬁlament as a function of microtube dimension, where θ is the angle of actin ﬁlament with respect to the tube length. 1 signiﬁes parallel alignment and 0 signiﬁes randomness in organization (n = 10 from ﬁve independent experiments). t-test has been performed between each microtube diameter and 25 μm, P < 0.05, P < 0.01, *P < 0.001, NS non-signiﬁcant. Data are presented as mean ± s.e.m. c Polar graph plotting the histogram of the cell orientation distribution inside microtubes (25, 100, and 250 μm (from left to right)). 90° means oriented along the microtube long axis, and 0o and 180o mean the cell is oriented perpendicular to the long axis a 0.8 1.0 0.6 0.4 0.2 Sa=<cos(2)> 0.0 0 Tube diameter (μm) 50 100 150 200 250 NS ** * * * b 40 30 20 10 50 0 Number ofcells 0 0 30 60 90 120 150 180 30 60 90 120 150 180 0 Flat 60 90 120 150 180 0 30 60 90 120 150 180 30 Φ = 25 μm Φ = 250 μm Φ = 100 μm c b c Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x 3 Epithelial cytoskeleton and cell orientation in different TCSs. a Phalloidin-stained MDCK TCSs exhibiting basal actin stress ﬁber organization in different tube dimensions (25 μm (left), 100 μm (middle), and 250 μm (right)). LC is the representative image of the cells at leading cell front of the tube and FC is the representative image of the following cells. Cyan arrow points in the direction of migration. White arrowheads point to the actin microﬁlaments oriented along the tube long axis. Scale bars: 15 μm. b Order parameter (Sa = < cos(2θ) > ) of the actin ﬁlament as a function of microtube dimension, where θ is the angle of actin ﬁlament with respect to the tube length. 1 signiﬁes parallel alignment and 0 signiﬁes randomness in organization (n = 10 from ﬁve independent experiments). t-test has been performed between each microtube diameter and 25 μm, P < 0.05, P < 0.01, P < 0.001, NS non-signiﬁcant. Data are presented as mean ± s.e.m. c Polar graph plotting the histogram of the cell orientation distribution inside microtubes (25, 100, and 250 μm (from left to right)). 90° means oriented along the microtube long axis, and 0o and 180o mean the cell is oriented perpendicular to the long axis are reminiscent of various epithelial ducts with a broad range of diameters, including those formed physiologically35,36 as well as those developed in 3D hydrogel10. was then used to stencil the shapes of the metal wires. After cross- linking, the metal wires were removed from the polymerized PDMS, resulting in PDMS blocks embedded with straight circular microtubes (Fig. 1b, c; Supplementary Fig. 2) of exact diameters of the metal wires. This technique can produce transparent and biocompatible microtubes with smooth inner surfaces for long- term cell culture34. We used the microtubes with diameters of 25–250 μm in our experiments because 25 μm represents the diameter of a distal tubule (~30 μm) in kidney nephrons35, while 250 μm is the approximate size of papillary collecting ducts (200–300 μm)36. The inner surfaces of the microtubes were functionalized with ﬁbronectin before placing the PDMS block in front of a cohort of advancing MDCK cells. The narrow gap between the bottom of the microtubes and the substrate enabled MDCKs to crawl into the microtubes (Fig. 1a, d). Results Formation of tubular epithelial cell sheet inside microtubes. We used smooth platinum or copper wires with various diameters as templates to fabricate circular microchannels (microtubes) in polydimethylsiloxane (PDMS) to study tubular collective epithelial migration (Fig. 1a). Brieﬂy, metal micro-wires were aligned in parallel just 1–2 μm above a silicon wafer using a precise stage (Supplementary Fig. 1). A PDMS polymer precursor To address these challenges, we provide a microtube platform for the study of collective epithelial dynamics that leads to the formation of hollow tubules. The microtubes have dimensions ranging from one to several cell lengths in dimension and serve as physical guiding cues for collective cell behaviors. We observe that epithelial Madin-Darby Canine Kidney (MDCK) and NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x 400 0.5 H1-GFP NS 0.4 0.3 0.2 0.1 0.0 25 50 75 100 150 250 25 μm 100 μm 250 μm Tube diameter (μm) 300 200 100 0 0 Time (min) Front edge displacement (μm) Average front velocity (μm/min) 100 200 300 400 500 600 700 800 00:00 02:00 04:00 Φ = 100 μm Φ = 25 μm Φ = 250 μm a b c d Fig. 4 Collective migratory speed of MDCK TCSs varies with microtube diameter. a Representative time-lapse montage showing the collective migration of H1-GFP MDCK cells inside a 100 μm microtube. (top: phase contrast; bottom: H1-GFP showing the nuclei). Scale bar: 100 μm. b 3D ﬂuorescent reconstruction of H1-GFP MDCK TCSs in different microtubes: top (left panel) and cross-sectional (right panel) views of the TCSs in each subﬁgure. Scale bars: 50 μm. c Displacement of MDCK cell fronts in different microtubes (25, 100, and 250 μm) as a function of time (n = 3 from three independent experiments). d Average velocity of cell front of H1-GFP MDCKs in tubes of different diameters (25, 50, 75, 100, 150, and 250 μm, from left to right), (n = 6–8 from 3–4 independent experiments in each condition). For each condition, t-test has been performed between each microtube diameter and 25 μm, unless otherwise indicated by line, P < 0.01, NS non-signiﬁcant. The plots represent the mean ± s.e.m. 00:00 02:00 04:00 a a Φ = 100 μm Φ = 25 μm Φ = 250 μm b b 400 25 μm 100 μm 250 μm 300 200 100 0 0 Time (min) Front edge displacement (μm) 100 200 300 400 500 600 700 800 c 0.5 H1-GFP NS 0.4 0.3 0.2 0.1 0.0 25 50 75 100 150 250 Tube diameter (μm) Average front velocity (μm/min) d d c Time (min) Fig. 4 Collective migratory speed of MDCK TCSs varies with microtube diameter. a Representative time-lapse montage showing the collective migration of H1-GFP MDCK cells inside a 100 μm microtube. (top: phase contrast; bottom: H1-GFP showing the nuclei). Scale bar: 100 μm. b 3D ﬂuorescent reconstruction of H1-GFP MDCK TCSs in different microtubes: top (left panel) and cross-sectional (right panel) views of the TCSs in each subﬁgure. Scale bars: 50 μm. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x c Displacement of MDCK cell fronts in different microtubes (25, 100, and 250 μm) as a function of time (n = 3 from three independent experiments). d Average velocity of cell front of H1-GFP MDCKs in tubes of different diameters (25, 50, 75, 100, 150, and 250 μm, from left to right), (n = 6–8 from 3–4 independent experiments in each condition). For each condition, t-test has been performed between each microtube diameter and 25 μm, unless otherwise indicated by line, *P < 0.01, NS non-signiﬁcant. The plots represent the mean ± s.e.m. curvature in all directions40,41 as is also observed in MDCK cells (Supplementary Fig. 6c). As the smallest microtubes have strong negative curvature in the circumferential direction, this could suggest that the cells spread less in these tubes and thus acquire a taller cell height. There is also a possibility that a signiﬁcant radial force exists pointing toward the center axis of the microtube that can stretch cell toward the center of the tube (Supplementary Fig. 6d). Assuming that acto myosin contractility generates a tension, T, in the circumferential direction of the tube through cell–cell junctions, the resulting force per unit length exerted on the epithelial cells surrounding the tube is Tρ~n, where ρ is the curvature and ~n is the normal vector to the border directed toward the interior of the tube. This radial force originated from tissue tension is proportional to curvature and may thus explain why cell height could be taller in smaller microtubes with higher curvature. Taken together, these ﬁndings identiﬁed a critical negative curvature of ~1/12.5–1/50/μm as the threshold for the MDCKs to respond to tubular constraint in a signiﬁcant manner. deeper into these small microtubes, hollow lumens enclosed by epithelial monolayers of single to multiple cells formed at the back of the clusters of leader cells (Fig. 2a; Supplementary Fig. 4). In addition, in such small tubes, the cell density decreased from roughly 3–4 cells/1000 μm2 at the leading edge to 1–2 cells/ 1000 μm2 about 250 μm away from the front (Fig. 2b; Supple- mentary Fig. 6b). The abrupt decrease in cell density at ca. 50–100 μm from the leading front is due to the change from the multilayered cluster to hollow lumen (Fig. 2a, b; Supplementary Figs. 4, 6b). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Epithelia probe external physical signals particularly through cell–substrate adhesions and the associated actin cytoskeleton, and thus adjust their organization and distribution according to the geometrical constraints12,25,37,38. In vivo extracellular matri- ces allow lumen formation of epithelial cells during which they undergo apicobasal polarization39. Thus, we ﬁrst sought to determine the TCS polarization by immunostaining the cells with Gp135—an apical marker. The TCSs were ﬁxed and imaged after the front had progressed inside the microtubes for 24–48 h to ensure the formation of lumens. The ﬂuorescent 3D reconstruc- tion conﬁrmed that MDCK TCSs polarized apicobasally in the tubes of all sizes with apical surfaces facing the lumens (Fig. 2a; Supplementary Fig. 4), recapitulating the polarized epithelial ducts in physiological conditions10. Notably, the cells at the tip of the smallest tubes (25–50 μm) lacked such polarity and instead of forming hollow TCSs, usually organized as multilayered epithelial structures (Fig. 2a; Supplementary Figs. 4, 5; Supplementary Movie 1), which plugged the microtube (Supplementary Fig. 6a). An analogous organization of multilayered epithelium at the ductal tip was also observed in both normal and neoplastic epithelia during morphogenesis6. Interestingly, after advancing NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x d Kymograph of the average velocity parallel to the longitudinal axis of different microtube (y-direction in a, from left to right: 25, 100, and 250 μm). e Kymograph of the average velocity along the tubular circumferential of different microtube (x-direction in a, from left to right: 25, 100, and 250 µm). The plots represent the mean ± s.e.m. For each condition, t-test has been performed between each microtube diameter and 25 μm, unless otherwise indicated by lines, P < 0.05, P < 0.01, P < 0.001, NS non-signiﬁcant Φ = 25 μm Φ = 100 μm Φ = 250 μm 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0.3 0.2 0.1 0 –0.1 –0.2 –0.3 0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8 0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8 50 μm 50 μm 50 μm Time (min) Cells advancing direction Velocity (long axis; μm/min) d 160 140 120 100 80 60 40 20 0 50 100 150 200 250 Tube diameter (μm) Longitudinal Circumferential Correlation length (μm) NS NS NS * * * ** * NS b d b Velocity (long axis; μm/min) Tube diameter (μm) Cells advancing direction e Tube diameter (μm) 0.9 0.8 0.7 0.6 0.5 0.4 0 50 100 150 200 250 Sv=<cos()> NS * * * * c c e Velocity (circumference; μm/min) Φ = 25 μm Φ = 100 μm Φ = 250 μm 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 0 0.2 0.1 0 –0.1 –0.2 0.3 0.2 0.1 –0.1 –0.2 –0.3 0.3 0.2 0.1 –0.1 –0.2 –0.3 50 μm 50 μm Time (min) Cells advancing direction Velocity (circumference; μm/min) e 50 μm Fig. 5 PIV analysis of MDCK cell tubes in microtubes of different diameters. a Direction of velocity ﬁelds showing backward and forward motion in 25 μm tubes (left), highly directed forward motion in 100 μm tubes (middle) and multiple leading edges (white arrows) in 250 μm tubes (right). Scale bars: 75 μm. b Longitudinal and circumferential correlation length of velocity vectors (n = 14 from ﬁve independent experiments). c Order parameter (Sv = < cos(ϕ) > ) of the velocity vectors in MDCK TCSs (n = 6 from three independent experiments). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x However, the slope of descending trend in cell count ﬂattened in 75 μm microtube and was reversed in 100 μm and larger microtubes. The low cell density and increasing trends were also reported in epithelia cultured on strips of different 2D constraints12, and this indicates that the epithelial organization in microtubes ≥100 μm approached that in planar tracks. More importantly, the transition in our experiment demonstrates the effect of tubular conﬁnement and curvature on epithelial organization, which starts to disappear as the tubular diameter exceeds 50 μm. Furthermore, the average cell height in the TCSs was found to be inversely proportional to the tubular diameter (Fig. 2c), where the averaged cell height in the largest microtubes (150–250 μm) was similar to that measured on the ﬂat substrates and the cells in the smallest microtubes were ~45% taller. Cells are known to spread less on bowl-shaped structures with negative Epithelial organization in microtubes. The cellular cytoskeleton in terms of contractility and dynamical properties has been shown to respond to the external mechanical cues for single cells42–44 and collectives12,15,45. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Therefore, we studied how the changes in tubular conﬁnement and curvature inﬂuenced the NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x 0.3 0.2 0.1 0 y x Φ = 25 μm Speed (μm/min) a a y x Φ = 100 μm Speed (μm/min) 0.8 0.6 0.4 0.2 0 y x Φ = 250 μm Speed (μm/min) 0.6 0.4 0.2 0 0.3 0.2 0.1 0 y x y x y x Φ = 25 μm Φ = 100 μm Φ = 250 μm Speed (μm/min) Speed (μm/min) Speed (μm/min) 0.8 0.6 0.4 0.2 0 0.6 0.4 0.2 0 a Φ = 25 μm Φ = 100 μm Φ = 250 μm Φ = 25 μm Φ = 100 μm Φ = 250 μm 160 140 120 100 80 60 40 20 0 50 100 150 200 250 Tube diameter (μm) Longitudinal Circumferential 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 0 0 200 400 600 800 1000 0.3 0.2 0.1 0 –0.1 –0.2 –0.3 0.2 0.1 0 –0.1 –0.2 0.3 0.2 0.1 –0.1 –0.2 –0.3 0.3 0.2 0.1 –0.1 –0.2 –0.3 0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8 0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8 50 μm 50 μm 50 μm 50 μm 50 μm Time (min) Time (min) Correlation length (μm) Cells advancing direction Cells advancing direction NS NS NS * * * * ** * NS Velocity (long axis; μm/min) Velocity (circumference; μm/min) b d Tube diameter (μm) 0.9 0.8 0.7 0.6 0.5 0.4 0 50 100 150 200 250 Sv=<cos()> NS * * * * c e 50 μm Fig. 5 PIV analysis of MDCK cell tubes in microtubes of different diameters. a Direction of velocity ﬁelds showing backward and forward motion in 25 μm tubes (left), highly directed forward motion in 100 μm tubes (middle) and multiple leading edges (white arrows) in 250 μm tubes (right). Scale bars: 75 μm. b Longitudinal and circumferential correlation length of velocity vectors (n = 14 from ﬁve independent experiments). c Order parameter (Sv = < cos(ϕ) > ) of the velocity vectors in MDCK TCSs (n = 6 from three independent experiments). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x d Kymograph of the average velocity parallel to the longitudinal axis of different microtube (y-direction in a, from left to right: 25, 100, and 250 μm). e Kymograph of the average velocity along the tubular circumferential of different microtube (x-direction in a, from left to right: 25, 100, and 250 µm). The plots represent the mean ± s.e.m. For each condition, t-test has been performed between each microtube diameter and 25 μm, unless otherwise indicated by lines, P < 0.05, P < 0.01, P < 0.001, NS non-signiﬁcant to the random orientation in MDCKs cultured on ﬂat surfaces. We further wanted to understand whether the basal stress ﬁber orientation had any correlation with the orientation of the cell body. To this end, we plotted the cell body orientation deﬁned as the angle formed between the major axis of the cell and the microtube longitudinal axis (Methods) as a function of the microtube diameter. As shown in the polar plot in Fig. 3c and Supplementary Fig. 7b, more cells aligned closer to the tube long axis for microtubes with diameters smaller than 100 μm. In contrast and as expected, a global isotropic cell orientation was observed in larger microtubes similar to that seen on ﬂat surfaces (Fig. 3c). Altogether, these data demonstrate a clear correlation between cell shape and actin cytoskeleton orientations. The alterations in cell orientation and actin organization appeared at a epithelial tissue organization by looking at high-resolution images of the cells’ basal actin stress ﬁbers (Fig. 3a). Parallel and aligned actin stress ﬁbers were observed in the leading cell fronts (LCs) in TCS of all diameters up to 75–100 μm (Fig. 3a; Supplementary Fig. 7a). Interestingly, in TCS smaller than 75 μm, actin stress ﬁbers were aligned longitudinally throughout the entire lumen (Fig. 3a; Supplementary Fig. 7a), while such alignment disappears in the follower cells of TCS in diameters ≥75 μm. To better quantify these ﬁndings, we computed the overall nematic actin order parameter deﬁned as Sa = <cos(2θ) > 25 (Methods), which indeed decreased by almost ﬁve-fold for tube diameters ranging from 25 to 250 μm (Fig. 3b). In larger microtubes (≥100 μm), instead of forming well-aligned actin microﬁlaments, an inter- crossing actin cytoskeletal arrangement was observed, comparable NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 6 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x threshold of ~75–100 μm in diameter, consistent with stress ﬁber orientation. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Upon computing the average velocity of the cell front (vf ) over the same period of time, we discovered that vf for smaller TCSs (≤75 μm) increased as a function of tubular diameter up to about four-folds and plateaued in TCSs of 100 μm and above. This observation is in sharp contrast to that on 2D conﬁned strips12 and cylindrical wires25, where vf decreases as the constraint reduces. The fact that tissue expansion speeds were lower for microtube diameters <100 μm could be related to cell jamming in these tubes due to higher cell densities and the additional plugging of the smallest microtubes by multilayered tissue structures49–51. We also noticed that vf of TCSs ≥100 μm was comparable to that on ﬂat tracks with a width larger than 100 μm12-18. This could be due to large TCSs having similar architectures (Figs. 2, 3 and see above) with epithelia on the broad 2D strips, and thus, their migratory pattern may resemble the planar counterparts. The vf vs. diameter trend with increasing microtube diameter was robust as experiments using wild type (WT) MDCK (Supplementary Fig. 9) and another epithelial cell line, MCF-10A (Supplementary Fig. 10) yielded similar results as that for H1-GFP MDCK, suggesting a shared behavior between epithelial tissues under such conditions. Velocity ﬁelds and migration phenotypes of epithelial TCSs. The previous observation motivated us to ask whether the var- iation of cell edge migratory velocity in different-sized TCSs arose from the differences in local and global velocity ﬁelds within the TCSs. The virtually opened tube images (Supplementary Fig. 8b) allowed the direct use of particle image velocimetry (PIV)52 to map the velocity ﬁeld (Fig. 5a), and in such images, x-axis represents the circumference of the tube, while y-axis denotes the tube length. Velocity vectors (arrows) in TCS of 25 μm in dia- meter revealed the presence of separate groups of cells migrating in opposite directions along the tubular long axis at a given instant (Fig. 5a, left), suggesting contraction–relaxation modes of migration. Similar behaviors had been reported previously12 as a mode of MDCK monolayer migration caused by the spatial constraint on ﬂat single cell-wide strips. However, the “tug-of- war” within the cell sheet on the highest curvature resulted in To further elaborate the dynamics of the TCS coordination, we investigated the variation in the velocity attained by the cell sheets in time and space. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Consequently, it appears that tubular conﬁnement/ curvature is sufﬁcient for both modulating the dynamics of epithelial cell–cell interaction and inducing alignment of cells in long distance. To conﬁrm this hypothesis, we calculated the order parameter of the velocity, Sv = < cos(ϕ) > , where ϕ is the angle between the local velocity vector and the tube long axis, (Methods) and the results demonstrated a similar incremental trend which plateaued at ~0.80 when Ф > 100 μm (Fig. 5c). This is again in contrast with planar conﬁnement where Sv decreases from a high value of 0.92 to ~0.80 as the width of the stripes increases12. We attribute the much lower order parameter in the smallest microtubes to the coexistence of the forward and backward velocity vectors, whose Sv cancels one another (Fig. 5a, left). In the case of intermediate-sized microtubes (Ф = 75 and 100 μm), PIV analysis demonstrated a velocity ﬁeld with many vectors parallel to x-axis (i.e., large ϕ, Fig. 5a, middle). This x- component of the velocity vector indicates that cells can migrate in the circumferential direction (circumferential swirling) and consequently, contributing to lower Sv. For the large microtubes (>100 μm), the TCSs exhibited similar order parameter and velocity ﬁeld patterns as epithelia on wide ﬂat stripes12. Although the transient vortices and circumferential migration frequently appeared, these were limited to small regions (mostly in the leading edge) of the sheets and the majority of cells migrated along the tubular long axis, bringing about a high Sv. Altogether, we conclude that the tubular conﬁnement with curvature has a profound inﬂuence on the collective epithelial migration, which is different from that of conventional 2D ﬂat constraint. p First, we investigated the displacement of the LC in the TCSs due to the critical role of leader cells in regulating collective cell migration48 and the relevance of different epithelial structures at the ductal tips (Fig. 2a and see above). As shown in Fig. 4c, the TCSs demonstrated linear progression with time. Interestingly, the displacement–time graphs for the larger TCSs (≥100 μm) exhibited a steeper slope than the smaller ones (≤75 μm) (Fig. 4c; Supplementary Fig. 8c), indicating that the TCSs under the highest constraint progressed with the slowest speed (see also Fig. 4d). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x much more prominent contracted–relaxed periods in the migratory patterns (Supplementary Movie 4). The periodic halt in the TCS advancement may explain the slow progression of the TCSs (Fig. 4c, d). In contrast, epithelial TCSs of 100 μm exhibited high longitudinal migration speed throughout the microtube (Fig. 5a, middle; Supplementary Movie 5). These high instanta- neous velocities (~0.8 μm/min, deep red arrows) were observed at the leading front of the TCS as well as in the following cells that are 5–6 cell lengths behind. Furthermore, in the largest micro- tubes (250 μm), several cohorts of cells migrated in different directions (Fig. 5a, right, white arrows). Groups of velocity vec- tors representing high or low instantaneous velocity as well as ephemeral velocity vortices were noted in these TCSs (Supple- mentary Movie 6), resembling ﬂat epithelial sheet behaviors. Tubular conﬁnement induces different migration speeds. Actin cytoskeletal organization is known to correlate with the migration dynamics of cell cohorts46,47. Thus, upon understanding the speciﬁc cell orientation and cytoskeletal organization in the TCS, we deemed it important to gauge the collective dynamics of the tissue migrating inside the microtubes. In order to study the collective migration patterns in TCSs, we cultured MDCKs that stably express Histone1-GFP (H1-GFP) in various sized micro- tubes (Fig. 4a; Supplementary Movie 2). The ﬂuorescent signal from the nuclei enabled the precise detection of individual cells within the tubular space (Fig. 4b; Supplementary Fig. 8a; Sup- plementary Movie 3). Moreover, we developed a Fiji code to virtually open the tube along its circumference into a planar equivalent (Supplementary Fig. 8b), such that we can quantify the collective movement on out-of-plane curvature. Variation in the velocity ﬁelds indicates the alternation in coordination within the cell sheets. Indeed, we quantiﬁed the spatial velocity correlation lengths in both longitudinal and circumferential directions increased up to Ф > 100 μm and plateaued at ~120–140 μm (Fig. 5b), which agrees with the values of 120–200 μm for planar unconﬁned substrates12. As expected, reducing conﬁnement and curvature leads to the epithelial dynamics resembling the one observed on 2D unconﬁned surfaces. Moreover, the change of correlation lengths in both directions reminisces the vf vs. diameter pattern (Figs. 4d, 5b), a phenomenon not seen in the 2D equivalents12. In addition, this is consistent with our previous observation of transition in the TCS architectures when the diameter increased beyond 75–100 μm (Figs. 2, 3). NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x ~60–80 min (Fig. 5d; Φ = 25 μm) were seen in the longitudinal vector kymograph, whereas the average transverse velocity was negligible over a long period (Fig. 5e; Φ = 25 μm). While the red/ yellow streaks represented the overall forward movement of the cells that corresponded to the TCS progression, blue/green ones accounted for the halts or low speed backward motion, which could be attributed to the relaxation of the tissue. This conﬁrms the contraction–relaxation modes under high conﬁnement. In contrast, in loose constrictions >75 μm, color coded for high x- axis velocity (0.6–0.8 μm/min) mainly localized in the frontier region and spanned more than 100 μm deep into the TCSs (Fig. 5d, middle and right). Such velocity ﬁeld kymograph in these microtubes agrees well with large-scale coordination among the cells. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x To quantify this, we plotted kymographs of the y- and x-components of the velocity averaged over the circumferential direction (Fig. 5d, e). Thus, the longitudinal vector (y-component) kymograph shows the spatiotemporal distribution of the local velocity in the direction of the planar surface (zero curvature), while the circumferential (x-component) kymograph presents the motion for an out-of-surface curved substrate. In highly conﬁned tube, the alternate hot color (red/ yellow) and cold color (blue/green) streaks with a period of NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x To understand why this is the case, we ﬁrst checked that the percentage of dividing cells in the tubes (over total number of cells in each tube) were similar across all tube diameters through EdU staining of proliferative cells54 (Fig. 6b). Next, we measured the average velocity in the longitudinal tube direction within a small ROI (radius of ~25 μm) centered on cell division events (Fig. 6c), before (deﬁned as negative time), during (deﬁned as t = 0 min), and after division over a period of ±70 min (t = 0 min is deﬁned as the ﬁrst instance when the two daughter cells were observed). The individual ROI curves for each micro- tube size were further averaged, and these ﬁnal curves showed that the local average longitudinal velocity always transiently reduced in magnitude around the division event (Fig. 6d, within ±30 min), before returning to the initial velocity values unper- turbed by the division events (Fig. 6d, −70 and +70 min). As previously suggested, cell divisions can lead to tissue ﬂuidization and increased viscosity55,56 that slow down local velocity. To further understand why tissue speed was lower for smaller microtubes from the aspect of cell division, we quantiﬁed the magnitude of the velocity disturbance due to division (Fig. 6d) by calculating the area of the dip in the velocity curve over a certain period around division (Fig. 6e, yellow region, A1) divided by the distance that should be traveled over the same period if there was no division (Fig. 6e, sum of the area of yellow and blue region, from –δt to +δt) (Methods). This quantity, which can be understood as the percentage of tissue local distance perturbed by division, showed that the perturbation magnitude increased for smaller microtube diameters ≤75 μm up to nearly 15% per single division (Fig. 6f), which can be expected as smaller microtubes comprised of fewer cells along the circumferential direction and should be more easily perturbed by each division event. The fact that smaller microtubes are perturbed more by division events is also consistent with our ﬁnding that the speed increase in division inhibited tissue (mitomycin C treatment) compared to normal tissue is higher for smaller microtubes, quantiﬁed by the speed ratio = vf without proliferation=vf with proliferation Role of cell–cell junction in collective migration dynamics. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x 11). Altogether, the results showed that the lower tissue speed trend in smaller microtubes was mainly inﬂuenced by the higher velocity perturbation in these tubes and not due to the difference in percentage of dividing cells in dif- ferent microtube sizes. intense in the latter (Fig. 5e, middle and right). Furthermore, the plateau in the large microtube corresponded to the duration it took for the tissue to form a complete TCS, before fast advancement with constant forward progression was achieved (Fig. 5d, e, right). We further generated velocity kymographs for the TCSs treated with mitomycin C. Consistent with the tissue front speed measurements, mitomycin C treatment enhanced the velocity along both the longitudinal and circumferential axes (Fig. 6g, h). Notably, the kymograph of longitudinal vector for small microtubes (Φ = 25 μm) demonstrated analogous contraction–relaxation patterns with frequent detachment of the leader cells from the TCS (the blank regions in the kymograph, Fig. 6g, h, left, n = 15/17 from six independent experiments for 25 μm tube vs. n = 0/16 from six independent experiments for 250 μm tube). This may indicate the build-up of tension within the epithelial sheets in the TCS under high conﬁnement with stress relaxation inhibited under these conditions. In TCSs ≥100 μm, we observed propagative waves opposite to the direction of migration (black dash lines in Fig. 6g, middle and right). These velocity waves traveled at ~3 and 10 μm/min inside 100 and 250 μm microtubes, respectively, corresponding to cell density alternations from 3 to 1 cells per 1000 μm2. Such propagating velocity waves in coordinated migration have been identiﬁed in epithelial monolayers on 2D surfaces57,58. Our results are in line with the reported observation but also show that the existence of these propagative signals depends on cell conﬁnement. Inﬂuence of cell proliferation on migration velocities. During the migration, cell division adds mass to the expanding TCSs but can also create disturbance to their mobility53 in packed condi- tions. To investigate how cell division impacted tissue expansion, we ﬁrst inhibited cell proliferation in MDCK tissues by mito- mycin C treatment and found that this increases vf in all con- ditions, but maintained the vf vs. diameter trend (Fig. 6a; Supplementary Fig. 9), showing that cell divisions lowered tissue speed. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x The difference between the intermediate (Φ = 100 μm) and large (Φ = 250 μm) microtubes was reﬂected by the high circumferential velocity in the former, but much less Φ = 25 μm Φ = 100 μm Φ = 250 μm 50 μm 50 μm 50 μm 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Cells advancing direction Cells advancing direction [+] Mitomycin C 0.3 0.2 0.1 –0.1 –0.2 –0.3 0 200 400 600 800 1000 0 200 400 600 800 0 0.3 0.2 0.1 –0.1 –0.2 –0.3 0 0.2 0.1 –0.1 –0.2 0 0.3 0.2 0.1 –0.1 –0.2 –0.3 0 Time (min) 50 μm 50 μm 50 μm [ ] Mitomycin C 1.2 1 0.8 0.6 0.4 0.2 –0.2 –0.4 0 1000 1.2 1 0.8 0.6 0.4 0.2 –0.2 0 Velocity (long axis; μm/min) Velocity (circumference; μm/min) 0.8 0.6 0.4 0.2 0.0 25 50 75 100 150 250 H1-GFP Mitomycin C(+) Tube diameters (μm) Tube diameters (μm) 80 60 40 20 0 25 50 75 100 150 250 Flat 0.0 0.1 0.2 0.3 25 μm 50 μm 75 μm 150 μm 250 μm 100 μm –80 –60 –40 –20 0 20 40 60 80 Time (min) Average velocity (μm/min) Average front velocity (μm/min) Appearance of two daughter cells A1 v Velocity –δt 0 +δt Time –10 min 0 min +10 min Cells advancing direction Tube diameters (μm) A1 v × δt 0.16 0.14 0.12 0.10 0.08 0.06 0 50 100 150 200 250 Distance perturbation factor * *** * NS NS NS * Percentage of proliferating cells a d g h e f b c 0.8 0.6 0.4 0.2 0.0 25 50 75 100 150 250 H1-GFP Mitomycin C(+) Tube diameters (μm) Tube diameters (μm) 80 60 40 20 0 25 50 75 100 150 250 Flat Average front velocity (μm/min) –10 min 0 min +10 min Cells advancing direction NS NS * Percentage of proliferating cells a b c 0.8 0.6 0.4 0.2 0.0 25 50 75 100 150 250 H1-GFP Mitomycin C(+) Tube diameters (μm) Tube diameters (μm) 80 60 40 20 0 25 50 75 100 150 250 Flat Average front velocity (μm/min) Cells advancing direction NS NS * Percentage of proliferating cells a b c 0.8 0.6 0.4 0.2 0.0 25 50 75 100 150 250 H1-GFP Mitomycin C(+) Tube diameters (μm) Average front velocity (μm/min) NS * a Tube diameters (μm) 80 60 40 20 0 25 50 75 100 150 250 Flat NS Percentage of proliferating cells b Cells advancing direction c b b a c –10 min 0 min +10 min Cells advancing direction Tube diameters (μm) A1 v × δt 0.16 0.14 0.12 0.10 0.08 0.06 0 50 100 150 200 250 Distance perturbation factor * * * NS f 0.0 0.1 0.2 0.3 25 μm 50 μm 75 μm 150 μm 250 μm 100 μm –80 –60 –40 –20 0 20 40 60 80 Time (min) Average velocity (μm/min) d Appearance of two daughter cells A1 v Velocity –δt 0 +δt Time e f f d e e Tube diameters (μm) Time (min) Φ = 25 μm Φ = 100 μm Φ = 250 μm 50 μm 50 μm 50 μm 0 200 400 600 800 1000 Cells advancing direction [+] Mitomycin C 0.3 0.2 0.1 –0.1 –0.2 –0.3 0 200 400 600 800 1000 0 200 400 600 800 0 Time (min) 1.2 1 0.8 0.6 0.4 0.2 –0.2 –0.4 0 1000 1.2 1 0.8 0.6 0.4 0.2 –0.2 0 Velocity (long axis; μm/min) g Φ = 25 μm 50 μm 0 200 400 600 800 1000 Cells advancing direction [+] Mitomycin C 0.3 0.2 0.1 –0.1 –0.2 –0.3 0 g Φ = 250 μm 50 μm 0 200 400 600 800 1000 1.2 1 0.8 0.6 0.4 0.2 –0.2 0 Velocity (long axis; μm/min) g h 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Cells advancing direction 0.3 0.2 0.1 –0.1 –0.2 –0.3 0 0.2 0.1 –0.1 –0.2 0 0.3 0.2 0.1 –0.1 –0.2 –0.3 0 50 μm 50 μm 50 μm [+] Mitomycin C Time (min) Velocity (circumference; μm/min) h NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 8 8 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x (Supplementary Fig. Fig. 6 Cell division perturbs local velocity ﬁeld more in smaller microtubes. a Average velocity of cell front of mitomycin C-treated H1-GFP MDCKs in tubes of different diameters (25, 50, 75, 100, 150, and 250 μm, from left to right), (n = 6 from three independent experiments in each condition) in comparison with that of H1-GFP MDCKs (taken from Fig. 4d). b Tubular conﬁnement does not affect MDCK proliferation. c Representative images of cell nucleus (H1-GFP MDCKs) in a 50 μm diameter tubular tissue moving upward (green arrow). Green circle shows ﬁxed region (radius ~25 μm), where velocity ﬁeld vectors are averaged to determine the perturbation by a cell division event at t = 0 min, deﬁned as the ﬁrst instance where two daughter cells (red arrows) emerged from a dividing cell (blue arrow). Scale bar: 50 μm. d Average velocity in ﬁxed region related to division as a function of time, further averaged over many division events. (25 μm: n = 79, 50 μm: n = 130, 75 μm: n = 166, 100 μm: n = 286, 150 μm: n = 311, 250 μm: n = 348, from two independent experiments per condition). e Schematic showing how the distance perturbation factor is calculated from the average velocity curves in d for a deﬁned radius of ﬁxed region, r. f Average distance perturbation factor as a function of tube diameter, where the factor is calculated for different pairs of parameters (r, δt), with r = 18.6, 21.1, 23.6, 26.0 μm and δt = 10, 20, 30 min. g, h Kymograph of the average longitudinal (g) and circumferential velocity (h) of mitomycin-treated H1-GFP MDCK TCSs migrating in different microtubes (from left to right: 25, 100, and 250 μm). Black dash lines indicate velocity waves that propagate opposite to the collective migration direction. t-test, P < 0.05, P < 0.01, *P < 0.001, NS non-signiﬁcant, for each condition, t-test has been performed between each microtube diameter and 25 μm, unless otherwise indicated by line. All data are presented as mean ± s.e.m. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x c Polar graph plotting the histogram of α-catenin KD MDCK cell orientation distribution under different conﬁned conditions (50 μm microtube, 100 μm microtube, and ﬂat substrate (from left to right)). d 3D reconstructed ﬂuorescent image of α-catenin KD MDCK TCS (phalloidin: red and DAPI: blue) in a microtube of 100 μm in diameter. e Average velocity of cell front of α-catenin KD MDCKs in tubes of different diameters (25, 50, 75, 100, 150, and 250 μm, from left to right), (n = 6 from three independent experiments in each condition) in comparison with that of H1-GFP MDCKs (taken from Fig. 4d). f Longtudinal velocity correlation length for α-catenin knock down and WT tissue in phase contrast movies of microtubes of diameters 50, 100, and 150 μm (n = 6 from three independent experiment per condition). g Bar graphs showing average of instantaneous speeds and h the instantaneous longitudinal velocities along Snail-overexpressed cell tracks, for different microtube diameters, (25 μm: n = 11, 50 μm: n = 12, 75 μm: n = 16, 100 μm: n = 16, 150 μm: n = 16, 250 μm: n = 16 cell tracks of varying duration between 100 and 800 min, from two independent experiments per condition). Data presented as mean ± s.e.m. For each condition, t-test has been performed between each microtube diameter and 25 μm, unless otherwise indicated by lines, P < 0.05, *P < 0.001, NS non-signiﬁcant. Scale bars: 50 μm in a and d, and 25 μm in b. Cyan arrows indicate the direction of collective migration only signiﬁcantly reduced vf in all diameters, but also changed the vf vs. diameter trend as the vf became consistent for all diameters (Fig. 7e). This indicates that such collective behaviors cannot be explained by purely physical contacts between cells but require proper cell–cell contacts, particularly cadherin-based adhesions, to be efﬁcient. Consistent with the low speeds in α-catenin KD tissue, the average velocity spatial correlation length for these tissues were smaller than those for WT tissue in microtubes of different sizes showing that they were also less coordinated in movement (Fig. 7f; Methods), indicating that collective dynamics remained crucial to explain cell expansion into the tubes. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x l i iﬁ l d d i ll di b l h d h M MDCK ll i S il i i f Φ = 50 μm Φ = 50 μm Φ = 100 μm Blue: Nucleus; Red: Actin Φ = 100 μm LCs FCs LCs FCs Cells advancing direction Tube diameter (μm) 0.5 0.4 0.3 0.2 0.1 0.0 25 50 75 100 150 250 Tube diameter (μm) Average front velocity (μm/min) H1-GFP α-catenin KD NS NS 25 50 75 100 150 250 Tube diameter (μm) 1.0 0.8 0.6 0.4 0.2 0.0 Snail NS * **** Average migration speed (μm/min) Snail MDCK 25 50 75 100 150 250 α-catenin KD MDCK WT 120 100 80 60 40 50 100 150 Tube diameter (μm) Longitudinal correlation length (μm) Instantaneous longitudinal velocity (μm/min) 2 0 –2 Φ = 100 μm Φ = 50 μm Flat 12 8 4 0 Number of cells 0 30 60 90 120 150 180 0 30 60 90 120 150 180 0 30 60 90 120 150 180 a c d f b e g h Fig. 7 Cell–cell junction has signiﬁcant inﬂuence on TCS migration. a 3D reconstructed ﬂuorescent images of phalloidin (red) and DAPI (blue)-stained α-catenin KD MDCK TCSs in a microtube of 50 μm. Left, side view, and right, cross-sectional view showing the hollow lumen structure at the ductal tip. b Fluorescent images of basal actin stress ﬁbers in different α-catenin KD MDCK TCSs. c Polar graph plotting the histogram of α-catenin KD MDCK cell orientation distribution under different conﬁned conditions (50 μm microtube, 100 μm microtube, and ﬂat substrate (from left to right)). d 3D reconstructed ﬂuorescent image of α-catenin KD MDCK TCS (phalloidin: red and DAPI: blue) in a microtube of 100 μm in diameter. e Average velocity of cell front of α-catenin KD MDCKs in tubes of different diameters (25, 50, 75, 100, 150, and 250 μm, from left to right), (n = 6 from three independent experiments in each condition) in comparison with that of H1-GFP MDCKs (taken from Fig. 4d). f Longtudinal velocity correlation length for α-catenin knock down and WT tissue in phase contrast movies of microtubes of diameters 50, 100, and 150 μm (n = 6 from three independent experiment per condition). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x g Bar graphs showing average of instantaneous speeds and h the instantaneous longitudinal velocities along Snail-overexpressed cell tracks, for different microtube diameters, (25 μm: n = 11, 50 μm: n = 12, 75 μm: n = 16, 100 μm: n = 16, 150 μm: n = 16, 250 μm: n = 16 cell tracks of varying duration between 100 and 800 min, from two independent experiments per condition). Data presented as mean ± s.e.m. For each condition, t-test has been performed between each microtube diameter and 25 μm, unless otherwise indicated by lines, P < 0.05, P < 0.001, NS non-signiﬁcant. Scale bars: 50 μm in a and d, and 25 μm in b. Cyan arrows indicate the direction of collective migration ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Φ = 50 μm Φ = 50 μm Φ = 100 μm LCs FCs LCs FCs Cells advancing direction a b Φ = 100 μm Φ = 50 μm Flat 12 8 4 0 Number of cells 0 30 60 90 120 150 180 0 30 60 90 120 150 180 0 30 60 90 120 150 180 c a c b FCs Blue: Nucleus; Red: Actin Φ = 100 μm d d d α-catenin KD MDCK WT 120 100 80 60 40 50 100 150 Tube diameter (μm) Longitudinal correlation length (μm) f 0.5 0.4 0.3 0.2 0.1 0.0 25 50 75 100 150 250 Tube diameter (μm) Average front velocity (μm/min) H1-GFP α-catenin KD NS NS e f e Tube diameter (μm) (μ ) 25 50 75 100 150 250 Tube diameter (μm) 1.0 0.8 0.6 0.4 0.2 0.0 Snail NS * ** Average migration speed (μm/min) g Tube diameter (μm) Snail MDCK 25 50 75 100 150 250 Instantaneous longitudinal velocity (μm/min) 2 0 –2 h h g Tube diameter (μm) Tube diameter (μm) 7 Cell–cell junction has signiﬁcant inﬂuence on TCS migration. a 3D reconstructed ﬂuorescent images of phalloidin Fig. 7 Cell–cell junction has signiﬁcant inﬂuence on TCS migration. a 3D reconstructed ﬂuorescent images of phalloidin (red) and DAPI (blue)-stained α-catenin KD MDCK TCSs in a microtube of 50 μm. Left, side view, and right, cross-sectional view showing the hollow lumen structure at the ductal tip. b Fluorescent images of basal actin stress ﬁbers in different α-catenin KD MDCK TCSs. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Since α-catenin is known to be a core mechanosensor of force transmission at cell–cell junctions59–61, together with previous observations of the signiﬁcant inﬂuence of α-catenin knock down (KD) on collective epithelial migration12, we then addressed the role of cell–cell junctions by performing similar assays on α-catenin MDCKs. We observed that the loss of cell–cell junction stability often coincided with the loss of the multilayered epithelial organization at the leading front in the most constricted microenvironments (Fig. 7a), which could be due to the inefﬁ- ciency of tension transmission between cells and thus the inef- fective build-up of inward pointing forces (Supplementary Fig. 6d). Other epithelial architectures and organizations were also disturbed in α-catenin KD, e.g., the lack of longitudinally aligned actin stress ﬁbers and more random cell orientation under the corresponding conditions (Fig. 7b, c). Moreover, due to unstable intercellular contacts, α-catenin KD MDCK cell sheets can sometimes break apart from the TCSs during ductal elon- gation (Fig. 7d; Supplementary Movie 7), reinforcing a crucial role of cadherin-based junctions in epithelial morphogenesis62. In terms of tissue dynamics, the loss of α-catenin in MDCK cells not 9 9 NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Cell front Cell front Φ = 150 μm Φ = 75 μm 0:00 0:20 0:40 1:00 0:00 0:10 0:20 0:30 Cells advancing direction 0.8 0.6 0.4 0.2 50 100 150 200 250 Tube diameter (μm) Parameter vp NS NS a b Fig. 8 Forward polarization is better established in bigger microtubes. a Representative images of PBD-YFP cells in a 75 μm diameter tubular tissue moving upward (cyan arrow). Green (red) arrows show the active PBD zones as marker of lamellipodia protrusion in (opposite to) the direction of tissue front (yellow dashed line) expansion. Time is given in h:min, scale bars: 20 μm. b Calculation of the average lamellipodia persistence parameter, vp ¼ P i vi= P i vi j j, as a function of tube diameter, where vi are all the velocity vectors falling into the zones marked by the active PBD signal (see Methods for how the zones are determined). Each point denotes an independent experiment. t-test: P < 0.01, NS non-signiﬁcant 0.8 0.6 0.4 0.2 50 100 150 200 250 Tube diameter (μm) Parameter vp NS NS b Cell front Cell front Φ = 150 μm Φ = 75 μm 0:00 0:20 0:40 1:00 0:00 0:10 0:20 0:30 Cells advancing direction a b a Cells advancing direction Fig. 8 Forward polarization is better established in bigger microtubes. a Representative images of PBD-YFP cells in a 75 μm diameter tubular tissue moving upward (cyan arrow). Green (red) arrows show the active PBD zones as marker of lamellipodia protrusion in (opposite to) the direction of tissue front (yellow dashed line) expansion. Time is given in h:min, scale bars: 20 μm. b Calculation of the average lamellipodia persistence parameter, vp ¼ P i vi= P i vi j j, as a function of tube diameter, where vi are all the velocity vectors falling into the zones marked by the active PBD signal (see Methods for how the zones are determined). Each point denotes an independent experiment. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x t-test: *P < 0.01, NS non-signiﬁcant F-actin pMLC Merge Φ = 25 μm Φ = 75 μm Φ = 250 μm 25 μm 50 μm 75 μm 100 μm 150 μm 250 μm 0.8 0.6 0.4 0.2 0 20 40 60 80 100 0.8 0.6 0.4 0.2 0 20 40 60 80 100 [Blebbistatin] (μM) 0.8 0.6 0.4 0.2 0 20 40 60 80 100 1.2 1.0 0.8 0.6 0.4 0.2 0.0 25 50 75 100 150 250 Tube diameter (μm) Ir = (It)/(If) Average front velocity (mm/min) a b c e d Fig. 9 Acto myosin contractility is different in various sized TCSs. a MDCK-WT TCSs ﬁxed and stained for actin (green) and pMLC (red). Scale bars, 25 μm for top panel and 40 μm for middle and bottom panels. Cyan arrow indicates the direction of collective migration. b Relative ﬂuorescent intensity, Ir, (pMLC, red ﬂuorescent intensity in TCSs as in a relative to ﬂat condition, see Supplementary Fig. 12) as a function of TCS diameters (n = 6 from three independent experiments in each condition). c–e Tissue expansion speed as a function of blebbistatin concentration can be separated into three groups. c About 25 μm with initial speed increase then decrease at higher blebbistatin concentration (n = 7 from three independent experiments in each condition). d About 50–100 μm with continuously fast increase in speed with increasing blebbistatin concentration (n = 6 from three independent experiments in each condition). e About 150 and 250 μm with slow increase in speed with increasing blebbistatin concentration (n = 6 from three independent experiments in each condition). Data are presented as mean ± s.e.m. F-actin pMLC Merge Φ = 25 μm Φ = 75 μm 1.2 1.0 0.8 0.6 0.4 0.2 0.0 25 50 75 100 150 250 Tube diameter (μm) Ir = (It)/(If) a b 1.2 1.0 0.8 0.6 0.4 0.2 0.0 25 50 75 100 150 250 Tube diameter (μm) Ir = (It)/(If) b F-actin pMLC Merge Φ = 25 μm Φ = 75 μm I = (I )/(I ) a b b a 50 μm 75 μm 100 μm 150 μm 250 μm 0.8 0.6 0.4 0.2 0 20 40 60 80 100 [Blebbistatin] (μM) 0.8 0.6 0.4 0.2 0 20 40 60 80 100 e d c [Blebbistatin] (μM) Fig. 9 Acto myosin contractility is different in various sized TCSs. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x Moreover, MDCK cells overexpressing Snail transcription factor that downregulates E-cadherin perturbed the collective cell behavior even more, and these cells failed to form TCSs even in the most constrictive microtubes as they exhibited single-cell migratory behaviors (Supplementary Movie 8). The instantaneous Snail- overexpressed cell speeds along their single-cell tracks increased with increasing microtube diameters, and reached a saturation point at microtube diameters ~75–100 μm (Fig. 7g), mirroring the trend found for tissue expansion speeds as a function of microtube diameter for collective tissues (Fig. 4d; Supplementary Figs. 9, 10a). However, the instantaneous velocity for Snail-overexpressed cells NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 10 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x a MDCK-WT TCSs ﬁxed and stained for actin (green) and pMLC (red). Scale bars, 25 μm for top panel and 40 μm for middle and bottom panels. Cyan arrow indicates the direction of collective migration. b Relative ﬂuorescent intensity, Ir, (pMLC, red ﬂuorescent intensity in TCSs as in a relative to ﬂat condition, see Supplementary Fig. 12) as a function of TCS diameters (n = 6 from three independent experiments in each condition). c–e Tissue expansion speed as a function of blebbistatin concentration can be separated into three groups. c About 25 μm with initial speed increase then decrease at higher blebbistatin concentration (n = 7 from three independent experiments in each condition). d About 50–100 μm with continuously fast increase in speed with increasing blebbistatin concentration (n = 6 from three independent experiments in each condition). e About 150 and 250 μm with slow increase in speed with increasing blebbistatin concentration (n = 6 from three independent experiments in each condition). Data are presented as mean ± s.e.m. along the microtube distributed mostly symmetrically about the zero value for all microtube sizes (Fig. 7h), which would lead to no average net movement anywhere, unlike other tissues which persistently migrated forward in the microtubes. Taken together, our results indicate the importance of cell–cell adhesion for the regulation of coordinated tissue migration and the maintenance of epithelial integrity and net tissue speed during tubulogenesis. tissue front63 or cryptic lamellipodia extension within the epi- thelium monolayer64 play an important role in collective migra- tion, we ﬁrst checked how well cells polarized within the microtubes of different sizes based on the velocity and direction of lamellipodia-based protrusions. To this end, we monitored the basal sections of cells in tubular tissues (virtually opened as described above) stably expressing YFP-tagged p21-binding domain (PBD-YFP) probe of activated Rac1 and Cdc4265, using the bright, activation zones as markers of lamellipodia structures (Fig. 8a, arrows). In general, the direction of lamellipodia pro- trusion correlated with the forward movement of the TCSs, but Lamellipodia protrusion under various tubular conﬁnement. As forward polarization induced by lamellipodia protrusion at the In contrast, even though the TCSs inside the microtubes may be at best considered as pseudo-3D due to the constrained movement on a curved surface, our assay mimics aspects of epithelial architecture and collective migratory dynamics leading to tubule formation that are highly pertinent to in vivo situations. g y p We have demonstrated that there is a transition in epithelial organization and migratory pattern due to the increasing tubular spatial constraint and/or curvature. In smaller microtubes with high surface curvature, cells are taller and thus TCSs can exhibit multilayered, fused structures that plug the smallest microtubes. This is in line with the observation that cells spread less on negative curvature surfaces, which can be due to a plausible interfacial stress that increases with the curvature. Due to this unique structure, cells could be more jammed in the smaller tubular tissues, which also consistently portrayed poor forward polarization, marked by haphazard lamellipodia protrusion directions. On ﬂat surfaces, MDCK cells within conﬂuent tissues started to transition from a more directed migration to a more jammed, diffusive motion with lower speeds around the cell density threshold of 3–4 cells/(1000 μm2)49–51. In contrast, the density threshold that separated smaller microtubes (≤100 μm diameter, with lower tissue speeds) from bigger microtubes was measured to be ~2 cells/1000 μm2 (Fig. 2b; Supplementary Fig. 6b), which suggested that surface curvature can shift the jamming transition to lower cell densities. The possibility of jamming in smaller tubes also coincided with the observation of backward–forward motion and swirling67,68 that predominantly happened in microtube diameters ≤100 μm (Fig. 5a, d, e). Contractility difference leads to migration speed variations. Then we investigated the role of acto myosin contractility, by studying phosphorylated myosin light chain (pMLC) distribution in the tubular tissues. pMLC signal was found to be diffuse, non- colocalizing with actin stress ﬁbers and weak in intensity (It) in the smallest tubes (diameter <50 μm, Fig. 9a, upper panel) compared with that on ﬂat substrate (If, and Supplementary Fig. 12). In contrast, we observed stronger signals of pMLC intensity that colocalized with actin stress ﬁbers in larger tubes (Fig. 9a, middle and lower panels), similar to those on ﬂat sur- faces. The changes in pMLC ﬂuorescent signal as a function of TCS diameter was quantiﬁed by the relative ﬂuorescent intensity, Ir = It/If (Fig. Lamellipodia protrusion under various tubular conﬁnement. As forward polarization induced by lamellipodia protrusion at the 9b) and the plot demonstrates an increasing trend from low Ir in small tubes to high values in large ones, where the Ir plateaus. This indicated that contractility level was lower in smaller tubes with higher conﬁnement/curvatures. g In order to gain a further understanding about contractility in TCS collective migration, we studied tissue front speed (vf ) as a function of varying blebbistatin concentrations (from 6 to 100 μM), which inhibited acto-myosin contractility to different degrees. Results showed that vf varied non-monotonically with blebbistatin concentration12,55. The contractility behavior can be separated into three distinct groups based on microtube size, for Φ = 25 μm, diameter ≤50–100 μm, and diameter > 100 μm (Fig. 9c–e, respectively), conﬁrming the differences in contrac- tility between smaller and bigger microtubes. Epithelial cell velocities in all microtubes increased in general with increasing blebbistatin concentration up to 50 μM (Fig. 9), possibly due to a decrease in tissue elasticity that allows the tissue to extend more easily55,66. Importantly, only tissue speed in the smallest tube (25 μm) presented a drastic drop when blebbistatin concentrations increased more than 50 μM, indicating the myosin-II contractile activity levels for such tubes were lowest and, such that they are the ﬁrst to be depleted of activity. This is consistent with the pMLC results showing that acto myosin activity in smallest TCSs was initially low. Overall, the analysis of pMLC staining and blebbistatin drug treatment results suggested that acto myosin contraction decreased with higher tube curvature. g Further, based on contractility results from pMLC staining and blebbistatin treatment, tubular tissues in the smallest microtubes can be deduced to have smaller active tissue propulsive stress consistent with less pronounced forward polarity, and could also have smaller traction forces. Tubular tissues in smaller micro- tubes were also found to experience more cell division-induced perturbations to their forward movement probably due to their smaller size. All these contributed to a mechanistic understanding of the decrease in tissue expansion speed in smaller microtubes. Importantly, the unique TCS migration strongly depended on the collectivity of the cells provided by a strong cell–cell junction, which could increase coordination between cells to induce better forward polarization. Consistent with this view, α-catenin KD in the MDCK cells portrayed lower tissue expansion speed in all microtube sizes and frequently disintegrated, indicating that cell–cell adhesion is an important driving force for tubule migration in highly constricted environments. Lamellipodia protrusion under various tubular conﬁnement. As forward polarization induced by lamellipodia protrusion at the Lamellipodia protrusion under various tubular conﬁnement. As forward polarization induced by lamellipodia protrusion at the NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x importantly, cells tended to portray numerous lamellipodia pro- trusion opposite to the movement of the tissue front (Fig. 8a, red arrows), which occurred more frequently in the smaller micro- tubes. To quantify this observation, we plotted a lamellipodia persistence parameter, vp deﬁned as P i vi= P i vi j j, where vi is the velocity associated with the direction of lamellipodia protrusion and ¼ j j denotes the absolute value (Method). This parameter would be equal to 1 (−1) if all the lamellipodia protruded in the same (opposite) direction as the tissue front movement, while its absolute value would decrease if lamellipodia protrusion in both directions occurred. Consistent with our observations (Fig. 8a), the persistence parameter was smaller for microtubes with dia- meter ≤100 μm (Fig. 8b), suggesting that tissue forward polar- ization was poorer in such tubes, in line with the measurement of taller cell height (Fig. 2c), lower tissue speeds (Fig. 4d; Supple- mentary Fig. 9), and contraction–elongation modes of migration (Fig. 5a, d). The fused multilayered structure observed at the migration front in smaller microtubes (Fig. 2a; Supplementary Fig. 4) could also explain the less pronounced forward polariza- tion there. signals, in-depth understanding of how constraint and out-of- plane curvature regulate tissue migration, including cell rear- rangement and organization, tissue velocity ﬁeld, and its order, is mostly lacking to date. 2D ﬂat assays have been previously evi- denced for the guidance of in-plane curvature and conﬁnement in wound healing24 and epithelial dynamics12. In addition, when plated on a micro-cylinder of positive curvature, epithelia orga- nize into tubular architecture of an inverted polarity and show a curvature-induced EMT25. However, both systems failed to recapitulate the processes of the formation of a hollow epithelial lumen. Though conventional soft lithography method has enabled the production of lumen networks, it is commonly lim- ited by a rectangular cross-section32. While most of our current understanding about epithelial tubulogenesis comes from 3D gel approaches8,10, very little information about the mechanisms of migration and tissue–environment interaction is obtained by such systems. Lamellipodia protrusion under various tubular conﬁnement. As forward polarization induced by lamellipodia protrusion at the On the other hand, the more coordinated velocity patterns in the larger microtubes suggested that once the conﬁnement is reduced, cells at the leading edge of the tube can transmit tissue stress much deeper into the tissue bulk. All these results showed that the emergence of different modes of collective cell migration in tubular NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications ARTICLE From the open images of PBD signals, the bright zones were speciﬁed automatically using the Auto Local Threshold plugin in ImageJ, with the midgrey and radius = 30 pixels options (resolution of images were 0.62 μm/pixel). These selected zones were visually compared with the original open images to verify that they mirrored reasonably the bright PBD zones, and only the velocity vectors falling within these regions were selected for further analysis. From these velocities, a lamellipodia persistence parameter, vp ¼ P i vi= P i vi j j was calculated, where vi is the velocity associated with the direction of lamellipodia protrusion and j ¼ j denotes the absolute value. This parameter would be equal to 1 (−1) if all the lamellipodia protruded in the same (opposite) direction as the tissue front movement, while its absolute value would decrease if lamellipodia protrusion in both directions occurred. A parameter closer to 1 would indicate that the cells within the tubular tissue are better polarized in the front–back direction. The easy combination of our microtube platform with high- resolution microscopy and simple construction have helped us to interrogate the pseudo-3D cellular behaviors and data with 2D technique and analysis. Such approach could be further gen- eralized and extended to more complicated 3D systems. With all these ﬁndings, we demonstrate the profound inﬂuence of spatial conﬁnement on epithelia and provide an in-depth characteriza- tion of collective cell migration on tubular negative curvature reminiscent of in vivo tubulogenesis. To quantify the disturbance that cell division events cause to the tissue expansion dynamics, the average longitudinal velocity vectors within a ﬁxed circular region (of radius, r) centered on each division event (between two daughter cells that ﬁrst emerged from the mother cell) were determined before (starting from –80 min), during (t = 0 min), and after (up to +80 min) the division (Fig. 6c, d). Such average velocity curves were further averaged for all division events, centered on their respective t = 0 min. A sharp dip in this average velocity curve around t = 0 min, indicative of the division-generated disturbance to local forward velocity ﬁelds, is seen superposed on a slowly decreasing velocity trend with increasing time at long-time scales (Fig. 6d). To quantify the dip as a way of numerating the disturbance, we ﬁrst leveled the slow tilt in the average velocity curve and approximated it as a horizontal, straight line. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x conﬁnement is in sharp contrast with the effect of 2D geometric constraints seen earlier12 and the positively curved cylindrical surface25. imaging. For myosin-II inhibition, cells were treated with 6, 12.5, 25, 50, and 100 μM blebbistatin (Cayman Chemical, 13013) 30 min before imaging. imaging. For myosin-II inhibition, cells were treated with 6, 12.5, 25, 50, and 100 μM blebbistatin (Cayman Chemical, 13013) 30 min before imaging. The effects of tubular constraints that we have observed on key migration features such as migration velocity, order parameters, and velocity ﬁeld distribution reﬂect the impact of spatial con- ﬁnement and curvature on epithelial lumen development. Along this line, the evidence of frequent detachment of front cells from the groups without proliferation in very narrow microtubes may be reminiscent of EMT19,56. Cell detachment events may result from a tug-of-war between cell–substrate and cell–cell interac- tions and as such, due to an increased tensile stress within the tissue under mitomycin treatment since cell proliferation can be seen as a source of stress dissipation56. It will be appealing to further investigate whether extreme constrictions, occurring during certain pathological conditions, could lead to aberrant morphogenesis. Image analysis. Virtual opening of the TCSs was performed after background subtractions on ImageJ. Time-lapse z-stacks of the tubes were ﬁrst resliced to obtain the xz plane. A circle was ﬁt to the circumference of the TCS, converted to line, straightened and resliced again to obtain a time-lapse image of virtually opened tube with the tube circumference (x-axis) and length of the tube (y-axis) as the new axes. This process was coded in ImageJ to help in converting a 3D time- lapse image into 2D and eased the process of analysis while conserving the relevant x, y, and z information. PIV was performed on the virtually opened tube images using PIVLab code48 on MATLAB. Angle between the velocity vectors obtained from PIVLab and the correlation length of the migrating TCS were calculated using a MATLAB code. Average velocity of cell migration was calculated on ImageJ. Orientation index of the cells was also calculated on ImageJ by considering indi- vidual cells as ellipses (n > 100). To analyze the velocity ﬁeld vectors related to activated (bright) PBD zones as a marker of lamellipodia protrusion, PIV was ﬁrst performed on the virtually opened, basal layer of the original confocal images of the PBD tissue. Methods The correction to the circumferential velocity was calculated by differentiating this formula, obtaining vcircumference ¼ v=p 1 x2 R2 , with v being velocity in the circumferential direction measured by PIV. The longitudinal parameters are correct as measured. The fabrication of bowl-shaped structures with negatively curved surfaces was done with SU8 coating on silicon wafer and common lithography methods, which eventually formed dome structures. PDMS was cast over the domes to create the bowl structures. Cell culture and imaging. MDCK-WT (Madin-Darby canine kidney wild type) cells, MDCK-H1-GFP (stable cell line transfected with H1-GFP), MDCK-PBD- YFP (stable cell line expressing YFP-tagged p21-binding domain probe of activated Rac1 and Cdc42, kindly provided by Fernando Martin-Belmonte, Universidad Autónoma de Madrid), MDCK-E-cadherin-GFP (stable cell line expressing E- cadherin-GFP), MDCK-Snail (cells overexpressing Snail transcription factor), and MDCK α-cat KD (stable cell line with α-catenin KD) were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin. MCF-10A cells (American Type Culture Collection, ATCC) were maintained in MEGM (Lonza) medium supplemented with Cholera toxin (100 ng/ ml). Microtubes were coated with 50 μg/ml Fibronectin (Sigma-Aldrich) for 1 h, washed with 1× PBS and then cells were seeded close to the edge of the microtubes. Once the migrating cell sheets entered the microtubes, the sample was placed on the confocal microscope (Nikon A1R or Zeiss LSM 710) and z-stacks (1–2 μm per step) covering the entire volume of the tubes were imaged every 10 min. Time-lapse images were performed over a period ranging from 12 to 24 h. For inhibiting cell division, mitomycin C (Sigma-Aldrich, M4287) was added at a concentration of 10 μM, incubated for 1 h, washed away and replaced with fresh medium before To quantify the relative pMLC ﬂuorescent intensity, we ﬁrst obtained a maximal intensity z-stack projection of the basal layers of original confocal images to avoid the curvature effect. The ﬂuorescent intensity in TCSs (It) was then measured over a rectangular shape of 5 × 50 μm2 along the tubular longitudinal direction and the intensity on ﬂat surfaces (If) was measured within the same ROI. The relative pMLC ﬂuorescent intensity (Ir) was deﬁned by the following formula: Ir = It/If. Sample ﬁxation and staining and confocal microscopy. Cells were grown and allowed to form tubular cell sheets inside microtubes. Methods Fabrication of circular microchannels. Smooth metal wires (typically made from copper or platinum) were aligned in parallel just one to two microns above a silicon wafer with a precise stage, which can be used to control the distance between each wire and the silicon wafer surface. A freshly mixed PDMS (a mixture of Sylgard 184 silicone elastomer base and Sylgard 184 silicone elastomer curing agent, 10:1 by weight) was poured on top of the silicon wafer covering the metal wires. The whole setup was then transferred into an 80 °C oven for 2 h or left at room temperature for 24 h to cure the PDMS. After the polymerization of the PDMS, the metal wire was pulled out from the PDMS microtubes during a sonication process in an acetone solution, which would extract unreacted elastomer curing agent and cause slight swelling in the polymer—loosening the PDMS–metal contact. The detached PDMS microtubes were then baked in an 80 °C oven for 30 min to remove any acetone remnant. The PDMS blocks containing straight microtubes were cut into small pieces with tubular microchannels of ~1 mm in length. The microchannels then had two openings, and the short length allowed the efﬁcient diffusion of nutrients into the microtubes. To allow collective epithelial cell migration into the microtubes, one piece of the PDMS block containing microtubes coated with ﬁbronectin was placed in front of a migrating MDCK cohort with one opening facing the moving cell sheet. Due to the very narrow step (~1–2 μm between the microtubes and the substrate, see above) MDCK cells were able to crawl into the microtubes with intercellular cohesion. The whole setup was immersed into complete medium and mounted on microscope for imaging. Phase contrast movies were used with α-catenin KD and Snail-overexpressing cells in microtubes as these cells have no ﬂuorescent markers. To better approximate the cell position and velocity components in the circumferential direction of tubes in phase contrast movies that only show a 2D projection of these values, the formula xcircumference ¼ R ´ sin1ðx=RÞ was used to make the positional conversion, where x is the position in the circumferential direction measured in phase contrast images, R is the microtube radius, with the coordinates taken to center around the midpoint of the tube axis taken as x=0. ARTICLE The area bounded by this straight line with the leveled average data, over a certain duration (−δt to δt around t = 0 min, Fig. 6e) quantiﬁes the perturbation to the distance cells would travel if there were no perturbation (positive if velocity drops, negative if velocity increases). A distance perturbation factor was quantiﬁed by normalizing this area with the area under the straight line within the same duration, such that data from different microtube sizes can be compared. To reduce bias, this factor was calculated for different pairs of parameters (r, δt) with r = 18.6, 21.1, 23.6, 26.0 μm and δt = 10, 20, 30 min, and all taken into account for statistics. Discussion The formation of epithelial tubules and cysts require a population of cells to coordinate their behaviors across space and time to interact with the microenvironment. While epithelia demonstrate extensive, collective migratory responses to external geometric NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications 12 NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications Sv ¼ < cosðϕÞ> The average correlation magnitude between two vectors that are separated by a distance j r j is given by the correlation function deﬁned as12: 18. Marel, A.-K. et al. Flow and diffusion in channel-guided cell migration. Biophys. J. 107, 1054–1064 (2014). 19. Wong, I. Y. et al. Collective and individual migration following the epithelial- mesenchymal transition. Nat. Mater. 13, 1063–1071 (2014). 20. Doxzen, K. et al. Guidance of collective cell migration by substrate geometry. Integr. Biol. 5, 1026–1035 (2013). Cu r * ¼ u r ′ þ r ; t ´ u r ′; t D E r ′ u r ′ þ r ; t 2 u r ′; t 2 1=2 + t Cv r * ¼ v r ′ þ r ; t ´ v r ′; t D E r ′ v r ′ þ r ; t 2 vð r ′; tÞ2 D E 1=2 + t Cu r * ¼ u r ′ þ r ; t ´ u r ′; t D E r ′ u r ′ þ r ; t 2 u r ′; t 2 1=2 + t Cv r * ¼ v r ′ þ r ; t ´ v r ′; t D E r ′ v r ′ þ r ; t 2 vð r ′; tÞ2 D E 1=2 + t 21. Segerer, F. J., Thüroff, F., Piera Alberola, A., Frey, E. & Rädler, J. O. Emergence and persistence of collective cell migration on small circular micropatterns. Phys. Rev. Lett. 114, 228102 (2015). y 22. Rausch, S. et al. Polarizing cytoskeletal tension to induce leader cell formation during collective cell migration. Biointerphases 8, 32 (2013). 23. Ng, C. K. M. & Yu, K. N. Proliferation of epithelial cells on PDMS substrates with micropillars fabricated with different curvature characteristics. Biointerphases 7, 21 (2012). 24. Ravasio, A. et al. Gap geometry dictates epithelial closure efﬁciency. Nat. Commun. 6, 7683 (2015). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x 7. Andrew, D. J. & Ewald, A. J. Morphogenesis of epithelial tubes: insights into tube formation, elongation, and elaboration. Dev. Biol. 341, 34–55 (2010). To stain for pMLC, samples were incubated overnight at 4 °C with the anti- pMLCT18+T19 antibody (Cell Signaling, #3674s, 1:50–100) and then stained with Alexa Fluor 568 secondary antibody (Invitrogen, A11011, 1:250) at room tem- perature for 1 h. Cells were then mounted with anti-bleaching medium (Vector Laboratories, H-1000) and z-stacks of the cell tubes were acquired on a confocal microscope (Nikon A1R or Zeiss LSM710). To stain for pMLC, samples were incubated overnight at 4 °C with the anti- pMLCT18+T19 antibody (Cell Signaling, #3674s, 1:50–100) and then stained with pMLC antibody (Cell Signaling, #3674s, 1:50 100) and then stained with Alexa Fluor 568 secondary antibody (Invitrogen, A11011, 1:250) at room tem- perature for 1 h. Cells were then mounted with anti-bleaching medium (Vector Laboratories, H-1000) and z-stacks of the cell tubes were acquired on a confocal microscope (Nikon A1R or Zeiss LSM710). 8. Lubarsky, B. & Krasnow, M. A. Tube morphogenesis: making and shaping biological tubes. Cell 112, 19–28 (2003). p g ( Laboratories, H-1000) and z-stacks of the cell tubes were acquired on a confocal microscope (Nikon A1R or Zeiss LSM710). 9. Wang, C.-C., Jamal, L. & Janes, K. A. Normal morphogenesis of epithelial tissues and progression of epithelial tumors. Wiley Interdiscip. Rev. Syst. Biol. Med. 4, 51–78 (2012). 10. O’Brien, L. E., Zegers, M. M. P. & Mostov, K. E. Building epithelial architecture: insights from three-dimensional culture models. Nat. Rev. Mol. Cell Biol. 3, 531–537 (2002). Calculation of the TCS parameters. Cells in TCSs were considered as ellipses, and the orientation of the cell was calculated as the angle between the major axis of the cell and the long axis of the microtubes. The order parameter for actin ﬁlament was deﬁned as cosine of twice the angle, θ of the ﬁlament with the tubular longitudinal axis. The overall actin order parameter for a certain TCS was calculated using the formula: Sa = <cos(2θ)>. The average order parameter for velocity vectors in PIV mapping was deﬁned by the following formula: 11. Angelini, T. E., Hannezo, E., Trepat, X., Fredberg, J. J. & Weitz, D. A. Cell migration driven by cooperative substrate deformation patterns. Phys. Rev. Lett. 104, 168104 (2010). 12. Vedula, S. R. K. et al. Sv ¼ < cosðϕÞ> 13. Kaliman, S., Jayachandran, C., Rehfeldt, F. & Smith, A.-S. Novel growth regime f C d l f b h ( ) 13. Kaliman, S., Jayachandran, C., Rehfeldt, F. & Smith, A.-S. Novel growth regime of MDCK II model tissues on soft substrates. Biophys. J. 106, L25–L28 (2014). of MDCK II model tissues on soft substrates. Biophys. J. 106, L25–L28 (2014). 14. Nelson, C. M., VanDuijn, M. M., Inman, J. L., Fletcher, D. A. & Bissell, M. J. where ϕ refers to the angle which the vector makes with the y-axis in the virtually opened TCS 2D image. Therefore, Sa = 0 refers to an isotropic alignment, Sa = 1 refers to a perfect orientation along tubular longitudinal axis, and Sa = −1 refers to a perfect alignment vertical to the tubular long axis. On the other hand, Sv = 0 corresponds to a random distribution of velocity vectors in the TCS, Sv = 1 corresponds to an overall forward movement (vectors parallel to the microtube length and directed along the direction of the migrating cell sheet) and Sv = −1 corresponds to an overall backward movement (vectors parallel to the microtube length and directed opposite to the direction of the migrating cell sheet). p y 14. Nelson, C. M., VanDuijn, M. M., Inman, J. L., Fletcher, D. A. & Bissell, M. J. Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures. Science 314, 298–300 (2006). 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A circular cross-section PDMS microﬂuidics system for replication of cardiovascular ﬂow conditions. Biomaterials 31, 3459–3464 (2010). 33. Wong, A. D. & Searson, P. C. Live-cell imaging of invasion and intravasation in an artiﬁcial microvessel platform. Cancer Res. 74, 4937–4945 (2014). 34. Zhang, W. et al. Elastomeric free-form blood vessels for interconnecting organs on chip systems. Lab Chip 16, 1579–1586 (2016). ARTICLE Emerging modes of collective cell migration induced by geometrical constraints. Proc. Natl Acad. Sci. USA 109, 12974–12979 (2012). Sv ¼ < cosðϕÞ> Sv ¼ < cosðϕÞ> Methods After the TCS progressed into the microtube for 24–48 h, the samples were ﬁxed with 4% paraformaldehyde for 15 min or −20 °C methanol for 5 min and then permeabilized with 0.2% Triton X-100 or 0.025% saponin for 15 min. Actin cytoskeleton was visualized after staining with Alexa-488 (Invitrogen, A12379, 1:250) or Alexa-568 (Invitrogen, A12380, 1:250) labeled Phalloidin for 1 h. To assess cell polarization, samples were incubated overnight in 2 μg/ml anti-Gp135 (DSHB, Q52S86) and then stained with 1:250–500 dilution of Alexa Flour 488 secondary antibody (Invitrogen, A11001). 13 NATURE COMMUNICATIONS\| 8: 1517 \| DOI: 10.1038/s41467-017-01390-x\| www.nature.com/naturecommunications Sv ¼ < cosðϕÞ> where Cu and Cv correspond to the correlation function in the directions that are vertical and parallel to the microtube longitudinal axis, respectively, u* and v* are the deviation of the velocity from the mean velocity in the directions vertical and parallel to the microtubular long axis, r * is the vector of the coordinates, and t is time. The spatial velocity correlation functions were computed by calculating the mean of the correlation coefﬁcient over all the directions such that Cu and Cv are now functions of k r k (the norm of r ). 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USA 109, 9342–9347 (201 Received: 23 December 2016 Accepted: 14 September 2017 Acknowledgements We thank Dr. Fang Kong from SMART, Singapore for helping to design and manu- facture the precise stage for the microtube fabrication as well as to make Supplementary Fig. 1, Wai Han Lau and Jun Liu from the microscopy core of Mechanobiology Institute Singapore (MBI) for imaging support, Hui Ting Ong and Dr. Hayri Emrah Balcioglu from MBI for help in programming and Dr. René-Marc Mège and Dr. Delphine Dela- cour from IJM, Paris, and Dr. Sham Tlili from MBI for helpful discussion. We thank Song Hui Tan and Bee Leng Tan from MBI laboratory facility for supporting in the lab work and Chun Xi Wong from MBI Science Communications Unit for help in making illustrations. We also thank MBI students—Yejun Wang, Zhe Wen, Yue Zhang, Anh Phuong Le, and Shreyansh Jain for support in the experiment. The MDCK cell lines were kindly provided by Sham Tlili, Shigenobu Yonemura, and W. James Nelson. 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The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 61. Benham-Pyle, B. W., Pruitt, B. L. & Nelson, W. J. Mechanical strain induces E- cadherin-dependent Yap1 and β-catenin activation to drive cell cycle entry. Science 348, 1024 (2015). 62. Maiden, S. L. & Hardin, J. The secret life of α-catenin: moonlighting in morphogenesis. J. Cell Biol. 195, 543–552 (2011). 63. Poujade, M. et al. Author contributions W.X., B.L. and C.T.L. conceived the project, W.X. designed the experiments. B.L. and C.T.L. supervised the study. W.X., S.S. and T.B.S. performed and analyzed all experi- ments. T.B.S. programmed codes for analysis. W.X. made the ﬁgures. W.X., S.S., T.B.S., B.L. and C.T.L. wrote the manuscript. All authors commented on and/or edited the manuscript and ﬁgures. 52. Petitjean, L. et al. Velocity ﬁelds in a collectively migrating epithelium. Biophys. J. 98, 1790–1800 (2010). 53. Doostmohammadi, A. et al. Cell division: a source of active stress in cellular monolayers. Soft Matter 11, 7328–7336 (2015). 54. Chaudhuri, P. K., Pan, C. Q., Low, B. C. & Lim, C. T. Topography induces differential sensitivity on cancer cell proliferation via Rho-ROCK-Myosin contractility. Sci. Rep. 6, 19672 (2016). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01390-x 40. Park, J. Y., Lee, D. H., Lee, E. J. & Lee, S.-H. Study of cellular behaviors on concave and convex microstructures fabricated from elastic PDMS membranes. Lab Chip 9, 2043–2049 (2009). 66. 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https://openalex.org/W2914028337	https://www.e3s-conferences.org/articles/e3sconf/pdf/2019/03/e3sconf_repar18_01002.pdf	English	null	US hegemony and Sino-Russia energy security cooperation	E3S web of conferences	2,019	cc-by	5,680	* Corresponding author: pangcw@126.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). US hegemony and Sino-Russia energy security cooperation Pang Changwei1,, Zhou Xiaojia2, and Sun Lu1 Pang Changwei1,, Zhou Xiaojia2, and Sun Lu1 1China University of Petroleum, Beijing, China 2Fudan University, Beijing, China 1China University of Petroleum, Beijing, China 2Fudan University, Beijing, China Abstract: Why China and Russia give up previous suspicious attitudes toward each other, and going toward close cooperation on Energy security? We argue the threat of American hegemony push China and Russia together. Facing US sanction and strategic pressure, Russia changed its perception about China, and regarded China rise as opportunity. As a consequence, Russia strengthened its ties with China, and completed the oil pipe line which favored China. In addition to this, Russia permitted China Energy company access to its upstream and downstream Energy industry. With the rise of China, the structural conflict between China and US intensified. Trump administration explicitly described China as adversary. The fact that China’s energy transportation line is under the control of US Navy is a source of vulnerability. Out of consideration of reducing risk of cutting off energy supply, China developed a close cooperation with Russia on Energy safety. , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 1 Introduction As neighboring countries, China and Russia had deeper grievances in the history of bilateral relations and had strong doubts about bilateral energy cooperation [1]. As a consequence, they achieved little in the progress of energy cooperation before 2008. Russia initially did not attach importance to the energy exports to the China in the east. After 2008, Sino-Russia energy security cooperation achieved a lot in the terms of the amount of contract and depth. In 2017, Russia became the No 1 oil exporting country for China, and Chinese capital also successively entered the upstream and downstream of Russian energy industry. The natural gas pipeline to China also completed more than half. Putin clearly pointed out that Russia should regard China’s rise as opportunity, not threat. Some scholars argue Sino-Russia energy cooperation has entered a stage of strategic partnership. For China, importing oil and gas from Russia is not the best choice, because China buys energy from Russia at the price above the international market level. At current stage, China could obtain energy from Middle East and Central Asia at reasonable price. Why Russian and China give up their realistic consideration, and choose to cooperate on energy security? g p p gy y Based on theory of energy security, we argue the dynamics of Sino-Russia cooperation is based on the common threat of US Hegemony. Although, Previous American , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 administration shows their willingness to cooperate with China, they never forget containing China’s influence. After Trump took office, he regarded China as explicit threat and adopted zero sum approach toward Sino-US relations. As the majority of China’s energy importation is through sea lane which is under the control of US and its allies, China spare no effort to diverse its importation of oil, in the hope of avoiding cut off energy supply in case of conflict with US. To defend its geopolitical influence sphere, Russia launched military intervention into Ukraine. As a result, Russia suffered the sanctions by US and its western allies. Diversification its energy exportation becomes the high priority of Russia Energy strategy agenda. China and Russia develop a comprehensive and deep cooperation relationship on Energy safety. This article can be divided into four sections. 2 Define Energy safety Thomas argues Energy safety involves economic, political, military, strategic security. The so called economic security means the state has sufficient resource, which also means state can access to goods and service at affordable price. Political security means maintaining domestic stability; strategic and military security refers to the military capability to detect threat and confront threat [2]. He argues that the oil crisis of the 1970s hit these three kinds of security. Oil shortages and soaring oil prices caused widespread inflation and caused Western countries to fall into economic crisis and political turmoil. Arms suppliers in industrialized countries have sold weapons to Middle Eastern countries in exchange for more Middle Eastern oil, threatening the military balance between the Arab countries and Israel, which led to a new round of arms race. At the same time, industrialized countries have chosen to strengthen their naval forces in order to protect the oil sources of international trade and transportation corridors. Daniel Yekin pointed out that “the goal of energy security is to ensure an adequate and reliable energy supply at reasonable price without compromising the country's main values and goals” [3]. Energy security includes not only the normal operation of the national economy, but also ensuring that the military force can obtain a reliable fuel supply to safeguard the country’s vital interests. Paul B. Stares summarized the above traditional energy security theory. He argues that traditional energy security concerns focus on the sudden suspension of existing energy supply agreements, Security threats caused by price by manipulation. Correspondingly, potential security threats such as political turmoil, economic threats, military conflicts and terrorist attacks are the most important traditional concerns. These safety concerns include not only the source of energy supply, but also the routes and means of energy transportation. Therefore, evaluating the level of energy security is, to a large extent, assessing whether a country is dependent on a certain type of energy, whether a country acquired energy in its territory, or whether a country’s energy need must be meet by importation. If its energy consumption has to rely on importation, then energy security involves the dependence on foreign energy sources, the diversification of energy sources, and the political turmoil or enemy control in the source. 1 Introduction In the first section, we define energy security through discussion. In the second section, we describe and analyze the motivation of cooperation of Russia and China. In the Third section, we introduce the progress of Sino- Russia Energy cooperation. The fourth section summarizes the full article. 3 Indo-Pacific strategy and China Energy security Indo-Pacific strategy is ideology oriented with its emphasis on confrontation, zero-sum game approach [4]. It originated from Japanese Prime minister Abe’s “Value diplomacy”, 2 2 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 which argue Asia Pacific countries with similar value and political regime should form coalition to strengthen security and economic cooperation, maintain international rule, and counter the rise of China. State secretary Tillerson argues, Indo-Pacific strategy geography scope should include Indian Ocean and Pacific Ocean, while Japan and Indian held the key strategic position in this strategy. He claim China’s Economic diplomacy undermine the freedom and sovereignty of developing countries. In response, US should provide developing counties with alternative finance mechanism. In addition, the 2017 US National Defense Strategy Report regards China as revisionist country that challenges US hegemony and undermines US security and prosperity. The 2018 US national defense strategy report continue the tone of 2017 National Defense strategy Report, describing China as an adversary, emphasizing Confrontation and Zero-sum game perspective. Both reports argue US Indo-pacific strategy should strengthen alliances, establish a security framework to deter China’s invasion, ensure stability of Asia-Pacific area, Freedom navigation on the high seas. Trump’s Indo-Pacific strategy has deviated from the basic positions of previous US government’s China policy. Both Bush administration and Obama administration China policy emphasize containment and cooperation. In the multilateral mechanism, the US tries to include China as much as possible. But Trump’s Indo-Pacific strategy emphasizes zero- sum game approach, regards China as adversary, ignore China’s contribution to the regional and world economy, and ignores the contribution of China’s AIIB and the “Belt and Road” construction to infrastructure construction in developing countries. Because the strategy emphasizes confrontation with China, it is difficult for China to cooperate with United States on the DPRK nuclear issue, the Iranian nuclear issue and climate warming issue. 4 Country’s reaction to the indo-pacific strategy The attitudes of the core countries of India-Pacific strategy are inconsistent with the United States. Although India supports US in its posture, it retains its view on the strategy. First of all, Indian stick to its non-alliance tradition and consistently opposed the adoption of alliances against third countries. Second, India thinks Indo-pacific strategy reflects US strategic value, not Indian strategic value. India thinks the geographic scope of Indo-pacific strategy is not large enough. India’s strategic influence is not limited to the Indian Ocean, but originates in Malacca and goes directly to the Middle East. India believes that the strategy emphasizes the containment of China, which is not in the interest of India. India believes that India lags behind China in both economic and military strength and is unwilling to be the main force against China. Cooperation with China is conducive to India's development. Finally, on the issue of exclusive economic zone, India and China hold a similar position. India also believes that foreign warships entering the exclusive economic zone of a country should obtain the consent of the country in advance. Japan basically supports the anti-China stance of the Indo-Pacific strategy, but it is limited in its support. First of all, Japan has a heavy dependence on the Chinese economy. To some extent, Japan has some kind of vulnerability to China. Second, the Japanese people opposed the revision of the peace constitution, which prohibits Japan act as a normal country and jointly carry out military operations with the United States in the Asia-Pacific region. The above two points have determined that Japan is unlikely to take a proactive and open military confrontation against China. Australia, the Philippines, South Korea, Thailand have close economic ties with China. The economic growth of these countries is partly driven by the Chinese economy. The Philippines has territorial disputes with China, but it seeks Chinese capital. South Korea has sought China’s help in the DPRK nuclear issue, and Thailand has been deeply integrated 3 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 into the Chinese economic circle. All Southeast Asian countries are reluctant to choose sides, because it is not in the interest of these countries. 4 Country’s reaction to the indo-pacific strategy A safe and stable external environment is a necessary condition for the economic development of all countries in the region. Finally, Cambodia and Laos, which have close ties with China, explicitly refused to join any alliance against China. into the Chinese economic circle. All Southeast Asian countries are reluctant to choose sides, because it is not in the interest of these countries. A safe and stable external environment is a necessary condition for the economic development of all countries in the region. Finally, Cambodia and Laos, which have close ties with China, explicitly refused to join any alliance against China. Trump's Indo-Pacific strategy has a strong ideological color, emphasizes confrontation, and views Sino-US relations in a zero-sum approach. This is not in line with the desire of most developing countries in the Indo-Pacific region which cherish development and stability. Constrained by their own national interest, the core countries of the strategy, like India, Australia, and Japan, can only provide limited support for Indo-Pacific strategy. No country is willing to play active role in confronting China. The key to the successful implementation of the India-Pacific strategy is whether the United States can persuade its allies to fight against China. From the current point of view, it is difficult for US to implement Indo-Pacific strategy, because neither its allies nor the small countries in the region are willing to fully confront China. Therefore, the safety of China's energy transportation sea lane depends mainly on Sino-US relations. As long as there is no direct military conflict between China and the United States, the United States will not attack Chinese oil carriers or block the Strait of Malacca against China. The Sino-US trade war and the Indo-pacific strategy will have great impact on the Sino-Russia relationship, Sino-US relationship, and Russia-US relationship. Under the pressure of the United States, China and Russia may form a quasi-alliance of military and politics, unite against the United States, and jointly resist the pressure of the United States and NATO. 5 Motivation behind Russia approaching China Due to historical reasons, Russia has deep security concerns about China. Russia worried that China-Russia energy cooperation will facilitate China’s economy which will transform into China’s relative advantage toward Russia. Such concern play important role in Russia’s decision on Sino-Russia energy cooperation. Before 2008, oil and gas cooperation between China and Russia achieve little progress. Around the route of oil and gas pipelines, China and Russia negotiated for a long time, and do not reach a deal. The amount and number of Sino-Russia energy contracts are relatively small. At that time, the Russian energy market was centered on Europe, while the economic construction in the Far East was backward. The Far East demand for energy was not large, and it was not the focus of Russian oil and gas development. However, the U.S.-led NATO has eroded Russia’s geopolitical interests, resulting in changes in Russia’s interest calculation in Sino-Russia energy cooperation. gy p On August 8, 2008, “five day war” broke out and Russian military intervened in Georgia. Due to military intervention in Georgia, Russia was sanctioned by the United States and Europe. Coupled with the decline in oil prices in the international market, Russia, which relies heavily on energy exports, suffered significant economic losses. At this point, Russia began to turn its attention to the East, seeking to diversification of its energy exportation. Russia allowed Chinese capital to enter upstream oil and gas energy industry which is previous forbidden, and the oil pipeline to China was finally completed. Putin began to see China's rise as an opportunity, not a threat [5]. In 2012, Putin re-elected as president. Based on the judgment of the international situation, he repeatedly emphasized the "eastward strategy" and improved the strategical level of Sino-Russia cooperation. Now, China has become the most important buyer in the Asian market for Russia. The completion of the oil pipeline to Daqing has further consolidated China's position in the Russian energy Asia strategy. 4 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 After the outbreak of the Ukrainian crisis in 2014, Russia’s relation with NATO continued to deteriorate, and Russia cooperation with NATO virtually stopped. In September 2015, Russia got involved in Syria civil war. 6 Motivation behind China approaching Russia China is full of doubts about the US-led international energy order. As a beneficiary of the current international order, China’s economic rise benefits a lot from the US-led international energy and security system. However, because China’s political regime is different from that of Western countries. The Western countries represented by the United States are full of doubts about China's rise. In order to prevent the lifeline of energy from being controlled by others, China has done its best to diversify its energy import channels, strengthen cooperation with Russia which is an energy powerhouse, and try to establish an international energy system that is independent of the United States. gy y p With the rise of China, the structural contradictions between China and the United States have become increasingly prominent. In 2010, China overtook Japan to become the world's second largest economy. Due to the rapid economic development, China's military modernization has also begun to accelerate. In the South China Sea dispute, China changed its diplomatic strategy, from the past “keeping a low profile” to “striving for a achievement”. China no longer endured the violation of China's maritime rights by Southeast Asian countries, and actively defended its rights. In order to access overseas markets and ensure the resources needed to develop the economy, China actively cooperates with third world countries to achieve mutual benefit and win-win results. The Chinese model has also been seen as an alternative to the development path of the West, and more and more underdeveloped countries have tried to follow suit. Since energy cooperation between China and the third world countries does not include ideology, there are no preconditions, and the third world countries have financing channels other than Western countries. All of this is seen by the West represented by the United States as a challenge to its hegemony and ideology. China's energy security is very vulnerable. First, China has become biggest consumer in terms of oil and gas in the world, but the self-supply rate is not high, and most rely on imports. Second, a large part of China's oil and gas imports come from the Middle East, and oil and gas transportation from the Middle East must pass through the Indian Ocean and the Straits of Malacca. With the current naval strength, China cannot guarantee the safety of its energy channel in the possible Sino-US military confrontation. 5 Motivation behind Russia approaching China The Syrian issue became another tipping point for Russia-West relation. In order to punish Russia, the US-led NATO jointly launched economic sanctions. Europe also continuously reduced its dependence on Russia's oil. The continuous development of shale oil technology has made the United States more capable of suppressing oil prices, which caused Russia to suffer major economic losses [6]. In response, Russia accelerated the pace of diversification of energy exports and signed a series of oil and gas cooperation contracts with China. Economic sanctions make it difficult for Russia to obtain Western capital and technology, which also creates opportunities for Chinese capital to enter Russia's oil and gas industry in depth. Until 2015, China has become Russia's largest oil buyer, and China imported more than 800,000 barrels of oil per day from Russia. After 2008, Russia, which has been continuously sanctioned and suppressed by the United States, has gradually accepted China as a strategic partner. In order to ensure the safety of energy exports that are related to its economic lifeline, Russia has adopt long term perspective toward Sino-Russia energy cooperation, choose the direction of the oil pipeline with priority to China, and sign a large number of long-term contracts with China to allow Chinese capital to enter its upstream and downstream oil and gas industry. Russia and China have formed a strategic partnership in the energy sector [7]. 6 Motivation behind China approaching Russia Finally, if China's energy imports rely on Western-led international energy markets, there will be great risk out of China’s control. 5 5 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 The energy cooperation between China and Russia has strategic implication, as energy cooperation can further strengthen the strategic mutual trust between China and Russia. As one of the few socialist countries in the world, China’s political system has been criticized by Western countries. Similarly, Russia’s choice of its national development path is also seen by the West as a heresy. China and Russia share common interests in safeguarding the autonomy of the country's development path. Secondly, in response to China's rise, the United States launched a strategic containment toward China, increased security cooperation with ASEAN countries, frequently conducted exercises in the South China Sea to demonstrated its power to deter China. Russian geopolitical influence sphere has also been undermined by US led NATO, and Russian Middle east ally Syria also suffer the suppression of western countries. In order to strengthen Sino-Russia tie, Russia supports China's “One Belt, One Road” initiative and is willing to link the “Eurasian economy” with the “Silk Road Economic Belt”. China and Russia have common position on the denuclearization of the Korean Peninsula, the development of Arctic resources and the use of waterways of Arctic. In fact, China is cooperating with Russia to establish an energy security system that is different from the West. First, at the United Nations, China stand in line with Russia to give support to the third world oil producing countries (Iran, Venezuela, and Sudan) that were excluded by the West, and to veto Western sanctions on these countries. Second, China has obtained Russian understanding and acquiescence, established close energy cooperation with Central Asian countries [8]. In order to prevent US influence from entering Central Asia, China and Russia reached a tacit agreement to establish a Shanghai Cooperation Organization. It is worth noting that the organization may also discuss the possibility of accepting the oil producing country Iran as its member state. 7 Sino-Russia cooperation on energy security After 17 years growth of energy consumption, China has become World biggest crude oil consumer. In 2017 China accounted for 33.6% of the total world Energy consumption growth. China has surpassed US becoming No 1 oil importing country in the world. The Chinese importation of oil reached 3.96 hundred million tons, increased 10.8% compared with last year. The percent of imported oil account for total china oil consumption increased 3 percent, and reach 67.4%. In 2017 Sino-Russia crude oil trade reached 59.8 million tons, and refined oil trade reached 2.1 million tons, total up 6.19 hundred million ton. The top five China’s oil importation countries were Russia, Saudi Arab, Angola, Oman. Russia is one of the world biggest countries in terms of Oil and gas reservation, production, and exportation. In 2017, Russia has oil and gas reservation about 14500 MT oil and 35 trillion cubic meters, which account for 6% and 18.1% of world reservation; In 2017, Russia oil production and gas production are 554.4 Million Ton, 635.6 billion cubic meters. Russia Exportation oil and gas are 423.8 MT and 230.9 billion cubic meters. (please see Table). Table. China Energy consumption and importation in 2017 Category Oil (Unit: million tons, increase) Gas (Unit: one hundred million cubic meters, increase) LNG (Unit: one hundred million tons, increase) Consumption 608.4（+3.9%） 2404（+15.1% Production by China 191.5（-3.8%） 1492（+8.5%） LNG importation 39（+48.4%） Importation from 4.3 362 6 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 Central Asia (oil through pipeline) (gas through pipeline) Importation from Russia 59.8（+14%） 0.44 Data Source: BP World Energy Outlook 2018, China Custom Russia turned its energy focus to the Far East area. Prime Minister Putin ratified Russian “2030 energy strategy” at November 13, 2009. According to the strategy, energy exportation to Asian market will account for 26%- 27% of total Russia energy production by 2030. Russia will speed up oil and gas exploitation of East area, and energy cooperation with China becomes top priority of Russia energy diplomatic agenda. In this document, Russia emphasizes the world class energy power status, and the ability to control risk of International energy market, establishes an International energy system independent of Western countries, and tries to diversify its energy exportation. 7 Sino-Russia cooperation on energy security y gy p On April 13, 2009, then Prime Minister Putin presided over a government meeting which approved the agreement on Sino-Russia cooperation in the oil field. Putin said that the agreement to build the Eastern Siberian-Pacific oil pipeline which connects with China's regional line will create a "stable and reliable market" for the sale of Eastern Siberia oil to the East. On April 21, 2009, China and Russia signed a set of agreement, like the "Intergovernmental Agreement on Sino-Russia Cooperation in the Field of Petroleum," and “cooperation agreements on Sino-Russia oil pipeline construction”, “300 million tons of crude oil trade”, and “$25 billion oil loan” etc. The move marks a major breakthrough in energy cooperation between the two countries. The agreement stipulates that Russia will supply 15 million tons of crude oil to China through pipelines within 20 years from January 1, 2011. In March 2013, Russia agreed to increase the supply of pipeline crude oil to 30 million tons per year. As a response, China initiated the construction of the Mohe-Daqing oil pipe line as supplement. p p pp The Sino-Russia crude oil pipeline has been fully expanded to increase transportation capacity. On January 1, 2018, China opened the inlet valve of Mohe Oil Station. Russian crude oil entered the second line of Sino-Russia crude oil pipeline and began to transport Russian crude oil from Mohe to Daqing Linyuan Refinery. The amount of pipeline oil imported from Russia will increase from 15 million tons per year to 30 million tons per year. On June 8, 2018, PetroChina and Gazprom (hereinafter referred to as the Russian Gas Company) signed the Supplementary Agreement on the Mutual Recognition and Cooperation Agreement on Standards and Conformity Assessment Results, marking the cooperation in the field of standard identification and mutual recognition. An important step has been advanced to help promote mutual recognition of national and industry standards between the two countries and to avoid repeated testing of products of mutual interest. At present, the construction of the Sino-Russia East Line natural gas pipeline project is progressing smoothly. Gazprom said that it is willing to cooperate with PetroChina to promote the Sino-Russia East-line natural gas pipeline, the Far East supply of gas to China, and underground gas storage, and so on, to create more economic and social benefits for both sides. 7 Sino-Russia cooperation on energy security In the context of the Ukrainian crisis and the United States, Europe and Japan jointly sanctioning Russia to withdraw the Crimean Peninsula, on May 21, 2014, China and Russia reached a long-term natural gas contract about Eastern line worth US$400 billion. The contract period is 30 years, 38 billion Cubic meters per year, the gas price is linked to the price of oil. According to the contract, China and Russia will invest 70 billion U.S. dollars to build infrastructure. The Chinese side will pay an advance payment of 25 billion U.S. dollars. During President Putin’s visit China in October 2004, PetroChina signed a strategic 7 7 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 cooperation agreement with the Gazprom. At this time, Sino-Russia gas cooperation has been completed after 10 years of negotiations. cooperation agreement with the Gazprom. At this time, Sino-Russia gas cooperation has been completed after 10 years of negotiations. The Sino-Russia Yamal LNG project was officially put into operation. In this project, PetroChina hold 20% stake, Silk Road Fund hold 9.9% stake, France total hold 20% stake, Russia Novatec hold 50.1% stake. On December 8, 2017, Russian President Vladimir Putin personally attended the first batch of LNG shipment celebrations and launched the loading button. The first phase of the Yamal LNG project was officially put into operation, with an annual production capacity of 5.5 million tons. After the completion of the third phase, the annual production capacity will reach 16.5 million tons and the condensate oil will be 1 million tons. According to the Sino-Russia contract, 54% of the project's output is sold to the Asian market, and about one-third of it will be supplied to the Chinese market. In March 2006, China and Russia signed the Memorandum of Understanding on Gas Supply. Russia is committed to building two natural gas pipelines, and plans to export 60 billion-80 billion cubic meters of natural gas to China every year for 30 years from 2011. In June 2009, the Chinese and Russian heads of state signed the Memorandum of Understanding on Natural Gas Cooperation. In October 2009, China and Russia signed the "Framework Agreement on Russia's Export of Natural Gas to China". 7 Sino-Russia cooperation on energy security The Russian side promised to export nearly 70 billion cubic meters of natural gas to China via the Western and Eastern Lines from 2014 to 2015. The Western line is 30 billion cubic meters, and the Eastern line is 38 billion cubic meters. In 2012 Gazprom and PetroChina confirm the intention to implement Western route. When in 2013 President Xi Jinping visited Russia, CNPC sign agreement with Gazprom. In October2013, Russia and CNPC reach a deal on the price calculation of gas supplied by Russia. In 2013, PetroChina and Rosneft signed a memorandum of understanding (MoU) on upstream cooperation in Eastern Siberia and on establishing a joint venture company that would develop oil and gas fields, securing future ESPO demand China hold 49% stake， Russia hold 51% stake in the new joint venture company. This agreement represent breakthrough of Sino-Russia cooperation, because China capital has entered the upstream Russian energy industry and Russian overcome its psychological barrier toward China. Before that, it is hard for China to convince Russia permit China take part in the development of Far East energy. In 2017, Gazprom and PetroChina signed a Supplementary Agreement of the Sales and Purchase for Russian gas supplies via the eastern route, which specifies the starting date for the deliveries. According to this document, gas supplies to China via the Power of Siberia gas trunk line will begin in December 2019. In 2013, PetroChina and Rosneft signed a memorandum of understanding (MoU) on upstream cooperation in Eastern Siberia and on establishing a joint venture company that would develop oil and gas fields, securing future ESPO demand China hold 49% stake， 8 Conclusions Although both China and Russia face the threat of US hegemony, the content and nature of threats vary. Russia’s traditional geographical sphere of influence in Europe has been undermined by US-led NATO. As a counterattack, Russia conducted military intervention against Ukraine and Georgia in 2004 and 2013 respectively. In retaliation, the United States took the lead in economic sanctions against Russia, Western European countries continued to reduce energy imports from Russia, and the United States used its shale oil technology to suppress oil prices. In order to ensure the safety of energy exports, Russia has changed its past practice of treating China as a potential strategic opponent and emphasizing relative returns. It regards China as a strategic partner and launches comprehensive and in-depth energy cooperation with China, allowing Chinese oil companies to enter the energy upstream and downstream industries. 8 8 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 , 0 (201 E3S Web of Conferences 77 1002 9) Regional Energy Policy of Asian Russia 2018 https://doi.org/10.1051/e3sconf /20197701002 With regard to the rise of China, the United States has deep doubts. Cooperation and contain are the basis of the previous US administration China policy. China's oil and gas is largely dependent on importation, which makes China's energy security very vulnerable. In particular, the sea lane of energy transportation goes through the Malacca strait which is under the control of US Navy. In order to insure energy safety, China has made every effort to diversify its oil import channels. Russia, which is geographically adjacent to China and has common interests with China in international affairs, has become a reliable partner for China to ensure energy security. The “Indo-Pacific Strategy” proposed by the Trump administration regards China and Russia as adversary, emphasizes confrontation and zero- sum approach. Thus, Sino-Russia energy security cooperation becomes more urgent. Under the threat of US hegemony, China and Russia no longer overemphasized the relative interests and carried out comprehensive and in-depth energy cooperation. Russia has become China's largest energy importer, and Siberian oil pipelines to China have also been completed. The Russian oil industry has opened up upstream and downstream industries to Chinese capital. Russian energy has changed from its focusing on the European market in the past to the current Far East market. 8 Conclusions At present, in order to avoid energy risks, China has established an energy system that is independent of the West. With the strategic threat of US become more and more severe, the energy cooperation between China and Russia will be further enhanced. References 1. B.Lo. Axis of convenience: Moscow, Beijing and the new geopolitics, Washington DC: Brookings (2008) 2. Raju G. C. Thomas. The Relationships among Energy, Security, and the Economy, in Raju G.C. Thomas & Bennett Ramberg (eds ), Energy and Securily in the Industrializing World, The UniversitPress of Kentucky, pp 1-5 (1990) 3. Daniel Yergin. Energy Security in the 1990s, Foreign Affairs, Vol. 67, No.1, pp 111- 132 (1988) 4. Michael D. Swaine, A Counterproductive cold war with China. https://www.foreignaffairs.com/articles/china/2018-03-02/counterproductive-cold-war- china. Accessed November 30, 2018. 4. Michael D. Swaine, A Counterproductive cold war with China. https://www.foreignaffairs.com/articles/china/2018-03-02/counterproductive-cold-war- china. Accessed November 30, 2018. 5. Vladimir Putin. “Russia and changing world”, https://sputniknews.com/analysis/20120227171547818/: Accessed November 30, 2018. 5. Vladimir Putin. “Russia and changing world”, https://sputniknews.com/analysis/20120227171547818/: Accessed November 30, 2018. https://sputniknews.com/analysis/20120227171547818/: Accessed November 30, 2018. https://sputniknews.com/analysis/20120227171547818/: Accessed November 30, 2018. 6. Panchangwei, 2018 nianguojiyoujiayuce: xiangtibodong zhendangshangyang, ouyajingji（Eurasian Economy, No.3, p. 9 (2018) 7. Tom Roseth. “Russia’s energy relations with China: passing the strategic threshold?”, Eurasian Geography and Economics, Vol. 58, No. 1, pp. 23-55 (2017) 7. Tom Roseth. “Russia’s energy relations with China: passing the strategic threshold?”, Eurasian Geography and Economics, Vol. 58, No. 1, pp. 23-55 (2017) 8. Ronald Danneruther, “China and global oil: vulnerability and opportunity”, International Affairs, Vol.87, No.6, p. 1358 (2011) 8. Ronald Danneruther, “China and global oil: vulnerability and opportunity”, International Affairs, Vol.87, No.6, p. 1358 (2011) 9 9
https://openalex.org/W2216017872	https://europepmc.org/articles/pmc4734453?pdf=render	English	null	Effects of spin–orbit coupling and many-body correlations in STM transport through copper phthalocyanine	Beilstein journal of nanotechnology	2,015	cc-by	7,156	Abstract The interplay of exchange correlations and spin–orbit interaction (SOI) on the many-body spectrum of a copper phtalocyanine (CuPc) molecule and their signatures in transport are investigated. We first derive a minimal model Hamiltonian in a basis of fron- tier orbitals that is able to reproduce experimentally observed singlet–triplet splittings. In a second step SOI effects are included perturbatively. Major consequences of the SOI are the splitting of former degenerate levels and a magnetic anisotropy, which can be captured by an effective low-energy spin Hamiltonian. We show that scanning tunneling microscopy-based magnetoconduct- ance measurements can yield clear signatures of both these SOI-induced effects. Effects of spin–orbit coupling and many-body correlations in STM transport through copper phthalocyanine Benjamin Siegert, Andrea Donarini and Milena Grifoni Full Research Paper Open Access Address: Institut für Theoretische Physik, Universität Regensburg, D-93040, Germany Email: Benjamin Siegert - benjamin.siegert@ur.de * Corresponding author Keywords: anisotropy; copper phthalocyanine; magnetotransport; spin–orbit interaction; scanning tunneling microscopy (STM) Beilstein J. Nanotechnol. 2015, 6, 2452–2462. doi:10.3762/bjnano.6.254 Received: 14 August 2015 Accepted: 27 November 2015 Published: 22 December 2015 This article is part of the Thematic Series "Molecular machines and devices". Guest Editor: J. M. van Ruitenbeek © 2015 Siegert et al; licensee Beilstein-Institut. License and terms: see end of document. Open Access Beilstein J. Nanotechnol. 2015, 6, 2452–2462. doi:10.3762/bjnano.6.254 Received: 14 August 2015 Accepted: 27 November 2015 Published: 22 December 2015 This article is part of the Thematic Series "Molecular machines and devices". Guest Editor: J. M. van Ruitenbeek © 2015 Siegert et al; licensee Beilstein-Institut. License and terms: see end of document. Open Access Beilstein J. Nanotechnol. 2015, 6, 2452–2462. doi:10.3762/bjnano.6.254 Received: 14 August 2015 Accepted: 27 November 2015 Published: 22 December 2015 This article is part of the Thematic Series "Molecular machines and devices". Guest Editor: J. M. van Ruitenbeek © 2015 Siegert et al; licensee Beilstein-Institut. License and terms: see end of document. Single-particle Hamiltonian for CuPc The one-body Hamiltonian , written in the atomic basis , reads (2) (2) To probe both SOI-induced splittings and magnetic anisotropy, we further investigated the current characteristics of a CuPc molecule in an STM configuration similar to the experiments in [13,14]: The molecule is put on a thin insulating layer grown on top of a conducting substrate. The layer functions as a tunneling barrier and decouples the molecule from the substrate. Hence the CuPc molecule acts as a molecular quantum dot weakly coupled by tunneling barriers to metallic leads (here the STM tip and the substrate). This quantum dot configuration should be favourable to experimentally probe SOI splittings and magnetic anisotropies when an external magnetic field is applied to the system, in analogy to the experiments in [6]. Indeed, we demon- strate that experimentally resolvable SOI splitting should be observed at magnetic fields of a few teslas. where α is a multi-index combining atomic species and orbital quantum number at position rα, see Figure 1a. For the ligand we consider the set of all 2s (1s for hydrogen), 2px and 2py orbitals as the σ-system, and consequently the set of 2pz orbitals as the π-system. On the metal, the 3dxy, , and 4s orbitals contribute to the σ-system, while the 3dzx and 3dyz belong to the π-system. This basis yields a total of 195 valence electrons for neutral CuPc. Atomic on-site energies εα and geometrical para- meters were taken from [7,15]. The hopping matrix elements bαβ in Equation 2 are obtained by using the Slater–Koster [16] and Harrison [17] LCAO schemes, similar to [18]. Numerical diagonalization of finally yields single particle energies εi, see Figure 1b, and molecular orbitals , cf. Supporting Information File 1. where α is a multi-index combining atomic species and orbital quantum number at position rα, see Figure 1a. For the ligand we consider the set of all 2s (1s for hydrogen), 2px and 2py orbitals as the σ-system, and consequently the set of 2pz orbitals as the π-system. On the metal, the 3dxy, , and 4s orbitals contribute to the σ-system, while the 3dzx and 3dyz belong to the π-system. This basis yields a total of 195 valence electrons for neutral CuPc. Atomic on-site energies εα and geometrical para- meters were taken from [7,15]. Introduction Fe4 and Mn12 [4]. Recently, magnetic anisotropy effects could be directly probed by magnetotransport spectroscopy for Fe4 in quantum-dot setups [5,6]. An interesting question is hence if other classes of metallorganic compounds, such as the widely studied metal phthalocyanines [7,8], exhibit sizeable magnetic anisotropy induced by the interplay of electronic correlations and SOI. Indeed, in an X-ray magnetic circular dichroism (XMCD) analysis copper phthalocyanine (CuPc) was found to exhibit enormous anisotropies in both spin and orbital dipole moments [9]. Furthermore, recent experimental findings for Spin–orbit interaction (SOI) can play a major role in molecular spintronics. For example, in combination with the configur- ation of the non-magnetic component (organic ligand), it is known to be essential in establishing magnetic anisotropy in high-spin molecular magnets [1], and it is quite generally expected in metalorganic compounds. Effective spin-Hamilto- nians are commonly used to describe this anisotropy, and usually capture well the low energy properties of these systems [1-3]. Such effective Hamiltonians have been derived micro- scopically for widely studied molecular magnets such as Fe8, 2452 2452 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. without magnetic fields are presented and SOI-induced signa- tures are analyzed. cobalt pththalocyanine in a scanning tunneling microscopy (STM) setup [10] suggest that many-body correlations play an important role in the interpretation of the transport measure- ments. In a recent work [11], we have explicitly investigated long-range and short-range electron–electron correlation effects in CuPc and found a singlet–triplet splitting of the former anionic groundstate of about 18 meV, and thus a triplet as anionic ground state. Results and Discussion The focus of this section is the establishment of a minimal model Hamiltonian for an isolated CuPc molecule capable to account for both electron–electron interaction and spin–orbit coupling effects. As discussed below, parameters are fixed such that experimental observations for the singlet–triplet splitting [8] as well as positions of anionic and cationic resonances [14] are satisfactorily reproduced. In its most general form and for a generic molecule such Hamiltonian reads In this work we add the SOI to our analysis. We find that it further removes the triplet degeneracy by inducing splittings of few tenths of millielectronvolts. Moreover, in combination with exchange correlations, it produces a magnetic anisotropy which can in turn be captured by an effective spin Hamiltonian. (1) (1) In general, the accurate calculation of the many-body prop- erties of metallorganic molecules, such as molecular magnets or our CuPc, is a highly nontrivial task. In fact, the number of their atomic constituents is large enough that exact diagonalization is not possible and standard density-functional schemes have diffi- culties in capturing short ranged electron–electron correlations [4]. In order to reduce the size of the many-body Fock space, we use a basis of frontier molecular orbitals as the starting point to include electronic correlations [11,12] and construct a gener- alized Hubbard Hamiltonian. Furthermore, the symmetry of the molecule greatly helps to reduce the number of matrix elements one has to calculate in this basis. where the single-particle Hamiltonian of the molecule is given by , describes electronic interactions and accounts for the spin–orbit interaction (SOI). Single-particle Hamiltonian for CuPc Single-particle Hamiltonian for CuPc (b) Single particle energies of relevant molecular orbitals. Black (grey) circles depict the π (σ) character of the corresponding orbital. The color (diameter) of the inner circles characterizes the type (weight) of the metal orbital contribution on the corresponding molecular orbital. (c) Depiction of the four frontier orbitals retained in this work: SOMO (S), HOMO (H) and LUMOzx/yz (Lzx/yz). Figure 1: (a) Geometry and atomic composition of CuPc. (b) Single particle energies of relevant molecular orbitals. Black (grey) circles depict the π (σ) character of the corresponding orbital. The color (diameter) of the inner circles characterizes the type (weight) of the metal orbital contribution on the corresponding molecular orbital. (c) Depiction of the four frontier orbitals retained in this work: SOMO (S), HOMO (H) and LUMOzx/yz (Lzx/yz). (3 Many-body Hamiltonian in the frontier orbitals basis In order to set up a minimal many-body Hamiltonian, we restrict the full Fock space to many-body states spanned by the SOMO (S), the HOMO (H) and the two LUMO (L±) orbitals and write Equation 1 in this basis. Hence, for neutral CuPc the number of electrons populating the frontier orbitals is N0 = 3. Single-particle Hamiltonian for CuPc The hopping matrix elements bαβ in Equation 2 are obtained by using the Slater–Koster [16] and Harrison [17] LCAO schemes, similar to [18]. Numerical diagonalization of finally yields single particle energies εi, see Figure 1b, and molecular orbitals , cf. Supporting Information File 1. The paper is organized as follows: We first derive a micro- scopic Hamiltonian for CuPc in the frontier orbital basis which includes exchange correlations and the SOI. This Hamiltonian is diagonalized exactly and used in further spectral analysis and transport calculations. Its spectrum is also used to benchmark the prediction of an effective spin Hamiltonian that captures well the low-energy properties of CuPc both in its neutral and anionic configurations. Finally, transport calculations with and Stemming from Hartree–Fock calculations for isolated atoms [15], the atomic on-site energies εα do not take into account the ionic background of the molecule and crystal field contribu- tions. Therefore, molecular orbital energies εi have to be renor- malized with parameters δi to counteract this shortage, yielding (cf. Supporting Information File 2) 2453 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. Figure 1: (a) Geometry and atomic composition of CuPc. (b) Single particle energies of relevant molecular orbitals. Black (grey) circles depict the π (σ) character of the corresponding orbital. The color (diameter) of the inner circles characterizes the type (weight) of the metal orbital contribution on the corresponding molecular orbital. (c) Depiction of the four frontier orbitals retained in this work: SOMO (S), HOMO (H) and LUMOzx/yz (Lzx/yz). (4) (4) where is the n = 3 metal orbital with angular momentum and magnetic quantum number m = ±1. To distinguish contributions from the pure phthalocyanine (Pc) ligand and the copper (Cu) center, we introduced and , respectively. Likewise, with cS ≈ 0.90, we can write for the SOMO: (5) (5) where is the n = 3 metal orbital with angular momentum and projection m = ± 2 onto the z-axis. Finally, the HOMO has no metal contributions and thus we have trivially . The representations introduced in Equation 4 have the advantage that the four frontier orbitals can then be characterized by the phases φi acquired under rotations of π/2 around the main molecular symmetry axis. For the SOMO φS = π, for the HOMO φH = 0 and for the two LUMOs φL± = ±π/2. Figure 1: (a) Geometry and atomic composition of CuPc. Many-body Hamiltonian in the frontier orbitals basis (3) In this work we use a constant shift δi = δ = 1.83 eV. In this work we use a constant shift δi = δ = 1.83 eV. Due to the odd number of valence electrons, in its neutral con- figuration CuPc has a singly occupied molecular orbital (SOMO). When the molecule is in its anionic groundstate, this orbital does not become doubly occupied [7]. Hence, the orbitals most relevant for transport (frontier orbitals) are the SOMO (S), the HOMO (H) and the two degenerate LUMOs (Lzx/yz), which transform according to the b1g, a1u and eg irredu- cible representations of the point group of CuPc (D4h), respect- ively. They are depicted in Figure 1c. The LUMO orbitals in their real-valued representations, and , have equal contributions cL ≈ 0.097 on both 3dzx and 3dyz orbitals of the metal. Due to their degeneracy, they can be transformed into their complex, rotational invariant representations: We exploit the distinct phases acquired by the frontier orbitals under 90° rotations to determine selection rules for the matrix elements Vijkl in , (6) (6) namely if , , cf. Support- ing Information File 2. Equation 6 in this basis then reads namely if , , cf. Support- ing Information File 2. Equation 6 in this basis then reads 2454 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. (7) (7) where creates an electron with spin σ on the copper atom in the orbital specified by . For an electron in the 3d shell of Cu we use ξCu ≈ 100 meV [20]. Projecting Equation 9 onto the minimal set of frontier orbitals then yields: (7) (10) (10) where the indices i, j, k now run over the set of frontier orbitals, and the notation [ijk] means that the sum runs only over unlike indices, i.e., i, j and k are different from each other in the corres- ponding sum. The abbreviations we introduced in Equation 7 are the orbital Coulomb interaction Ui = Viiii, the inter-orbital Coulomb interaction Uij = Viijj, the exchange integral , the ordinary pair hopping term and the split pair hopping term . Contributions with four different indices are found to be very small (of the order of microelectronvolts) and thus omitted in this work. Many-body Hamiltonian in the frontier orbitals basis The matrix elements Vijkl are calculated numerically using Monte Carlo integration [19] and renormalized with a dielectric constant εr = 2.2 in order to account for screening by frozen orbitals [12]. A table with the numerically evaluated interaction constants is found in Supporting Information File 2. where λ1 = 1/2ξCu\|cL\|2 = 0.47 meV and λ2 = ξCu(cScL)/ = 6.16 meV are now effective spin–orbit coupling constants. A similar analysis of SOI in CuPc, laying more focus on the central Cu atom, can be found in [21]. Finally, many-body eigenenergies ENk and eigenstates , labeled after particle number N and state index k, are obtained by exact numerical diagonalization of in the frontier orbitals basis. Despite numerically tractable, the problem described by is still highly intricate, as the Fock space has dimension 44 = 256. In reality, though, only few low-lying many-body states are relevant at low energies. This enables further simplification and even an analytical treatment, as discussed in the next subsection. basis In the following we will analyze the neutral and anionic low- energy part of the many-body spectrum of CuPc and establish an effective Hamiltonian which enables us to analyze the low- energy behaviour in a more lucid way. To this extent, we start by observing that (in the considered particle number subblocks) contains different energy scales, in particular, U > J > λ, which suggests a hierarchy of steps. We use U, J and λ to denote the set of all Hubbard-like parameters (Ui, Uij), all exchange parameters ( ) and all SOI parameters (λi), respectively. As a first step we set both the exchange (J) and SOI (λ) contributions to to zero and determine the neutral and anionic groundstates. In a second and third step exchange and SOI are added, respectively. A perturbative contribution to the bare one-body Hamiltonian relevant in molecular systems is provided by the SOI. In the following we derive an effective spin–orbit coupling operator acting on the subset of frontier orbitals. The atomic SOI oper- ator reads (8) (8) where α and run over all atoms and shells, respectively. By evaluating Equation 8 only on the central copper atom, i.e., and α = Cu, in second quantization is given by Anionic low-energy spectrum Continuing with the anionic low-energy part of the spectrum of , we find an eightfold degenerate ground- state: (13) (13) Figure 2: Lowest lying anionic states of CuPc, together with their grade of degeneracy d. Without exchange and SOI, the anionic groundstate is eightfold degenerate. When exchange interaction between SOMO and LUMOs is introduced, the degeneracy is lifted, yielding two triplets and two singlets because of the orbital degen- eracy of the LUMO. SOI further splits the triplet states, generating a twofold degenerate anionic groundstate consisting of the states and . with corresponding energy . The eightfold degeneracy comes from the two unpaired spins in either SOMO or LUMO and the orbital degeneracy of the LUMO orbitals. In order to make the anionic eigenstates also eigenstates of the spin oper- ators and , they can be rewritten as Finally, to analyze in a third step how affects the low- energy part of the anionic part of the spectrum, in particular which degeneracies are lifted, we treat it as a perturbation and apply second-order perturbation theory to obtain the energy shifts. To this end, some additional states have to be considered. They are listed in Supporting Information File 3. Finally, to analyze in a third step how affects the low- energy part of the anionic part of the spectrum, in particular which degeneracies are lifted, we treat it as a perturbation and apply second-order perturbation theory to obtain the energy shifts. To this end, some additional states have to be considered. They are listed in Supporting Information File 3. (14) (14) The states and experience a downshift due to and become the groundstates. Measuring energies with respect to , we get (17) The orbital degeneracy of the LUMOs, expressed by the index τ, is responsible for the two sets of singlets (total spin S = 0) and triplets (total spin S = 1). Considering exchange interaction in a second step, we find that only the term in Equation 7, (17) see Figure 2. Note that in our numerical calculations and are mixed and the degeneracy of the resulting states is lifted by a small shift in the range of some μeV. A more detailed discus- sion concerning the mixing of and can be found in Sup- porting Information File 3. Neutral low-energy spectrum (9) Neutral low-energy spectrum In the neutral low-energy part of the spectrum, we retain the two spin-degenerate groundstates of , (11) with corresponding energy . We defined . Th d t t i E ti 11 ith ff t d b In the neutral low-energy part of the spectrum, we retain the two spin-degenerate groundstates of , (11) (9) (11) with corresponding energy . We defined . The groundstates in Equation 11 are neither affected by 2455 2455 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. nor by the exchange terms in Equation 7. Trivially, the effective Hamiltonian in the basis of reads: (16) nor by the exchange terms in Equation 7. Trivially, the effective Hamiltonian in the basis of reads: (16) nor by the exchange terms in Equation 7. Trivially, the effective Hamiltonian in the basis of reads: (16) nor by the exchange terms in Equation 7. Trivially, the effective Hamiltonian in the basis of reads: (16) (16) (12) (12) for the singlets and triplets, respectively. for the singlets and triplets, respectively. for the singlets and triplets, respectively. In principle Equation 7 also contains terms that act on the neutral groundstate, such as for example pair hopping terms proportional to , and cause admixtures with other many- body states. However, according to our full numerical calcula- tions, these admixtures are rather small and do not affect trans- itions between neutral and anionic states. Figure 2: Lowest lying anionic states of CuPc, together with their grade of degeneracy d. Without exchange and SOI, the anionic groundstate is eightfold degenerate. When exchange interaction between SOMO and LUMOs is introduced, the degeneracy is lifted, yielding two triplets and two singlets because of the orbital degen- eracy of the LUMO. SOI further splits the triplet states, generating a twofold degenerate anionic groundstate consisting of the states and . Interaction with magnetic fields (19) An experimentally accessible way to probe magnetic anisotro- pies is to apply external magnetic fields. In order to account for interactions of orbitals with magnetic fields, the atomic hopping matrix elements bαβ in Equation 2 have to be corrected with Peierls phase factors, (20) where for we omitted smaller additional contributions from other states. The energies change according to (25) (25) (21) (22) (21) (22) where, using the gauge , the phase is given by (26) (21) where, using the gauge , the phase is given by (26) Here (xα, yα) are the in-plane atomic coordinates. Owing to the planar geometry of CuPc, depends only on the z-component Bz of the magnetic field B. In Figure 3 we show the dependence of the energies of the frontier molecular orbitals on the strength of the magnetic field in z-direction, Bz. For the two LUMOs we observe a linear dependence on the magnetic field, yielding an effective orbital moment of μorb = 33.7 μeVT−1, while the LUMO−(+) goes down (up) in energy with Bz (Figure 3a). The energies of the HOMO and the SOMO, however, scale quadratically with the magnetic field at a much lower scale (Figure 3b). This behaviour is expected, since the a1u and b1g representations have characters +1 under rota- tions, which transform Bz to −Bz. Thus the energies of HOMO and SOMO can not depend on the sign of Bz and must move at least quadratically with Bz. The two-dimensional eg representa- For further details we refer to Supporting Information File 3. Finally, the singlets S+ and S−, similar to and , change very little (with respect to ): For further details we refer to Supporting Information File 3. Finally, the singlets S+ and S−, similar to and , change very little (with respect to ): (23) (23) By introducing , an approximate Hamiltonian up to first order in can be given for the N0 + 1 particle subblock: By introducing , an approximate Hamiltonian up to first order in can be given for the N0 + 1 particle subblock: (24) (24) Equation 24 is one major result of this work. It shows that, similar to the well-studied molecular magnets [3-6], the inter- play of spin–orbit coupling and exchange interactions yield magnetic anisotropies that can be captured by effective spin Hamiltonians. Anionic low-energy spectrum The next states are and with (15) (15) directly determines the low-energy structure of the anionic low- energy part because of the singly occupied SOMO and LUMOs: The degeneracy between singlets and triplets is lifted, see Figure 2, and we obtain (18) (18) 2456 2456 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. Due to their quadratic dependence on λ1 and λ2, these states change very little with . The degeneracy of the states and is lifted by the mixing of these states through . We find Due to their quadratic dependence on λ1 and λ2, these states change very little with . The degeneracy of the states and is lifted by the mixing of these states through . We find dots with comparable SOI and hopping integrals the Dzyaloshinskii–Moriya interaction is sizeable and produces interesting effects on magnetization [24] and transport charac- teristics [25]. (19) (20) Interaction with magnetic fields Noticeably, because Equation 24 was derived from the microscopic molecular Hamiltonian , it was possible to check that deviations are in range of microelectron- volts and only of quantitative nature by comparison of the spec- trum to the numerically evaluated one. Another source of magnetic anisotropy is the Dzyaloshinskii–Moriya interaction [22,23]. Although the latter is also linear with respect to the SOI, it does not appear in our model. The fundamental reason for neglecting it is the large ratio between the hopping integrals (of the order of electronvolts) and the SOI (ξCu ≈ 100 meV), which also justifies our perturbative analysis in terms of molecular orbitals. However, for molecular quantum Figure 3: (a) Dependence of the single particle orbital energies on the magnetic field strength. From this, the effective orbital moment of the LUMOs, here depicted in their complex representation, can be extracted as μorb = 33.7 μeVT−1. The energies of the SOMO and HOMO orbitals depend quadratically on the magnetic field and involve a much lower scale than the LUMOs, as seen in the close-up in panel (b). Figure 3: (a) Dependence of the single particle orbital energies on the magnetic field strength. From this, the effective orbital moment of the LUMOs, here depicted in their complex representation, can be extracted as μorb = 33.7 μeVT−1. The energies of the SOMO and HOMO orbitals depend quadratically on the magnetic field and involve a much lower scale than the LUMOs, as seen in the close-up in panel (b). 2457 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. (31) tion on the other hand has zero character under rotations, which implies that the constituents of eg transform under such rotations either with different signs or into each other; indeed under a rotation LUMO+ is mapped onto LUMO− and vice versa. (31) where creates an electron in lead η with spin σ and momentum k. The tunneling Hamiltonian finally is given by Finally, the interaction of electronic spins with magnetic fields is represented by adding a Zeeman term to Equation 1, by (32) (32) (27) (27) It contains the tunneling matrix elements , which are obtained by calculating the overlap between the lead wavefunc- tions and the molecular orbitals [26]. They yield the tunneling rates where gS = 2 and S is the total spin operator on the molecule written in the frontier orbital basis. Effective low-energy Hamiltonian Effective low-energy Hamiltonian Putting everything together, an effective low-energy Hamilto- nian including magnetic interaction terms for both orbital and spin degrees of freedom can thus be given. It reads which are of the order of 10−6 eV and 10−9 eV for the substrate and the tip, respectively. Finally, the dynamics of the transport itself is calculated by evaluating the generalized master equa- tion, (28) (28) where is the Hamiltonian for the corresponding low-energy N-particle subblock as given by Equation 12 and Equation 24. (33) (33) (33) Dynamics and transport Reduced density operator and current for the reduced density operator [26,29] ρred = TrS,T(ρ). The Liouvillian superoperator The transport calculations for the molecule in an STM setup are done by using the formalism introduced in earlier works [18,26,27]. For the sake of clarity, in the following we briefly discuss the main steps to obtain the current through the molecule. The full system is described by the Hamiltonian (34) (34) contains the terms and describing tunneling from and to the substrate and the tip, respectively. To account for relaxation processes leading to de-excitation of molecular excited states, we included a relaxation term , analogously to [30]: (29) (29) (35) where describes the isolated molecule, see Equation 1. To incorporate image charge effects in our model, leading to renor- malizations of the energies of the system’s charged states [28], we included a term [11], (35) It depends on the deviation of ρ from the thermal distribution ρth,N of the N-particle subblock, which is given by a Boltzmann distribution: It depends on the deviation of ρ from the thermal distribution ρth,N of the N-particle subblock, which is given by a Boltzmann distribution: (30) (30) (36) where is the particle number operator on the molecule. Elec- trostatic considerations regarding the geometry of the STM setup yielded δic ≈ 0.32 eV [11]. The Hamiltonians and corresponding to substrate (S) and tip (T), respectively, are describing noninteracting electronic leads. They read (36) 2458 2458 2458 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. (38) with β = (kBT)−1. Since acts separately on each N-particle subblock, it conserves the particle number on the molecule and thus does not contribute to transport directly. In this work, the relaxation factor 1/τ is around the same order of magnitude as the mean tip tunneling rate onto the molecule. In particular, we are interested in the stationary solution for which . Finally, the current through the system in the stationary limit can be evaluated as (38) where e is the electron charge and αT accounts for the fact that in STM setups the bias voltage drops asymmetrically across the junction. Electrostatic considerations yielded αT = 0.59 for the tip and αS = −0.16 for the substrate [11]. If given without indices, Vres denotes the bias voltage corresponding to the groundstate-to-groundstate resonance. (37) The negative differential conductance at large negative bias in Figure 4 is caused by blocking due to population of excited states of the molecule. Dynamics and transport Reduced density operator and current This has already been discussed in a previous work [27] and will not be of further interest here. yielding the current operator for lead η as . Transport characteristics In this work, a tip–molecule distance of 5 Å was used and simu- lations were done at the temperature T = 1 K. We assumed a renormalization of the single particle energies δi = δ =1.83 eV (cf. Equation 3), an image-charge renormalization δic = 0.32 eV and a dielectric constant εr = 2.2 in order to fit our spectrum to the experiment of Swart et al. [14], which was carried out with CuPc on a NaCl(3 ML)/Cu(100) substrate with a workfunction of = 4.65 eV. With this, we find a triplet–singlet separation of the anionic ground state of 18 meV, which is in good agree- ment with experimental measurements of 21 meV [8]. Numer- ical results for the current and the differential conductance, according to Equation 37 and using the full Hamiltonian in Equation 29, are shown in Figure 4. Anionic (cationic) reson- ances at positive (negative) bias voltages are clearly seen. Transport simulations at finite magnetic fields In Figure 5 we show the splitting of the anionic resonance with applied magnetic field in a dI/dV map. In the upper panel SOI is switched off, whereas in the lower panel it is switched on. One striking difference at first glance is the zero-field splitting for non-vanishing SOI, which is proportional to λ1 but enhanced by the bias drop, cf. Equation 38. For vanishing SOI, when Sz is a good quantum number, we can readily identify the corres- ponding transitions by using the effective spin Hamiltonian introduced in Equation 28. In the following, transitions from the neutral groundstate will be denoted by arabic numbers: Figure 4: Current and differential conductance curves exhibiting the anionic (cationic) resonance at positive (negative) bias voltage. Note that in contrast to all other results in this work, this curve is taken at a temperature of 60 K to emphasize the resonances in the dI/dV curve. while transitions from the neutral excited state will be denoted by Roman numerals: Figure 4: Current and differential conductance curves exhibiting the anionic (cationic) resonance at positive (negative) bias voltage. Note that in contrast to all other results in this work, this curve is taken at a temperature of 60 K to emphasize the resonances in the dI/dV curve. Other transitions are forbidden due to the selection rule for Sz, ΔSz = ±(1/2). The reason for the splitting into four lines observed in the upper panel of Figure 5 is that the orbital moment of the LUMO is not of the same size as the Bohr magneton. Notice that, in our model, the bias voltage at which a tip-medi- ated transition from the m-th neutral state to the n-th anionic state of the molecule is happening is 2459 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. anisotropy due to the orbital moment of the LUMOs can be observed, although still blurred by temperature. Again, at finite SOI in Figure 6e there is a much more pronounced dependence on θ. The high conductance areas at θ = 0° and θ = 180° for Vb − Vres ≈ 0.8 meV correspond to the high conductance area in the middle of Figure 5 bottom, where many transitions are taking place at the same time. Conclusion We established a model Hamiltonian for CuPc which accounts for electron–electron, spin–orbit and magnetic interactions in a minimal single particle basis represented by four frontier orbitals; the SOMO, the HOMO and two degenerate LUMOs. The distinct properties of these orbitals under rotations allowed us to deduce selection rules for matrix elements of the Coulomb interaction, which drastically reduce the number of nonvan- ishing terms and simplify the numerical diagonalization of the full many-body Hamiltonian. For the low-energy parts of the neutral and anionic blocks of the many-body spectrum we could further derive an effective spin Hamiltonian, capturing both SOI-induced splittings and magnetic anisotropy. Analogous Hamiltonians accounting for the effect of atomic SOI in molecular systems with orbital degeneracies have been derived for example in carbon nanotubes [31]. Figure 5: Differential conductance maps as a function of the strength Bz of the magnetic field in z-direction. Upper (lower) panel: Spin–orbit interaction switched off (on). Solid and dashed lines depict the add- ition spectrum as calculated from the effective spin Hamiltonian, cf. Equation 28. Transitions starting from the neutral groundstate are denoted by solid lines, those from the neutral excited state by dashed lines. For non-vanishing SOI, see lower panel of Figure 5, the definite assignment of transitions is not straightforward, at least for small magnetic fields. Since and are shifted downward by SOI, transition (2) now is the lowest lying transition, whereas transition (1) is shifted upward due to the positive contribution +λ1 to . Furthermore, transition (iv) is the only excited-state transition which can be definitely assigned to a line in the lower panel in Figure 5. In order to study fingerprints of the SOI under realistic experi- mental conditions, we have studied the magnetotransport char- acteristics of a CuPc based junction in an STM setup. To this extent, a generalized master equation for the reduced density matrix associated to the full many-body Hamiltonian had to be solved in order to numerically obtain both the current and the differential conductance. Noticeably, by using the effective spin Hamiltonian, it was possible to reconstruct the nature of the many-body resonances observed in the numerical calcula- tions. Figure 6 finally shows dI/dV maps as a function of the angle θ between the magnetic field and the z-axis. Panels (a), (b) and (c) show results obtained with vanishing SOI and panels (d), (e) and (f) are for finite SOI. Transport simulations at finite magnetic fields At B = 8 T, the magnetic field is dominating and a characteristic double cosine-like behaviour of the resonances can be observed, for both the case with no SOI, Figure 6c, and finite SOI, Figure 6f. For vanishing SOI, this behaviour is caused by the orbital moment of the LUMOs, since they interchange their positions when going from Bz to −Bz. The overall splitting between the double cosines, most evident at θ = 90°, is caused by the Zeeman term. The results for B = 8 T in Figure 6f at finite SOI are similar to those in Figure 6c, with the only difference that the cosine at large biases is more stretched, the one at low bias more compressed. Figure 5: Differential conductance maps as a function of the strength Bz of the magnetic field in z-direction. Upper (lower) panel: Spin–orbit interaction switched off (on). Solid and dashed lines depict the add- ition spectrum as calculated from the effective spin Hamiltonian, cf. Equation 28. Transitions starting from the neutral groundstate are denoted by solid lines, those from the neutral excited state by dashed lines. Conclusion Again, the results were fitted using the effective spin Hamiltonian introduced in Equation 28 with good agreement. The respective transitions can be identified by checking the assigned transitions in Figure 5 at the corres- ponding field strength. In summary, we believe that our work significantly advances the present understanding of spin properties of CuPc. Moreover, the flexibility of our model Hamiltonian approach opens new perspectives for the investigation of other configurationally similar metallorganic compounds. Already at \|B\| = B = 1 T, cf. panels (a) and (d), the influence of SOI can be clearly seen. While for vanishing SOI any aniso- tropy of the dI/dV map is hidden beneath the temperature broad- ening, for finite SOI a slight θ-dependence can be observed. For B = 3 T, now also in the vanishing SOI case, Figure 6b, a slight 2460 Beilstein J. Nanotechnol. 2015, 6, 2452–2462. Figure 6: Differential conductance maps vs the angle θ, formed by the applied magnetic field with the z-axis. Left (right) panels are without (with) SOI. Upper, middle and lower panels are calculated for a magnetic field strength of 1, 3 and 8 T, respectively. Solid and dashed lines depict the addition spectrum as calculated from the effective spin Hamiltonian, cf. Equation 28. Transitions starting from the neutral groundstate are denoted by solid lines, those from the neutral excited state by dashed lines. Figure 6: Differential conductance maps vs the angle θ, formed by the applied magnetic field with the z-axis. Left (right) panels are without (with) SOI. Upper, middle and lower panels are calculated for a magnetic field strength of 1, 3 and 8 T, respectively. Solid and dashed lines depict the addition spectrum as calculated from the effective spin Hamiltonian, cf. Equation 28. Transitions starting from the neutral groundstate are denoted by solid lines, those from the neutral excited state by dashed lines. Supporting Information Supporting Information Supporting Information File 1 Transformation from the atomic to the molecular orbital basis. [http://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-6-254-S1.pdf] Supporting Information File 2 Symmetries in the frontier orbitals basis. [http://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-6-254-S2.pdf] Supporting Information File 3 Details on the perturbative treatment of the SOI. [http://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-6-254-S3.pdf] Supporting Information Supporting Information File 1 Transformation from the atomic to the molecular orbital basis. [http://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-6-254-S1.pdf] Supporting Information File 2 Symmetries in the frontier orbitals basis. [http://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-6-254-S2.pdf] Supporting Information File 3 Details on the perturbative treatment of the SOI. [http://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-6-254-S3.pdf] The authors thank Thomas Niehaus, Jascha Repp and Dmitry Ryndyk for fruitful discussions. Financial support by the Deutsche Forschungsgemeinschaft within the research program SFB 689 is acknowledged. 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https://openalex.org/W4386389337	https://greenationpublisher.org/index.php/JGIA/article/download/9/9	Indonesian	null	Pengaruh Kepemimpinan, Tata Ruang Kantor dan Motivasi Kerja Terhadap Kinerja Pegawai pada Dinas Pengendalian Keuangan dan Aset (DPKA) Kota Padang	Jurnal Greenation Ilmu Akuntansi	2,023	cc-by	6,123	Corresponding Author: aurorafebrianda@yahoo.com Abstract: This study aims to determine (1) the effect of leadership on employee performance at the financial and asset management service (DPKA) Padang city, (2) the influence of office layout on employee performance at the financial and asset management service (DPKA) Padang city, (3) the effect of leadership on work motivation at the financial and asset management service (DPKA) in Padang City, (4) the effect of office layout on work motivation in the Padang City Financial Asset Management Service (DPKA), (5) the effect of work motivation on performance at the financial management service and assets (DPKA) of the city of Padang. This type of research is descriptive associative. The results of the study show that (1) there is a significant influence between leadership variables on employee performance at the financial and asset management office (DPKA) in Padang City, (2) there is a significant influence between office layout variables on employee performance at the financial and asset management office (DPKA) Padang City, (3) There is a significant influence between leadership variables on work motivation in the financial and asset management service (DPKA) Padang City, (4) There is a significant influence between office layout variables on work motivation in the financial management service assets (DPKA) Padang City, (5) There is a significant influence between work motivation variables on performance in the financial and asset management service (DPKA) Padang City. Pengaruh Kepemimpinan, Tata Ruang Kantor dan Motivasi Kerja Terhadap Kinerja Pegawai pada Dinas Pengendalian Keuangan dan Aset (DPKA) Kota Padang Pengaruh Kepemimpinan, Tata Ruang Kantor dan Motivasi Kerja Terhadap Kinerja Pegawai pada Dinas Pengendalian Keuangan dan Aset (DPKA) Kota Padang Aurora Febrianda Fakultas Ekonomi, Universitas Negeri Padang, Indonesia, aurorafebrianda@yahoo.com Corresponding Author: aurorafebrianda@yahoo.com https://greenationpublisher.org/JGIA, https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 Keyword: Leadership, Office Layout, Work Motivation, Employee Performance. Abstrak: Penelitian ini bertujuan untuk mengetahui (1) pengaruh kepemimpinan terhadap kinerja pegawai pada dinas pengelolaan keuangan dan aset (DPKA) kota padang, (2) pengaruh tata ruang kantor terhadap kinerja pegawai pada dinas pengelolaan keuangan dan aset (DPKA) kota padang, (3) pengaruh kepemimpinan terhadap motivasi kerja pada dinas pengelolaan keuangan dan aset (DPKA) kota padang, (4) pengaruh tata ruang kantor terhadap motivasi kerja pada dinas pengelolaan keuangan aset (DPKA) kota padang, (5) pengaruh motivasi kerja terhadap kinerja pada dinas pengelolaan keuangan dan aset (DPKA) kota padang. Jenis penelitian ini adalah deskriptif asosiatif. Hasil penelitian menunjukkan bahwa (1) Terdapat pengaruh yang signifikan antara variabel kepemimpinan terhadap kinerja 32 \| P a g e https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 pegawai pada dinas pengelolaan keuangan dan aset (DPKA) Kota Padang,(2) Terdapat pengaruh yang signifikan antara variabel tata ruang kantor terhadap kinerja pegawai pada dinas pengelolaan keuangan dan aset (DPKA) Kota Padang,(3) Terdapat pengaruh yang signifikan antara variabel kepemimpinan terhadap motivasi kerja pada dinas pengelolaan keuangan dan aset (DPKA) Kota Padang, (4) Terdapat pengaruh yang signifikan antara variabel tata ruang kantor terhadap motivasi kerja pada dinas pengelolaan keuangan aset (DPKA) Kota Padang, (5) Terdapat pengaruh yang signifikan antara variabel motivasi kerja terhadap kinerja pada dinas pengelolaan keuangan dan aset (DPKA) Kota Padang. Kata Kunci: Kepemimpinan, Tata Ruang Kantor, Motivasi Kerja, Kinerja Pegawai. https://greenationpublisher.org/JGIA, https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 4 Lain – lain pendapatan asli daerah yang sah 100% 82.28% 80.47% 82.35% Jumlah rata – rata penyelesaian 80.67% 78.56% 78.28% Sumber : Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang tahun 2012 Berdasarkan data pada tabel 1 terlihat pelaksanaan kegiatan atau program-program yang ada pada Dinas Pengelolaan Keuangan dan Aset (DPKA) selama 3 tahun terakhir dari tahun 2010 sampai dengan tahun 2012,dimana sepanjang tahun 2010 tingkat penanganan atau penyelesaian kegiatan atau program pekerjaan yang ada rata-rata sekitar 80.67%, kemudian sepanjang tahun 2011 menurun menjadi 78.56% dan pada tahun 2012 kemampuan penanganan atau penyelesaian pekerjaan menurun lagi menjadi 78.28%. Disini terlihat kalau setiap tahun target realisasi kegiatan/program selalu mengalami penurunan. Seorang pemimpin juga tidak luput dari sorotan kenapa pada setiap tahunnya target realisasi mengalami penurunan. Menurut Mulyadi dan Rivai, (2009) dalam suatu organisasi ada dua pihak yang saling tergantung dan merupakan unsur utama dalam organisasi yaitu pimpinan sebagai atasan, dan pegawai sebagai bawahan. Dan berdasarkan observasi awal yang telah penulis lakukan pada Dinas Pengelolaan Keuangan dan Aset Kota Padang, mengenai kepemimpinan ada terdapat beberapa masalah yaitu Pemimpin kurang peduli dengan karyawan, terlihat kurang memberikan teguran kepada karyawan kantor yang datang tidak tepat waktu dari waktu yang di tetapkan, Kurang melibatkan karyawan dalam proses pengambilan keputusan, dan Pemimpin tidak menjalin komunikasi yang baik dengan karyawan. Hal ini tentunya akan menyebabkan ketimpangan sehingga apa yang diinginkan pemimpin tidak sesuai dengan apa yang dilakukan oleh karyawannya. Hal ini dikarenakan tidak lancarnya komunikasi antara karyawan dengan pimpinan sehingga tujuan dari organisasi tersebut tidak tercapai dengan baik. Selain melihat dari Kepemimpinan penulis juga melakukan wawancara kepada beberapa pegawai yang ada di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Menurut Berdasarkan hasil dari wawancara tersebut bahwa penurunan pelaksanaan kegiatan kerja ini terjadi juga karna akibat bencana alam yang melanda Kota Padang pada tahun 2009. Bencana alam yang terjadi pada tahun 2009 menyebabkan sarana dan prasarana yang mendukung pekerjaan banyak mengalami kerusakan,sehingga sampai sekarang hal inilah yang menyebabkan suasana kerja kurang kondusif. Kondisi kerja dikatakan baik apabila memungkinkan seseorang untuk dapat meningkatkan produktifitas kerjanya, baik kondisi fisik maupun psikologis. Kondisi fisik berupa pemanfaatan dan penataan ruangan kantor secara langsung dan nyata berkaitan erat dengan peningkatan kinerja serta memberikan kontribusi untuk prestasi kerja yang efektif dan efisien. Penataan ruangan kantor juga menjadi hal yang penting karena menyangkut kenyamanan individu dan kelompok dalam bekerja. PENDAHULUAN Manusia sebagai sumber daya merupakan faktor penting yang harus diarahkan dan digerakkan serta dibina agar dapat melaksanakan tugas dan fungsinya untuk dapat mencapai tujuan utama organisasi. Seperti diketahui bahwa didalam suatu organisasi segala macam aktivitas yang berhubungan dengan produktivitas dan hasil kerja pegawai selalu menjadi sorotan yang tidak pernah luput dari pengawasan pimpinan organisasi. Suatu tindakan pegawai akan mempengaruhi kestabilan keadaan organisasi yang akan berdampak pada produktivitas organisasi itu sendiri. Oleh karena itu pimpinan organisasi harus mampu menilai dan memperhatikan kondisi dinamika organisasi serta perilaku pegawai yang dapat mengganggu produktivitas organisasi tersebut. Disamping itu untuk menciptakan kondisi lingkungan organisasi yang optimal diperlukan penilaian kinerja pegawai oleh pimpinan dengan tujuan terciptanya hasil kerja yang baik. Penilaian kinerja pada dasarnya merupakan faktor kunci guna mengembangkan suatu organisasi secara efektif dan efisien. Penilaian kinerja individu sangat bermanfaat bagi dinamika pertumbuhan organisasi secara keseluruhan, melalui penilaian tersebut maka dapat diketahui kondisi sebenarnya tentang bagaimana kinerja karyawan. Kinerja menurut Mangkunegara (2001:67) adalah hasil secara kualitas dan kuantitas yang dicapai seorang pegawai dalam melaksanakan tugasnya sesuai dengan tanggung jawab yang diberikan kepadanya. Dengan bertitik tolak dari pendapat diatas dapatlah dikatakan bahwa kinerja merupakan kesuksesan pegawai didalam melaksanakan suatu pekerjaan yang sesuai dengan tugas dan tanggung jawab yang dibebankan kepadanya. Berkenaan dengan Kinerja Pegawai tersebut, Dinas Pengelolaan Keuangan dan Asset Kota Padang, juga tidak luput dengan permasalahan kinerja yang dihasilkan dalam memberikan pelayanan yang baik kepada masyarakat. Berikut gambaran kinerja yang telah dicapai berdasarkan kegiatan atau Program Kerja sepanjang 3 tahun terakhir. Tabel 1 : Laporan Realisasi pelaksanaan kegiatan pemungutan pajak Kota Padang Dinas Pengelolaan Keuangan dan Aset Kota Padang Periode 2010 - 2012 No Kegiatan / program Target Realisasi Realisasi kegiatan (%) 2010 2011 2012 1 Pajak daerah 100% 77.69% 76.95% 75.47% 2 Retribusi daerah 100% 85.19% 81.40% 80.05% 3 Hasil pengelolaan kekayaan daerah yang dipisahkan 100% 77.54% 75.45% 75.25% bel 1 : Laporan Realisasi pelaksanaan kegiatan pemungutan pajak Kota Padang Dinas Pengelolaan Keuangan dan Aset Kota Padang Periode 2010 - 2012 33 \| P a g e https://greenationpublisher.org/JGIA, Agar arus kerja dari pegawai dapat berjalan dengan lancar dan baik maka diperlukanlah penataan ruangan kantor yang baik pula. Tata ruang kantor merupakan penentuan mengenai kebutuhan-kebutuhan ruang dan tentang penggunaannya secara terinci dari ruangan tersebut untuk menyiapkan suatu susunan praktis dari faktor-faktor fisik yang dianggap perlu bagi pelaksanaan kerja perkantoran dengan biaya yang layak. Berdasarkan observasi peneliti bahwa tata ruang kantor dan kenyamanan dalam bekerja di Dinas Pengelolaan Keuangan dan Aset Kota Padang belum memiliki susunan yang baik. Seperti yang terlihat pada lampiran 8 pada gambar 1 terlihat File-file atau dokumen 34 \| P a g e https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 Tidak diletakan dimana mestinya,dokumen banyak yang menumpuk di meja. di bawah meja dan tidak tersusun dengan rapi. Gambar 2 terlihat juga banyak kertas-kertas diletakan di dalam karung dan beberapa galon yang di letakan di tepi jalan dan dibagian pojok kanan ruangan yang bersebelahan di file kabinet. Dan penyusunan file kabinetnya juga tidak teratur. gambar 3 terlihat kursi tamu kepala seksi pengawasan terletak di tengah-tengah pintu masuk Kepala bidang Pengendalian. Pada gambar 4 terlihat letak file yang jauh menyebabkan pegawai berjalan mondar-mandir,sehingga mengganggu pegawai lainnya,dan file file banyak menumpuk di atas lemari dan di bawah lemari. Pada gambar 5 terlihat bukan hanya meja yang berantakan, tapi juga adanya dapur kecil di pojokan belakang yang sangat mengganggu pemandangan orang yang akan masuk kedalam ruangan tersebut. Pada gambar 6 terlihat Letak lemari dan file-file berserakan sehingga memperlambat pegawai untuk menemukan lagi file yang di perlukan dan kecepatan pegawai dalam bekerja sangat lambat. Setelah peneliti melakukan pengamatan pada tiap-tiap bagian di Dinas Pengelolaan Keuangan dan Aset Kota Padang. disini terlihat pada masing-masing bagian hampir terdapat masalah yang sama pada penataan ruangan kantor, dan tidak dapat dipungkiri lagi bahwa penataan ruangan kantor menjadi begitu penting dalam meningkatkan kinerja pegawai guna mencapai prestasi organisasi dan meningkatkan hasil kerja. Adapun sikap dan perilaku pegawai yang juga menunjukan mengapa target realisasi pelaksanaan kegiatan selalu tidak sesuai dengan realisasi kegiatan yang dilaksanakan setiap tahunnya,yaitu adanya pegawai yang sering tidak masuk kerja. https://greenationpublisher.org/JGIA, Hal ini dapat dilihat dari angka kemangkiran pegawai Dinas Pengelolaan Keuangan dan Aset Kota Padang yang dapat dilihat pada tabel 2 dibawah ini : Tabel 2 : Absensi pegawai Dinas Pengelolaan Keuangan dan Aset Kota Padang Bulan Jumlah Pegawai Hari kerja Jumlah absen Januari 149 21 14 Februari 149 20 13 Maret 149 19 4 April 149 22 10 Mei 149 22 10 Juni 149 19 8 Sumber : Dinas Pengelolaan Keuangan dan Aset Kota Padang Berdasarkan tabel 2 diatas dapat kita lihat bahwa motivasi kerja pegawai di Dinas Pengelolaan Keuangan dan Aset Kota Padang masih rendah. Disini terlihat masih banyak pegawai yang tidak hadir pada hari kerja. Oleh karena itu Pimpinan harus bisa berusaha meningkatkan Motivasi kerja pegawai hingga dapat meningkatkan kinerja sesuai dengan target realisasi. g Menurut Hasibuan (2006:141) salah satu faktor penting dalam kinerja itu adalah Motivasi,karena motivasi adalah hal yang menyebabkan, menyalurkan dan mendukung perilaku manusia supaya mau bekerja giat dan antusias mencapai hasil yang optimal. Oleh karena itu seorang pemimpin seharusnya mampu memberikan motivasi kepada karyawannya. untuk kemajuan organisasi, agar organisasi yang dijalankan mampu bersaing dengan perusahaan lain. Motivasi semakin penting karena pimpinan membagikan pekerjaan pada bawahannya untuk dikerjakan dengan baik dan terintegrasi kepada tujuan yang diinginkan. Kepemimpinan Kepemimpinan sangat erat berkaitan dengan kemampuan seseorang untuk mempengaruhi sekelompok orang yang menjadi anggota organisasi untuk mencapai tujuan organisasinya. Beberapa pengertian yang dikemukakan para ahli tentang gaya kepemimpinan diantaranya. y Menurut Terry dalam Thoha (2003:5) merumuskan kepemimpinan itu adalah aktivitas untuk mempengaruhi orang-orang supaya diarahkan mencapai tujuan organisasi. Sedangkan kepemimpinan menurut Hasibuan (2006:169) adalah seseorang yang mempergunakan wewenang dan kepemimpinannya, mengarahkan bawahan untuk mengerjakan sebagian pekerjaannya dalam pencapaian tujuan organisasi. Tata Ruang Kantor Menurut Gie,T.Liang (2000:186) mengemukakan bahwa tata ruang kantor adalah :“Tata ruang kantor dapat dirumuskan sebagai penyusunan perabot dan alat-alat perlengkapan pada luas lantai yang tersedia. Tata ruang kantor merupakan penyusunan alat-alat kantor pada letak yang tepat serta pengaturan tempat kerja dan faktor yang menimbulkan kepuasan dalam bekerja, arus pekerjaan menjadi lancar, serta keteraturan yang ada disekitar pegawai. Tata ruang kantor dapat juga sebagai pengaturan penggunaan ruangan untuk menyiapkan suatu susunan yang praktis dari faktor fisik yang dianggap perlu bagi pelaksanaan kerja kantor dengan biaya yang layak”. Pendapat lain dikemukakan oleh Nitisemito (1982:183) bahwa tata ruang kantor merupakan suatu kegiatan yang mengatur segala sesuatu yang ada disekitar para pekerja dan yang dapat mempengaruhi dirinya dalam menjalankan tugas yang dibebankan. Dengan kata lain, arti tata ruang kantor dapat pula diutarakan sebagai pengaturan dan penyusunan seluruh mesin kantor, alat perlengkapan kantor serta perabot kantor pada tempat yang tepat, sehingga pegawai dapat bekerja dengan baik, nyaman, leluasa dan bebas untuk bergerak sehingga tercapai efisiensi kerja. Kinerja Menurut Schermerhorn, Hunt dan Osborn dalam Rivai (2005:15), kinerja merupakan kualitas dari pencapaian tugas-tugas, baik yang dilakukan oleh individu, kelompok maupun 35 \| P a g e https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 perusahaan. Menurut Stolvitch and Keeps dalam Rivai (2005:14), kinerja merupakan seperangkat hasil yang dicapai untuk menuju pada tingkatan pencapaian serta pelaksanaan suatu pekerjaan yang diminati. perusahaan. Menurut Stolvitch and Keeps dalam Rivai (2005:14), kinerja merupakan seperangkat hasil yang dicapai untuk menuju pada tingkatan pencapaian serta pelaksanaan suatu pekerjaan yang diminati. Berdasarkan pengertian diatas,maka penulis mendefiniskan kinerja pegawai sebagai sebagai suatu hasil dari apa yang telah dicapai dalam setiap pelaksanaan kegiatan yang telah dilakukan oleh pegawai selama periode waktu tertentu untuk mencapai prestasi kerja yang diinginkan oleh organisasi. Setelah seseorang diterima,ditempatkan pada suatu organisasi atau unit kerja tertentu, mereka harus dikelola agar menunjukan kinerja yang baik. Setiap pemimpin pada semua tingkat, bertanggung jawab terhadap kinerja bawahannya dan organisasi atau unit kerja yang dipimpinnya. Sejak seseorang terpilih atau diangkat mempimpin suatu organisasi kerja, tugasnya yang pertama dan utama adalah merencanakan kinerja karyawan dan organisasi yang dipimpinnya. METODE Jenis Penelitian ini difokuskan pada Pengaruh kepemimpinan, tata ruang kantor dan motivasi kerja terhadap kinerja. Penelitian ini tergolong pada jenis penelitian deskriptif asosiatif. Tempat penelitian dilakukan pada Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang, Sedangkan untuk waktu penelitian dimulai bulan Maret selama 1 bulan 2014.Populasi dalam penelitian ini adalah seluruh pegawai di DPKA sebanyak 149 orang. Penelitian kali ini menggunakan metode propotional random sampling, yang mengambil populasi untuk menjadi responden sebanyak 60.Jenis dan sumber data yang digunakan dalam penelitian ini adalah: Data primer Data Sekunder.Teknik Pengumpulan datastudi dokumentasi dan kuesioner. Instrumen Penelitian merupakan suatu alat pengumpulan data, adapun bentuk instrumen pengumpulan data primer dengan menggunakan kuesioner/angket. Kuesioner yang digunakan dalam penelitian ini disusun dengan berpatokan dari variabel yang ada, kemudian temukan indikator lalu terakhir dikembangkan menjadi item-item pernyataan, dengan menggunakan skala likert.Sebelum melakukan penelitian, terlebih dahulu dilakukan uji coba angket penelitian, yang dimaksudkan untuk menguji kesahihan (validitas) dan keterandalan (reliabilitas) instrument sebelum digunakan sebagai alat pengumpul data pada responden yang sebenarnya, dengan hasil sebagaimana berikut :Uji Validitas, setelah melakukan uji coba terhadap 30 responden maka terdapat 7 item pernyataan yang tidak valid yaitu 2 item pernyataan dari variabel X1, 2 item pernyataan dari variabel X2, 1 item pernyataan dari variabel X3 dan 2 item pernyataan dari variabel Y. Uji Reliabilitas dari hasil uji coba, seluruh variabel dinyatakan relibel dengan nilai cronbach alpha pada variabel X1 0,858, X2 0,890,X3 0,872 dan Y 0,876. Motivasi Kerja Motivasi kerja merupakan faktor psikis (jiwa) yang bersifat non-intelektual. Motivasi kerja mempunyai peran dalam menumbuhkan gairah kerja. Dengan adanya motivasi maka seseorang akan merasa senang dan bersemangat untuk bekerja. Pegawai yang memiliki motivasi kuat, akan mempunyai banyak energi untuk melakukan kegiatan kerjanya. Menurut Keller (dalam Indriani 2005:14), ada 4 kategori yang bermuatan motivasi yaitu; minat, relevansi, harapan dan kepuasan. Minat yaitu berkenaan dengan apakah keingintahuan seseorang terbangun atau tidak dan apakah semangat kerja seseorang dapat bertahan untuk jangka waktu yang diperlukan atau tidak. Kemudian, Relevansi yaitu 36 \| P a g e https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 berkenaan dengan persepsi seseorang (Pegawai) mengenai seberapa jauh kebutuhannya terpenuhi oleh aktivitas kerjanya. Harapan yaitu mengacu pada persepsinya mengenai kemungkinan untuk berhasil dan seberapa jauh hasil tersebut dapat dikontrol seseorang. Sedangkan kepuasan mengacu pada kombinasi ganjaran eksternal dan motivasi internal serta apakah semua itu sesuai dengan antisipasi yang dilakukan seseorang. Sedangkan menurut Blote (dalam Fauzan 2002:56) ada tiga aspek motivasi yaitu: affect (sikap), self concept (konsep diri) dan effort (usaha). Sikap merupakan kecenderungan seseorang untuk bertingkah laku dalam melakukan aktivitas kerja. Misalnya, kegiatan dalam melakukan pekerjaan dengan sebaik mungkin. Konsep diri merupakan kemampuan Pegawai (seseorang) dalam mengetahui kemampuannya. Konsep diri yang positif dapat terlihat misalnya jika seseorang mendapat kesulitan memecahkan suatu permasalahan maka dia tidak mau menyerah begitu saja karena dia merasa mampu untuk menyelesaikan permasalahan tersebut. Usaha, maksudnya seberapa besar upaya yang dilakukan untuk mencapai tujuan dalam bekerja. Dari pendapat beberapa ahli di atas dapat disimpulkan bahwa motivasi kerja memegang peranan penting dalam memberikan semangat, gairah dan rasa senang dalam bekerja sehingga Pegawai mempunyai kemampuan untuk mengikuti dan melaksanakan fungsinya sebagai bawahan pada organisasi. Pegawai yang memiliki motivasi kerja yang tinggi tentu akan mampu menjalankan perannya dengan baik sehingga ia mampu mencapai tujuan visi dan misi alam organisasinya. HASIL DAN PEMBAHASAN Pembahasan hasil penelitian dilakukan berdasarkan hasil analisis deskriptif dan analisis jalur yang telah diuraikan dalam bentuk deskripsi variabel penelitian serta analisis hasil penelitian tentang pengaruh Kepemimpinan (X1), Tata ruang kantor (X2) dan Motivasi 37 \| P a g e https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 kerja (X3) terhadap Kinerja (Y) pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. kerja (X3) terhadap Kinerja (Y) pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. g Pengaruh Kepemimpinan (X1) terhadap Kinerja (Y) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. garuh Kepemimpinan (X1) terhadap Kinerja (Y) di Dinas Pengelolaan Keuanga Aset Kota Padang. Hasil penelitian ini menunjukan bahwa terdapat pengaruh yang signifikan Kepemimpinan terhadap Kinerja Pegawai. Dengan Kepemimpinan yang baik akan meningkatkan kinerja pegawai begitu pula sebaliknya. Kepemimpinan yang baik seperti memberi teguran kepada karyawan apabila terlambat, memberi kesempatan kepada karyawan mengeluarkan pendapat, dan memotivasi karyawan dalam bekerja. Akan membantu pegawai untuk lebih fokus dalam menjalankan tanggung jawab yang di bebankan Pemimpin kepadanya. Hasil deskriptif penelitian memperlihatkan rata-rata skor Kepemimpinan kerja di Dinas Pengelolaan Keuangan dan Aset Kota Padang diperoleh hasil sebesar 2,24 dengan persentase tingkat capaian responden (TCR) adalah sebesar 44,9%, angka ini berada pada kriteria kurang baik. Sementara itu rata-rata skor Kinerja diperoleh hasil sebesar 2,27 dengan tingkat capaian responden (TCR) adalah sebesar 45,40%, angka ini berada pada kurang baik. g p p g p g Berdasarkan penelitian diatas kepemimpinan adalah aktivitas untuk mempengaruhi orang-orang supaya diarahkan mencapai tujuan dalam suatu perusahaan agar sesuai dengan waktu yang di tetapkan. Sesuai dengan pendapat terry dalam thoha (2003:5) yang merumuskan kepemimpinan itu adalah aktifitas untuk mempengaruhi orang-orang supaya diarahkan mencapai tujuan organisasi. Dan juga sesuai dengan pendapat hasibuan (2006:169) yang mengatakan seseorang yang mempergunakan wewenang dan kepemimpinannya, mengarahkan bawahan untuk mengerjakan sebagian pekerjaannya dalam pencapaian tujuan organisasi. Kepemimpinan yang baik akan menghasilakan kinerja yang di harapakan oleh organisasi tersebut. hasil penelitian ini sesuai dengan penelitian yang di lakukan oleh Harkemela renat (2007) tentang pengaruh kepemimpinan dan motivasi kerja terhadap kinerja pegawai di dinas perhubungan komunikasi dan informatika provinsi Sumatra barat. Dimana pengaruh kepemimpinan di Dinas Perhubungan Komunikasi dan Informatika Provinsi Sumatera Barat berpengaruh terhadap kinerja pegawai. Semakin baik kepemimpinan maka kinerja juga akan semakin baik. Dari penelitian di atas berarti kepemimpinan dan kinerja saling berpengaruh. Kepemimpinan yang baik akan menghasilkan kinerja yang baik untuk organisasinya. HASIL DAN PEMBAHASAN Yang dapat dilihat dari bagaimana seorang pemimpin dapat mempengaruhi bawahannya untuk bekerjasama menghasilkan pekerjaan yang efektif dan efisien. Pengaruh Tata ruang kantor (X2) terhadap Kinerja (Y) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Pengaruh Tata ruang kantor (X2) terhadap Kinerja (Y) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Hasil penelitian ini menunjukan bahwa terdapat pengaruh yang signifikan Tata Ruang Kantor terhadap Kinerja Pegawai. Dengan tata ruang kantor yang baik akan meningkatkan kinerja pegawai dan begitu pula sebaliknya. Fasilitas yang tersedia seperti perabot-perabot kantor disusun atau ditata sesuai dengan yang dibutuhkan sehingga tidak mengganggu kelancaran pekerjaan pegawai,pencahayaan atau penerangan yang cukup dalam jumlah dan mutu yang tepat dan dalam tempat yang tepat pula, peredaraan udara yang cukup dan bersih dalam ruangan kerja, alat-alat kantor yang tersusun dengan rapi ,menciptakan suasana kerja yang nyaman bagi pegawai,sehingga kinerja pegawaipun dapat ditingkatkan. Hasil deskriptif penelitian memperlihatkan rata-rata skor Tata ruang kantor di Dinas Pengelolaan Keuangan dan Aset Kota Padang diperoleh hasil sebesar 2,386 dengan persentase tingkat capaian responden (TCR) adalah sebesar 47,738%, angka ini berada pada 38 \| P a g e https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 kriteria kurang baik. Sementara itu rata-rata skor Kinerja diperoleh hasil sebesar 2,27 dengan tingkat capaian responden (TCR) adalah sebesar 45,40%, angka ini berada pada kurang baik. kriteria kurang baik. Sementara itu rata-rata skor Kinerja diperoleh hasil sebesar 2,27 dengan tingkat capaian responden (TCR) adalah sebesar 45,40%, angka ini berada pada kurang baik. Tata ruang kantor sangat perpengatuh terhadap kelancaran aktifitas suatu pekerjaan,membuat pegawai yang bekerja dalam suatu perusahaan nyaman dalam setiap tanggung jawab yang di berikan kepadanya, dengan penataan tata ruang kantor yang baik, pegawai akan menyelesaikan kerjanya sesuai dengan ketentuan yang telah di tetapakan seperti yang dikatakan Nitisemito (1982:183) tentang “tata ruang kantor merupakan suatu kegiatan yang mengatur segala sesuatu yang ada disekitar para pekerja dan yang dapat mempengaruhi dirinya dalam menjalankan tugas-tugas yang dibebankan. Oleh sebab itu tata ruang kantor merupakan salah satu faktor yang juga dapat mempengaruhi kinerja pegawai, hal ini disebabkan karena pegawai senang bekerja pada ruang yang ditata dengan baik Penataan ruangan yang membuat pegawai nyaman seperti jarak meja masing-masing karyawan, sirkulasi udara di ruangan, warna ruangan dan pengedapan suara pada ruangan tersebut, yang membuat nyaman pegawai dalam menyelesaikan pekerjaannya. Seperti yang di jelaskan Gie,T.Liang (2000) yang mengatakan ada beberapa azas dalam tata ruang kantor, diantaranya :1). Azas jarak terpendek 2). Azas rangkaian kerja 3). Azas segenap penggunaan segenap ruangan dan 4). Azas perubahan susunan tempat kerja. Adapun faktor-faktor yang mempenharuhi tata ruang kantor dalam gie,T.Liang (2000) diantaranya : 1). Warna 2). Tata cahaya 3). Pengaturan udara dan 4). Suara. Pengaruh Tata ruang kantor (X2) terhadap Kinerja (Y) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Penataan ruang kantor yang baik akan mengasilkan kinerja yang baik pula pada perusahaan. hasil penelitian ini sesuai dengan penelitian yang dilakukan oleh Rilnawati eka (2011) tentang pengaruh tata ruang kantor terhadap kinerja pegawai pada lembaga permasyarakatan terbuka pasaman. Yang menunjukan bahwa terdapat pengaruh yang signifikan antara pengaruh tata ruang kantor terhadap kinerja pegawai dilembaga permasyarakatan terbuka pasaman. Dari pendapat diatas terlihat pentingnya tata ruang yang baik pada suatu kantor. Kurang baiknya tata ruang kantor juga akan berakibat pada rendahnya kinerja pegawai. Pengaruh Motivasi Kerja (X3) terhadap Kinerja (Y) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Pengaruh Motivasi Kerja (X3) terhadap Kinerja (Y) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Hasil penelitian ini menunjukan bahwa terdapat pengaruh signifikan Motivasi terhadap Kinerja pegawai. Dengan Memberikan motivasi kepada karyawan suatu perusahaan atau organisasi akan mencapai tujuan perusahaan tersebut. Dan begitu pula sebaliknya, apabila karyawan atau pegawai tidak termotivasi dalam melakukan pekerjaannya maka suatu perusahaan atau organisasi itu tidak akan mencapai tujuan yang telah di tetapkan tadi. Seperti yang kita ketahui Motivasi merupakan sebuah keahlian dalam mengarahkan karyawan pada tujuan organisasi agar mau bekerja dan berusaha sehingga keinginan para karyawan dan tujuan organisasi dapat tercapai. Motivasi seseorang melakukan suatu pekerjaan karena adanya suatu kebutuhan hidup yang harus dipenuhi. Kebutuhan ini dapat berupa kebutuhan ekonomis yaitu untuk memperoleh uang, sedangkan kebutuhan nonekonomis dapat diartikan sebagai kebutuhan untukmemperoleh penghargaan dan keinginan lebih maju. Dengan segala kebutuhan tersebut, seseorang dituntut untuk lebih giat dan aktif dalam bekerja, untuk mencapai hal ini diperlukan adanya motivasi dalam melakukan pekerjaan, karena dapat mendorong seseorang bekerja dan selalu berkeinginan untuk melanjutkan usahanya. Oleh karena itu jika pegawai yang mempunyai motivasi kerja yang tinggi biasanya mempunyai kinerja yang tinggi pula. Hasil deskriptif penelitian memperlihatkan rata-rata skor Motivasi di Dinas Pengelolaan Keuangan dan Aset Kota Padang diperoleh hasil sebesar 2,585 dengan persentase tingkat capaian responden (TCR) adalah sebesar 51,85%, angka ini berada pada 39 \| P a g e https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 kriteria kurang baik. Sementara itu rata-rata skor Kinerja diperoleh hasil sebesar 2,27 dengan tingkat capaian responden (TCR) adalah sebesar 45,40%, angka ini berada pada kurang baik. kriteria kurang baik. Sementara itu rata-rata skor Kinerja diperoleh hasil sebesar 2,27 dengan tingkat capaian responden (TCR) adalah sebesar 45,40%, angka ini berada pada kurang baik. g p p g p g Salah satu faktor yang mempengaruhi hasil kinerja suatu organisasi itu adalah faktor motivasi, motivasi berfungsi sebagai memberikan dorongan seseorang untuk melakukan suatu pekerjaan untuk mencapai tujuan yang telah ditetapkan oleh organisasi itu sendri seperti yang dikemukakan oleh Suharto dan Cahyono (2005) dan Hakim (2006) menyebutkan ada salah satu faktor yang mempengaruhi kinerja yaitu faktor motivasi, dimana motivasi merupakan kondisi yang menggerakan seseorang berusaha untuk mencapai tujuan atau mencapai hasil yang diinginkan.dan Rivai (2004) juga menunjukan bahwa semakin kuat motivasi kerja, kinerja pegawai akan semakin tinggi. Hal ini berarti bahwa setiap peningkatan motivasi kerja pegawai akan memberikan peningkatan yang sangat berarti bagi peningkatan kinerja pegawai dalam melaksanakan pekerjaannya. Jadi, hubungan antara motivasi dengan kinerja adalah Motivasi berpengaruh positif terhadap kinerja karyawan. Pengaruh Motivasi Kerja (X3) terhadap Kinerja (Y) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Hasil kinerja pegawai tidak lepas dari motivasi pegawai itu sendiri. Termotivasinya pegawai dalam melakukan pekerjaanya akan memberikan hasil kinerja yang di inginkan dalam organisasi tersebut. Hasil penelitian ini sesuai dengan penelitian yang dilakukan oleh Septia Reni (2007) tentang pengaruh tata ruang kantor dan motivasi kerja terhadap kinerja pegawai di dinas perhubungan komunikasi dan informatika provinsi sumatra barat. Yang menunjukan bahwa terdapat pengaruh yang signifikan antara pengaruh motivasi terhadap kinerja pegawai di dinas perhubungan komunikasi dan informatika provinsi Sumatra barat. Dari hasil penelitian diatas berarti Motivasi terhadap Kinerja dapat meningkatkan hasil yang telah di tetapkan pada perusahaan atau organisasi tersebut. Semakin baik motivasi yang diberikan pimpinan kepada pegawai maka semakin tinggi kinerja yang dihasilkan pegawai tersebut untuk perusahaan itu sendiri. https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 kompleks melaksanakan fungsi konstruktif atau perubahan adaptif melalui tiga subproses. Kepemimpinan yang efektif akan meningkatkan motivasi kerja pegawai dalam melaksanakan tugasnya. Demikian sebaliknya, kepemimpinan yang kurang efektif akan mengakibatkan berkurangnya motivasi kerja pegawai dalam bekerja. kompleks melaksanakan fungsi konstruktif atau perubahan adaptif melalui tiga subproses. Kepemimpinan yang efektif akan meningkatkan motivasi kerja pegawai dalam melaksanakan tugasnya. Demikian sebaliknya, kepemimpinan yang kurang efektif akan mengakibatkan berkurangnya motivasi kerja pegawai dalam bekerja. Kepemimpinan dalam suatu organisasi perpengaruh terhadap motivasi kerja, apa bila seorang pemimpin mampu memberikan motivasi kepada pegawai dengan baik, maka pegawai akan termotivasi untuk melakukan setiap tanggung jawab pekerjaanya dengan baik dan benar. Dan hasil peneilitan ini sesuai dengan penelitian yang di lakukan oleh Harkemela renat (2007) tentang pengaruh kepemimpinan dan motivasi kerja terhadap kinerja pegawai di dinas perhubungan komunikasi dan informatika provinsi Sumatra barat. Dimana pengaruh kepemimpinan di Dinas Perhubungan Komunikasi dan Informatika Provinsi Sumatera Barat berpengaruh terhadap motivasi kerja pegawai. Semakin baik kepemimpinan maka motivasi kerja pegawai juga akan semakin baik. j p g j g Hal ini dapat disimpulkan bahwa kepemimpinan berpengaruh terhadap motivasi kerja. Semakin baik kepemimpinan maka motivasi kerja juga akan semakin baik. Pengaruh Tata ruang kantor (X2) terhadap Motivasi Kerja (X3) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. il li i i i j k b h d h i ifik Pengaruh Kepemimpinan (X1) terhadap Motivasi Kerja (X3) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Pengaruh Kepemimpinan (X1) terhadap Motivasi Kerja (X3) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Hasil penelitian ini menunjukan bahwa terdapat pengaruh yang signifikan Kepemimpinan terhadap Motivasi Kerja. Dengan Kepemimpinan yang baik karyawan akan termotivasi untuk melakukan tugasnya dengan baik dalam suatu perusahaan dan akan menjngkatkan kinerja perusahan itu sendri. Hasil deskriptif penelitian memperlihatkan rata-rata skor motivasi kerja di Dinas Pengelolaan Keuangan dan Aset Kota padang diperoleh hasil sebesar 2,585 dengan persentase tingkat capaian responden (TCR) adalah sebesar 51,85%, angka ini berada pada kriteria kurang baik. Sementara itu rata-rata skor kepemimpinan di Dinas Pengelolaan Keuangan dan Aset Kota Padang diperoleh hasil sebesar 2,24 dengan tingkat capaian responden (TCR) adalah sebesar 44,9%, angka ini berada pada kriteria kurang baik. Seorang pemimpin harus mampu memotivasi pegawainya untuk mencapai tujuan yang di harapkan pada suatu organisasi tersebut. Dan suatu organisasi tanpa pemimpin pasti mengalami kekacauan, ketidak teraturan dan tidak mempunyai tujuan untuk membuat organisasi itu maju dan berkembang seperti yang di jelas Danim (2004:14) mengemukakan bayak faktor yang mempengaruhi motivasi kerja seseorang diantaranya iklim kerja, kepemimpinan, insentif dan persaingan positif. Sedangkan Keith Davis dalam Danim (2004:18) mengatakan bahwa tanpa kepemimpinan, organisasi hanya merupakan kelompok manusia yang kacau, tidak teratur, dan tidak akan dapat melahirkan perilaku tujuan. Dari hasil diatas bahwa pengaruh kepemimpinan terhadap motivasi kerja adalah positif. Ini dapat diartikan jika terjadi kenaikan koefisien skor kepemimpinan akan mengakibatkan motivasi kerja. Sebaliknya, jika terjadi penurunan skor kepemimpinan juga akan membawa penurunan motivasi kerja pegawai. Hal ini sesuai dengan pendapat Kotler dalam Tika (2006:89) yang menyatakan bahwa kepemimpinan dalam suatu organisasi yang 40 \| P a g e https://greenationpublisher.org/JGIA, KESIMPULAN Berdasarkan hasil Penelitian dan Pembahasan maka dapat ditarik Kesimpulan sebagai berikut : 1. Kepemimpinan berpengaruh positif dan signifikan terhadap Kinerja Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. Semakin baik kepemimpinan maka akan dapat meningkatkan kinerja pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. 1. Kepemimpinan berpengaruh positif dan signifikan terhadap Kinerja Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. Semakin baik kepemimpinan maka akan dapat meningkatkan kinerja pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. 2. Tata ruang kantor berpengaruh positif dan singnifikan terhadap Kinerja Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. Semakin baik penataan ruangan Kantor maka akan semakin meningkat kinerja Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. 3. Kepemimpinan berpengaruh positif dan signifikan terhadap Motivasi Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. Semakin baik kepemimpinan maka akan dapat memotivasi pegawai untuk bekerja lebik baik lagi. 3. Kepemimpinan berpengaruh positif dan signifikan terhadap Motivasi Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. Semakin baik kepemimpinan maka akan dapat memotivasi pegawai untuk bekerja lebik baik lagi. 4. Tata ruang kantor berpengaruh positif dan singnifikan terhadap Motivasi Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. Semakin baik penataan ruangan Kantor maka akan semakin meningkat Motivasi pegawai bekerja lebih baik lagi karna sudah mendapatkan kenyamanan pada saat bekerja. 4. Tata ruang kantor berpengaruh positif dan singnifikan terhadap Motivasi Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. Semakin baik penataan ruangan Kantor maka akan semakin meningkat Motivasi pegawai bekerja lebih baik lagi karna sudah mendapatkan kenyamanan pada saat bekerja. 5. Motivasi Kerja berpengaruh positif dan signifikan terhadap Kinerja Pegawai di Dinas Pengelolaan Keuangan dan Aset (DPKA) Kota Padang. Semakin baik Motivasi yang diberikan kepada pegawai, akan meningkatkan Kinerja Pegawai itu sendiri dalam menjalankan tugas-tugas yang diberikan. https://greenationpublisher.org/JGIA, Vol. 1, No. 1, Februari. 2023 pegawainya untuk bekerja dan menyelesaikan pekerjaan yang di berikan kepadanya. Karna segala sesuatu yang membuat seorang termotivasi itu salah satunya kenyaman dalam lingkungan kerja itu sendiri. pegawainya untuk bekerja dan menyelesaikan pekerjaan yang di berikan kepadanya. Karna segala sesuatu yang membuat seorang termotivasi itu salah satunya kenyaman dalam lingkungan kerja itu sendiri. Pengaruh Tata ruang kantor (X2) terhadap Motivasi Kerja (X3) di Dinas Pengelolaan Keuangan dan Aset Kota Padang. Hasil penelitian ini menunjukan bahwa terdapat pengaruh yang signifikan Tata Ruang Kantor terhadap Motivasi Kerja. Dengan penataan Tata Ruang Kantor yang baik akan meningkatkan Motivasi kerja karyawan untuk bekerja lebih baik lagi dan begitu pula sebaliknya, Apabila penataan Tata Ruang Kantornya tidak baik maka karyawan tidak akan termotivasi untuk bekerja dan menyelesaikan pekerjaan yang di tanggung jawabkan kepadanya. Hasil deskriptif penelitian memperlihatkan rata-rata skor motivasi kerja di Dinas Pengelolaan Keuangan dan Aset Kota Padang diperoleh hasil sebesar 2,585 dengan persentase tingkat capaian responden (TCR) adalah sebesar 51,85%, angka ini berada pada kriteria kurang baik. Sementara itu rata-rata skor Tata ruang kantor diperoleh hasil sebesar 2,386 dengan tingkat capaian responden (TCR) adalah sebesar 47,738%, angka ini berada pada kurang baik. Penataan ruang kantor yang baik akan menghasilkan motivasi kerja yang baik untuk pegawainya, dengan mendapatkan kenyamanan bekerja apapun yang dikerjakan akan terasa ringan.seperti yang di kemukakan hasibuan (2003:163) menyatakan seseorang mempunyai kebutuhan yang berhubungan dengan tempat dan suasana lingkungan tempat ia bekerja yang terdiri dari lingkungan fisik dan lingkungan sosial atau non fisik. Dimana lingkungan kerja yang baik akan membangun motivasi pegawai dan dengan adanya motivasi ini maka pekerjaan yang dilakukan akan terlaksana dengan baik. Dan pendapat Herrera dalam Djumadi (2006) bahwa lingkungan tempat kerja dan budaya kerja yang baik akan memotivasi karyawan untuk meningkatkan kinerja. Berpengaruhnya tata ruang kantor terhadap motivasi kerja pegawai sangat membantu suatu organisasi dalam pencapaian hasil kerja yang telah di tetapkan pada organisasi tersebut.Dan hasil penelitian ini sesuai dengan penelitian yang dilakukan oleh Septia Reni (2007) tentang pengaruh tata ruang kantor dan motivasi kerja terhadap kinerja pegawai di dinas perhubungan komunikasi dan informatika provinsi sumatra barat. Yang menunjukan bahwa terdapat pengaruh yang signifikan antara pengaruh tata ruang kantor terhadap motivasi pegawai di dinas perhubungan komunikasi dan informatika provinsi Sumatra barat. Dari hasil penelitian diatas berarti Tata Ruang Kantor dan Motivasi dapat meningkatkan kinerja pegawai. Semakin baik Tata Ruang ruang kantor pada suatu perusahaan atau organisasi, akan memberikan kenyaman kepada pegawai itu sendiri untuk bekerja. Dengan penataan Tata Ruang Kantor yang baik juga akan meningkatkan motivasi 41 \| P a g e https://greenationpublisher.org/JGIA, REFERENSI Akirmen. 2008. Statistik 1. Padang: Universitas Negeri Padang Arikunto, Suharsimi. 2002. Prosedur Penelitian Suatu pendekatan Praktek. Jakarta: PT. Rineka Cipta. Danim, Sudarman. 2004. Motivasi, Kepemimpinan, dan Efektifitas kelompok. Jakarta: PT. Rineke Cipta. Djumadi. 2010. Pengaruh Kondisi kerja dan Kepuasan Kerja terhadap Kinerja Karyawan Lembaga Pendidikan Nonformal. Fanti, Nike Yuri. 2005. Dasar – Dasar Manajemen. Bandung: Rosda Karya. Fauzan, Ahmad (2002). Pengaruh Motivasi Terhadap Hasil Belajar Geografi Di SMP N 3 Padang. Padang: FIS Padang. g g Griffin, Ricky W. & Ebert, Ronald. J. 2002. Bisnis. Jakarta : Prenhallindo. Hasibuan, Melayu. 2006. Manajemen Personalia dan sumber daya manusia.CV.Haji masingging . 2007. Manajemen personalia dan Sumber daya manusia. CV. Haji masingging. Hasibuan, Melayu. 2006. 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Manajemen SDM untuk perusahaan dari Teori ke Praktek. Jakarta: PT. raja Grafindo Persada Rivai, veithzal. 2005. Manajemen Sumber daya manusia untuk perusahaan. Jakar Raja Grafindo Persada. Rivai, veithzal. 2006. Kepemimpinan dan Perilaku Organisasi. Jakarta : PT. Raja Grafindo Persada. Robbins, Stephen. 2002. Prinsip-prinsip Perilaku Organisasi. Edisi Kelima. Jakarta : Erlangga. Sadirman (2004). Interaksi dan Motivasi Belajar Mengajar. Jakarta: PT. Raja Grafindo Persada Sedarmayanti. 2009. Sumber Daya Manusia dan Produktifitas Kerja. Bandung : CV.Mandar Maju Siagian, S.P. 2003. Manajemen Sumber Daya Manusia. Jakarta: Bumi Aksara. Solikhin (2006). Analisis Pengaruh Motivasi Kerja Terhadap Kinerja Pegawai Negeri Sipil di Badan Kepegawaian Daerah Kabupaten Tegal. Tegal: Kantor BKD. http://elibrary.mb.ipb.ac.id.gdl.php 3 Sepetember 2013 p y p g p p p Suharto dan Cahyo. 2005. Pengaruh Budaya Organisasi, Kepemimpinan Dan Motivasi Terhadap Kinerja Sumber Daya Manusia di Sekretarian DPRD Provinsi Jawa Tengah. JRBI. Vol 1. No 1. Hal 13-30. Suharto dan Cahyo. 2005. REFERENSI Pengaruh Budaya Organisasi, Kepemimpinan Dan Motivasi Terhadap Kinerja Sumber Daya Manusia di Sekretarian DPRD Provinsi Jawa Tengah. JRBI. Vol 1. No 1. Hal 13-30. Sugiyono. 2009. Metode Penelitian Administrasi. Bandung: Alfabeta. The liang gie. 2000. Administrasi Perkantoran Modern. Yogyakarta: Super Sukses. Thoha, Miftah. 2003. Kepemimpian Dalam Manajemen. Jakarta: PT. Grafindo Persada. Timpe, A Dale. 2000. Kinerja. Jakarta: Gramedia Umar, Husen.2005. Metodologi Penelitian. Jakarta : Rineka Cipta. Purwanto, Ngalim (2004). Psikologi Pendidikan. Jakarta: PT. Remaja Rosdakarya Wahjosumidjo. 1992. Kepemimpinan dan Motivasi Kerja. Jakarta: Sinar Harapan. . 1994. Kepemimpinan Dan Motivasi. Jakarta: Graha Indonesia. Winardi. 2001. Memotivasi dan pemotivasian dalam manajemen. Jakarta: PT. Raja Grafindo Persada. Suharto dan Cahyo. 2005. Pengaruh Budaya Organisasi, Kepemimpinan Dan Motivasi Terhadap Kinerja Sumber Daya Manusia di Sekretarian DPRD Provinsi Jawa Tengah. JRBI. Vol 1. No 1. Hal 13-30. Sugiyono. 2009. Metode Penelitian Administrasi. Bandung: Alfabeta. The liang gie. 2000. Administrasi Perkantoran Modern. Yogyakarta: Super Sukses. Thoha, Miftah. 2003. Kepemimpian Dalam Manajemen. Jakarta: PT. Grafindo Persada. Timpe, A Dale. 2000. Kinerja. Jakarta: Gramedia Umar, Husen.2005. Metodologi Penelitian. Jakarta : Rineka Cipta. Purwanto, Ngalim (2004). Psikologi Pendidikan. Jakarta: PT. Remaja Rosdakarya Wahjosumidjo. 1992. Kepemimpinan dan Motivasi Kerja. Jakarta: Sinar Harapan. . 1994. Kepemimpinan Dan Motivasi. Jakarta: Graha Indonesia. Winardi. 2001. Memotivasi dan pemotivasian dalam manajemen. Jakarta: PT. Raja Grafindo Persada. g y g The liang gie. 2000. Administrasi Perkantoran Modern. Yogyakarta: Super Sukses. hoha, Miftah. 2003. Kepemimpian Dalam Manajemen. 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https://openalex.org/W2315939579	https://zenodo.org/records/1675137/files/article.pdf	English	null	LIII.—<i>On the South-African</i> Curculionidæ <i>of the Genus</i> Cossonus, <i>Clairv</i>	Annals & magazine of natural history	1,905	public-domain	6,564	On: 01 February 2015, At: 16:17 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, On: 01 February 2015, At: 16:17 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, To cite this article: Guy A. K. Marshall F.Z.S. (1905) LIII.—On the South-African Curculionidæ of the Genus Cossonus, Clairv , Annals and Magazine of Natural History: Series 7, 15:88, 392-405, DOI: 10.1080/03745480509443068 To link to this article: http://dx.doi.org/10.1080/03745480509443068 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions 392 3Ir. G. A. K. 3lnrsliall 011 thc fl:inks, whilc thc lower segment of tlic limb3 is iienrly wliitc. Tlte tail, too, is shorter and wliitc below, instcad of ntiiformly dusky. y It is tltiis obvious that in respect of colour tlm Bonin deer comes niiicli nearer to tlic Formosan than it does to tlic dusky Pliilippiiie sarnhnr (I liavc not R spccitticn of tlic larger Luzon samb;rr .with n4iicli to coinpnrc it). Accordingly, for all I ran say to the contrary, it rnay bc nii itiiportcd brccd of tlic Formosan sarnbnr, dwarfed by tlio smn\l area of its habitat. 13ut as therc is it0 evitlencc of this, it may bc provisioiinlly rcgarded afi B distinct island race under the nnrnc of Ceruus (Ritsa) uniwlor loniriensis, with tho female skull in the llritisl~ bluscuni as the type. Downloaded by [University of Calgary] at 16:17 01 February 2015 LIII.-On the S o d d f r i c a t a Curculionidm of the Genus Cossonus, Ckiiru. By GUY A. K. ~IAIWIIALL, F.Z.S. THE genus Cossonus has practically a yorld-widc distribution, but, as at present known, is Lcttcr represetrted in Anicrica tlian i n \ylicre clse. Ody ciglit species liavc liitlicrto beon recorde d froin Southern Africa, and six niore arc described in titc present pnpr. But even with this .incrcnso it seems probable that tlie numbrr of Soutli-Africnti species niiglit readily be doubled wlioii tlic nrcn is iiiore thorouglily workcd. Tltc timber-belts of tlrc 'l'rnnskci and Pondolnnd, the yellor- wood forests of Natal, tltc ltenvily wooded regions wliicli edjoiii tlie eastern littoral from Zuliilaittl tiortIi\sni.d into tlic tropics, arc all practically virgin ground for tlic colcopterist, and when adcquatcly csplored will doiibtlcss yield ninny iicw forms. 1 ltesc irtsects seem, as B rule, to bc but poorly ropresctitcd in collectioiis, for unless specially searched for tltcy arc not likely to be niet with by tlrc casual collector. A11 our species kith whose habits I am acquaiiited livc under the bark of decajitig trces ; but tlicy scciii to require dinip surroundings, niid thus in tho drier parts of tlic country thoy arc only to bo ibtind in trees wlticli are subject to moist decay, such as Eupliorbins niid n fow soft-\vooded spcies like tlic Erxtlirinas. In t l k c lnttcr I linve only found them witen tlie trecs wcre being killed by tlic nttncks of certain Longicorn or hiprestid larva., tlie Cossoni bciiig usually found in tlic moist drcnying nl;\tter left in tlic tracks of tliesc larux just beneath tlic bark. Jj'itli tlieiii also ocenl- n few otltcr Curculionidx, sucli ns ' I 303 South-iIJricnii Species of Co- SSOllUJ. Coymiiieriis, P?t?ct-opAnpis, &., ns wclI as various Nitidulich, Stnlhyliiiitlrc, aiid some dipterous Inrvm. Tiicsc in turn attract tlic predaceous IIist erdm of such gcncra as Ilololepfa, Yachjcrarus, Placodes, niid Kpicchinus, wliicli doultlcsa prey on tho Cossoiii 3s well ns o n tlic otlicr iiijecta. Coymiiieriis, P?t?ct-opAnpis, &., ns wclI as various Nitidulich, Stnlhyliiiitlrc, aiid some dipterous Inrvm. Tiicsc in turn attract tlic predaceous IIist erdm of such gcncra as Ilololepfa, Yachjcrarus, Placodes, niid Kpicchinus, wliicli doultlcsa prey on tho Cossoiii 3s well ns o n tlic otlicr iiijecta. p y j It will probably lo found that iiiost of tlic specie3 liavc n fairly wide distribution. Syriopfic Key for the S o d - A fricarr Species of Co-. >>011113. Syriopfic Key for the S o d - A fricarr Species of Co-. >>011113. 1. (24.) Roatruiu mare or leas strongly dilated in tlic n y i d M f . 2. (7.) Intervnla on clytra bcnriig rows of punctured. 3. (4.) Protliorns vcr deeply inciscd ncnr npes, bipartite ; t i c interval3 near the su- turc ccirinate, much narroner tlrair the slri:c ........................ (3.) l’rotliorns simply conetrictcd n e x nprs, not inciecd; tlic interviile nenr the siituru plnne, IW broad 113 tlrc stria. (G.) Protlar.ar willout auy discal iniprca- siui:a or n central carina ; roatruur very gradually dilatcd from b s e to apes . . (6.) l’rothornx with n distinct cariiia nnd n loiigitudinal basal iniprejriori on each side of it ; rostrum suddeuly nnd rery strongly dilated nt npcs ............ 7. (2.) Intervald on e l p inrpunctntc. [l:ALr. 8. (21.) l’rotliorilx distiirctly iuryrcered or cari- nnte on the disk. 9. (20.) Rostrum without nny ccntrnl furron nbovc. 10. (10.) I3 tra with only Ocouipleto shin! and n siort sub1iuiiir.ral olio ; thc centnil iui- presrion 0x1 pratliomx riot iiiterrupted 1. C. nbscissur, Boll. 4. 5. 2. C, Shrppard:, sp. n. 0. 3. C. cariicuUis, finclx iind sp.ir:ely puirctured. 12. (13.) Protkornz sulpyriforni, the rides gradu- nlly dilated from npcs to Lo~oud iiiidd!c ; uictnsternuui cvrnly but sparsely piiiictiircd ................ 13. (19.) l’~11t11uras subqudmte, thc dcsrupidly diliited frour apes to bduru riiiddlc, t11113 formin;: 11 distinct slioulilct; ;uetnsturiium with n large ccntral iin- punctate nrca .................... 5. C! iiicicilis, IGhr. 4. C d r i c w i i t s , ndi. Awii LL’ Ifuy rV Ilisf Ser 7 JW XY 27 1. (24.) Roatruiu mare or leas strongly dilated in tlic n y i d M f . y 2. (7.) Intervnla on clytra bcnriig rows of punctured. p 3. (4.) Protliorns vcr deeply inciscd ncnr npes, bipartite ; t i c interval3 near the su- turc ccirinate, much narroner tlrair the slri:c ........................ (3.) l’rotliorns simply conetrictcd n e x nprs, not inciecd; tlic interviile nenr the siituru plnne, IW broad 113 tlrc stria. 4. p , (G.) Protlar.ar willout auy discal iniprca- siui:a or n central carina ; roatruur very gradually dilatcd from b s e to apes . . 5. Downloaded by [University of Calgary] at 16:17 01 February 2015 This is certainly tlic C:HC with C! nbscissiis, 13011., rvliicli rirngcs from Algon lhy 10 tlic Equator ntid acro~s tlic continent to Ciitncrliir. C. sulitmlis, Boh., lias also been foand Loth iii Natal niid OIL the Coiigo, but lias not liitlierto Lccii rccorded from any iiitcriiiedintc localities. oaded by [University of Calgary] at 16:17 01 February 2015 1. C. nbscissur, Boll. 0. C. ImnarauknsiJ, [sp. n. 394 14. (11.) Pfothorns with tho sniootlr discs1 nreas irnponctnte. 14. (11.) Pfothorns with tho sniootlr discs1 nreas irnponctnte. p 16. (18.) Fnrehend with n distinct centrnl fovca ; centrnl cnrinn ofprothorax vergnnrrow, obsolcto or incourplcte. p 16. (17.) Forrhend with n shallow trnnsversc im- iression; thoracic imprec' -ion nmroiwr, i e e c'r, more rbnrply V-shpcd, nud mitiorit n eentrnl cnrina ; elyin blnck. ; y 17. (la.) Forehend without n tmuversr inrprcs- sion ; thoracic iNprcaJio11 lrrnnder nnd ehdloscr, and with on irrrgulrr ccn- tral earinu ; elgtrn ferruginous brown, witli tbe suture and lntenl margin3 blnck ............................ 7. C. autrcmlis,Doh. 18. (16.) Forelend with no cmtml forcn; cen- tral carin3 of prothorax complete, trond nod impunctnte.. ............ 8. C. IlJnldn, sp. 11. 19. (10.) EIjtm with 10 coniplrtr: strim ; tho ccn- trnl iniprrssiori on pothorns trnna- p verscly ruterrupted nbout middle .... [sp. n. [ p 10. C'. c h p t u l u s , F. I). C. chirinclrmis, 20. (0.) Iiostrunr n-ith n central furrow at b w o . y p ( ) 21. (8.) l'rothorns plnno nnd mootli, n-ithout n carinn or iiny distinct inipreesions; body irrucli depressed. y p 22. (23.) Rostrum lonncr thnu hcad and only sliglitl dieted; hcad short, sob- couiraI1'; el trn witlr 9 E~inl~orn stris, the intervnfs being broad and planc . . g p g , 23. (22.) Rostrum shorter tlinn hcnd and broadly dihtud ; head e16ng~tc~ siibpundmte ; eljtra with 10 decp strim, the interrnh narrow nnd subrnrinnto ............ 18. C. cornzplunalu, g p 23. (22.) Rostrum shorter tlinn hcnd and broadly dihtud ; head e16ng~tc~ siibpundmte ; eljtra with 10 decp strim, the interrnh narrow nnd subrnrinnto ............ zp , 24. (1.) llostrurn subcjlindrical, not dilatcd nt [up. n. npcx. 24. (1.) llostrurn subcjlindrical, not dilatcd nt npcx. p 25. (20.) Rostrum impunctnte dorsnlly ; centrnl impression on prothoras nt bnse only, the medinn cariun indistinct anterio.rly. 13. C. immeritirs, y , 26. (2.5.) nostrum punctate dorsally; c e n t d nu- >ion on prothornr rcnchinm from [Filhr. [::.to npex, tho median cnrinn &tinct [sp. n. throughout ...................... 14. c. rcrob~co~fu, Syriopfic Key for the S o d - A fricarr Species of Co-. >>011113. g y p (6.) l’rothornx with n distinct cariiia nnd n loiigitudinal basal iniprejriori on each side of it ; rostrum suddeuly nnd rery strongly dilated nt npcs ............ 0. g y p 7. (2.) Intervald on e l p inrpunctntc. 8 (21 ) l’ li il di ii l i d 8. (21.) l’rotliorilx distiirctly iuryrcered or cari- nnte on the disk. 8. (21.) l’rotliorilx distiirctly iuryrcered or cari- nnte on the disk. 9. (20.) Rostrum without nny ccntrnl furron nbovc. 9. (20.) Rostrum without nny ccntrnl furron nbovc. 10. (10.) I3 tra with only Ocouipleto shin! and n siort sub1iuiiir.ral olio ; thc centnil iui- presrion 0x1 pratliomx riot iiiterrupted 10. (10.) I3 tra with only Ocouipleto shin! and n siort sub1iuiiir.ral olio ; thc centnil iui- presrion 0x1 pratliomx riot iiiterrupted finclx iind sp.ir:ely puirctured. p y p 12. (13.) Protkornz sulpyriforni, the rides gradu- nlly dilated from npcs to Lo~oud iiiidd!c ; uictnsternuui cvrnly but sparsely piiiictiircd ................ 12. (13.) Protkornz sulpyriforni, the rides gradu- nlly dilated from npcs to Lo~oud iiiidd!c ; uictnsternuui cvrnly but sparsely piiiictiircd ................ 4. C d r i c w i i t s , ndi. p y p 13. (19.) l’~11t11uras subqudmte, thc dcsrupidly diliited frour apes to bduru riiiddlc, t11113 formin;: 11 distinct slioulilct; ;uetnsturiium with n large ccntral iin- punctate nrca .................... 5. C! iiicicilis, IGhr. 27 Awii. LL’ .Ifuy. rV. Ilisf. Ser. 7. JW. XY. 1. Cossonur Q ~ S C ~ S U J , Bolt. Cnssonrrr absciaais, Doh. Sch8n. Gen. Curc. iv. p. 1036 (1833) ; Gent. \\'iegnr. Arch. 1871, i. p. 76. Cnssonrrr absciaais, Doh. Sch8n. Gen. Curc. iv. p. 1036 (1833) ; Gent. \\'iegnr. Arch. 1871, i. p. 76. Long. 53-7, lat. It-2 mm. Long. 53-7, lat. It-2 mm. €l d €lead subconical, obsoletely punctnte on vcrtcx, forelrcad atrotigly puiicturcd mid with a broad central impression con- taiuing n deop round fovea; cyce sliglitly prominent. Ros- trum stout, about as long as broad, scarcely curved, strongly dilated from base to apcr; uppcr surface with R centr:rl impression (continued from forehead) at the base only, platie South-African Species of Cossonus. 395 anteriorly, with distinct confluent punctuation and occasionally a faint central carina; antcnnz inserted about middle, piceous. Profhoras bipartito, longer than broad, apex truncate and much iinrro\wr tli:m base, which is deeply bisinuntc ; n very deep coiistricfiori i m r apes ; the posterior portion slinhtly coiivcs, evciily sct witli large deep puricturcs aiid wi& nn obsoletc median carina ; the anterior portion somen-liat raised and much less coarsely unctured ; colour dull black, bare. Myfra scparatcly rouiidefat base, 3 little broadcr than pro- thorax at slioulders, wliich arc slopinn sitlos subparallel to beyond niit1dle ; upper surface slig1iiIi coiivcs, witli only eight complete stria+ confaining r o w ot' very large and deep foveolic, the third and fourtli rows each divided into two at base by narrow c a r i m cxteiiding only a short distancc, tile intervals very narrow, each with a row of iiidistinct and distant punctures bearing miuutc setx, wliicli can only bs perceived with clifljculty ; colour dull black. Legs closely punctured and with sparse pnlc pubescence, feinora black, tibia niid tarsi piceous. p CAPE COLONY : Algoa Bay (Ur. H. Brauris) ; NATAL llalvcrn (C. A'. Burker) ; '~'RASSVAAL: Loydenburg (Ur. J. W. ,!I. Qrinriiry) j UASIIOXALAND : Victoria. J , Q y) j TYPE missing ; in Ecklnn niid Zeylier's collection. !"is species may be readily recognized froin a11 its South- African congeners by the structuro of the tliorar. Tlrc only specimen I liavo takcri was fouritl under tlic bark of n largo doad acacia. 'l'hc species lias been recorded by Faust from Cameruri aiid by Gerstaecker from B r u s h in German East Africa. 1. Cossonur Q ~ S C ~ S U J , Bolt. Some speciriicris are covered with a fine earthy indumentum, which can be made to scale off; it is not quite clear whether this is a natural covering or merely advcntitious. 2. Cossonus Sheppnrdi, sp. n. Long. 5-6, lat. 14-14 mm. Long. 5-6, lat. 14-14 mm. g , l e a d subconical, almost im unctatc on vertex, forehead fovea, encli puncture bcing filled with n short, depressed, scale-like, white setn j e p prominent. Rosfririti stout, about as long us Iiead, scarcely curved, gradually dilated from base to apes; up er surface plniie, with punctures and setw similnr to those on P orelread ; anteiiiix ftxrugiiious, inserted in front of middle. Profhoraz longer tlinri broad, aprs trilricatc, base faiiitly bisinuatc, sides eliglitly roliiided, broadest rather behind middle and strongly constricted near apes ; upper eurfnce slightly convex, rritliout nny central firrrow or closcly and coarsely puiicture s and with A small central 27* 27* Xr. G. A. R. Nnrslinll on tAe 3DG cnrina, evciily set with large slinllow punctures, cncli con- tniiiiiig a very short white sctn ; colour dull black. E Z j h subtruiicate at hnsc, x little broader tlinn prothorns at shoulders, whicli arc rounded, sides subpnrnllcl to bryond rniddlc ; upper surface sonicwlint convcs, with ten cornplcte strim contaiiiing deep closely-set puncturcu, tlic intcrvnls plane, as broad ns the strix iicnr tlic suture, hut tliose bctwccii tlic third and fourth strin: niid between the fifth niid sistli arc vcry iinrrow and niicyeii, so tlint tlic piii~cturcs in tlicso striz arc respcctively npprosiiiiatcd and often irrcgulnr or cvcn more or less coIifuscd togctlirr (TYPE form) ; the brondcr intcrvnls Iinvc encli x TOW of sninll Lilt distinct arid close punctures bcnriiig iiiiiiiite depressed wliitc An: j colour dull black. L c p punctntc, iyitli scnttcred t1ol)ressed wliitc set;\', D~CCOUS, with thc tarsi fcrruginaus. , g I'ORTUaUESE E. AFRICA : &irn (1'. 11. Shq)]lnrrl). TYPE TYPE in tlic Tlritisli IIuseiim. TYPE in tlic Tlritisli IIuseiim. Allied to C. a6scissus, noIi., from wlticli it differs, Iiowcvcr, in iiiniiy c1inr;ictera. Its iienrcst ally is C. towidris, Boli., from Sciicgnl, but tlic Inttcr, wliicli is II niore robust iiisrct, Iins its rostrum vcry strongly niid qundraiigiilnrly dililted nt tlic apes and with n low central cnriiin; tlic thoracic coiistric- tion is similar, hut the \insa\ iiinrgin is worc dccply lisinuatc, niid tlic iiitervala on the cljtra arc niucli more conraoly pu nc t ii rcd. p I hnve niucli pleasure in dcilicnting tliis spccics to Xr. P. A. Sliclqinrd, of Beira, to whom I ;iin iiidcltcd for niucli valuallo ninterial froxu that liitlicrto uii\\-orlrcd locnlity. 3. 2. Cossonus Sheppnrdi, sp. n. Cossoiras cnrinicollis, Ftlir. Co'oJsonrts mriiricollis, I.'&hr. Loiig. 5-5, lnt. lg-24 nim. I<. l'et.-Ab-ad. Forb. 1871, p. 284. 3. Cossoiras cnrinicollis, Ftlir. g , g 1JE~ld subconical, closcly arid strongly pu!ictiired ; forclicnil with n dccp cciitrd fovcn j cycs sliglitly Iironiinciit. Rostriini rntlirr longcr tlinn Iic;id, ~1~r:i11~l-sitlccl to about middlc, tlieiicc nbriiptly aiid quadraiigulerly dilated ; uppcr surface pluic, closcly niid distinctly puiicturcd, but with n vnrinllo inipunc- tnte riiedinii liiic o r pntcli ; niitciiiix inscrtctl iic:ir apes. P r o ~ h o r m scnrccly loiiger tlinii broad, npcs truncate niid niucli narrower tlinii bas?, wliicli is sliglitly bisinuntc, side3 rnodcrntoly roundcd, broatlcst n t iiiiddlo, s iglltly constrictml nt nprs, postcrior niiglea witli n vcry srnnll acute projrctioii ; uppcr suifnce nh03t plniir, closely niid dihiictly Iiullcturod tlirougliout, and with n snrootli central cnriiin, irIiicIi 11:1; n trond slinllow impression on each sidc nt bnsc ; colour LL;& SoulA-Africaii S'iecies of Cossonus. 307 or ferrugiiious, with cnrina and margins black. Elylm a littlc brondcr tlinn protliorax nt slioulders, which are sloping, base subtriincnte, sidcs pnrnllel to beyond iiiiddle ; upper surfncc slightly coiivcs, with tcn coinplcte broad stri,?: con- taining rows of li\rge decp puuct1lrcs, tllc iiitcrvala ~ ~ n r r o \ v siibcoiivcs, niinutcly punctiilnte ; colour citlior b1:rck or fcrruginous, witli the suture and margins black or eiitircly f'erruginons. Lcgs cornpnrntively 1011g, closcly punctulnto a i d sparsely pubescent, wrying in colour from black to fcrriigiiious. g NATAL: Durban. 'l'si-c in tlic Stockliolni Niiscuni. Nucli tlic largest of our South-Africnn spccics niirl with 110 11c:1r ally. 4. cosso~lrls africnafrs, I3011. CUWJ~XIU rfricrinus, Uoh. Schiin. Gcn. Curc. ciii. p. 278 (1845). CUWJ~XIU rfricrinus, Uoh. Schiin. Gcn. Curc. ciii. p. 278 (1845). Lnng. 4-44, Lit. 1i-14 niiii. Lnng. 4-44, Lit. 1i-14 niiii. g , Ilead stibcoiiicnl, \,crtcs iiiipiirictntc, forclrcal fiiiely pniic- tiired iilld with :L distiiict fovcn ; cycs slightly proinincnt. Rostrurrt scnrccly longcr tlinn licnd, narrow ;\lid subcylindricnl ;it Law, strongly and qundrnrigulnrly dilatctl iii tlic npicnl lialf ; upper siirface smooth, flattcncd towards apcs, with finc scnttcrctl Iwiictuation ; antenna stout, frrrugiiious. 3. Cossoiras cnrinicollis, Ftlir. Pro- thoras nbout ns long as broad, npcx truncate, mocli nnrrowcr tlinn base, ~Iiicli is Iiglitly bisinuntc, sides slightly rouiidcd, and gradiinlly dilated from apcs to just bcliintl middlc, apex v i t l i n slight constriction j upper surface sonie\vliat conves, with a distiiict cciitrnl cnrinn aid n slinllow loiigitudiiinl iru- prcssion ~ i i cnch sido of it ; tlie puiicturcs nre close arid dcep iicnr tlic cnriiiiil becoining firicr OH the disk 3rd stronger agniii townrd3 tlic sidcs ; colour bl:ick, sliining. E(ytm scpnrntcly roaiitled ;it base, n little broader tliau 1)rotliori~r at sliouldcrs, which nrc roundedly proriiinent, sides pnrallcl to Lcjoiid niiddle ; upper surfncr? sliglrtly convex, with 9 coin- pleto fine striic containing rows of siiiall closely-sct punctures, tho iritcrvnl3 conipnr:rtivcly broad, alrrioat plaiic, iriipunctntc, but faintly cori:iccous ; coloiir black, modcrotely sliining. Legs piccous, bare, fiiicly punctcite ; tibix nnc\ tarsi fcrrugiiious. CAPE OF GOOD HOPE (Ii'dtlberg) Cnpc 'l'owii (A g p y p g CAPE OF GOOD HOPE (Ii'dtlberg), Cnpc 'l'owii (A. R ~ ~ I Y z J ) . ) 'IWE in the Stockliolni Xuscum. Th The puiictuntioii of tlic discnl nrcns of tlic protlioras is varinblc, being- inuclr closcr and nroro distinct ill sonic spccinieiis. Tlicsc lnttcr arc thus iiot unlike very small Blr. G. A. K. Marsha\\ on the 398 cxamplcs of C. eZoo,rptuhrs, F., in gcncral appearaiice ; but the rostra1 structure of that spccies is very diffcrcnt and tlic elytra are more dceply striate. 5. CusSo~IIIs i,rcit.ilis, F311r. Coasoniis irrn'viliJ, Fahr. CEfv. R. VetAlind. Forh. 1871, p. 2% Long. 4-4$, Int. I.k-15 nlm. Long. 4-4$, Int. I.k-15 nlm. g , Head suhconicnl, inipunctatc on vcrtcs, forelicad purictu- late arid with a variablo central foven; eyes rntlicr IarFe, slightly prominent. Rostrumabout ns long as liead, iiarrovving gradunlly from base to middle, tlience IJroadly and qundmii- gulnrly dilated to npcs ; upper surfacc own, closcly mid fiiiely punctured ; nntcnrizc short and stout, ferrugiaous, inserted in front of niiddlc. Prothorax rat.licr longer than broad, apes truncate arid inucli iinrrower than basc, wliicli is strorigly bisinunte, sides slightly rounded, broadest about riiidtlle, faintly constricted :it npcr ; uppcr surfiicc sliglitly coiives, with n strong central iinprcssion, iiiirrow from near apcs to middle, tliericc brondly ntid triari,nuliirly dilnted to basc, t l i stiterior pnrt Lcing usiinlly shallow ; tlir. imprcssioii is strongly puncturcd and contailis n very narrow iindulnting carinn, the disk sinootli arid with very fine E ~ ~ T S C puiictuntioii, Lcyond ~vliicli tlic sidcs arc dceply a i d coarsely piilicturcd ; colour black, sliining. Elytra sepnratcly roundcd at 1x1~0, very littlc broader tlinli protlioras at shoulders, which arc sloping, sides prnllel to bejond niiddlc ; upper surface iiiorleratcly COI~VCX', with nine complctc strongly piinctiircd strirc, wliicli are vcig Jecp near the sutui,c, but beconio sliallowcr Iatcrally, intervals nlniost ns broad as strix, coiivcx and impuiictatc; colour black, sliining:. Ltgs short, piccous, him, arid obso- letely punctured, tarsi paler. Sorith-Afiicarr Species of Cossonus. 309 Sorith-Afiicarr Species of Cossonus. 6. Cossonus traiisvnalensis, sp. 11. TAong. 33, Int. I min. g flead subcoiricnl, black, sliiriing, impunctate, with n distinct sliallou- triiiisverse iinpressioii Loiiiiid tho ej-cs ; fore1ie:itl with a sm:ill ceiitrnl fovea ; eycs slightly proiiiiiient. Rostrum &out ns long ns Iicad, moderiitely curved, cyliiitlrical nt b a ~ tlieiice gl-nduolly dilated a i d flattened to\\nrds apes, uppcr surtiicc with very fiiic fniiit puiictuntion, tlic sidcs comcly pncturcd ; nntennir: ferru~inous, iiisertcd about iniddle. I’rothornz n litlle longer tlinn broad, sides slightly roundcd, Lronrlcst nLoot niiddlc, cmstrictcd near apes, Lase bisinuate ; upper sorfnro plniio, wit 11 a deep, sliarply dcfined, V-slinped ceiitrnl irirltrezsioii, wliicli is tillcd with coarse punctures nnd lins no disliiict central cnrinn; discnl nrca impunctate, tho ~itlcs closely and decply punctured; colour black, glabrous. Hytra roundcd at basc mid scarcely broader tliari the thorax, sliouldcrs rouiided, sidcs pnrnllel to loyond middle ; upper surface sliglitly coiivcx, scarcely striate except closc to base, but with nine complete rows of dccp scparntc puncture, wliicli arc smaller apically and laternlly, tlic iritcrvnls plane and imjiunctstc ; colour black, shining, glabrous. Legs piccous, olsolotely punctured. p , TRAXSYA TYPE in the British Museum. Nearly d i e d structurally to C. sufurnZis, 13011., but the present species is a distinctly narrower and slightly more convex insect, and, in addition to tlic characters nieritioried in the key, it may bc distiriguislied Ly the coaraor punctuation of the rostrum and tlic diffcrcricc in thc strintion of tlic elytra. y p p n' ATAL : Vcrulam, Umgeni ; ~ASIIONALAND : Salisbury. 'I'YPE in tlic Stockholm IIuscum. (' y p p n' ATAL : Vcrulam, Umgeni ; ~ASIIONALAND : Salisbury. 'I'YPE in tlic Stockholm IIuscum. (' 'I'YPE in tlic Stockholm IIu Tii i l i k Tiic typical spccimens taken by Walilberg in (' Caffraria JJ are apparcntly immnture, all my O \ Y ~ exniiiplcu being of a shining black colour. 'l'ho species in general form a i d sculp- turc is nllicd to suturalis, Dolt., and trmst.nule,,sis, llsIiI.J but it is distinctly morc convex, the rostruni is much more dilatcd and more evidcntly punctured abovc, and tlie thoracic irnprcssion is of a diffcrcnt shape. In Xatal I found tliis iiiscct in decaying Eupliorbias, and at Salisbury under the rotting bark of what is locally linorvu as tlie cabbage-trce." 7. Cossonus srrturalis, l3oli. &880?ittS rulurn&, I)oh. Schbn. Qcn. Curc. ir. p. 1033 (1833). Long. 4-5, Inr. 1-11 nim. g , Head subconical, black, glabrous, minutely punctured, nrid with a deep obloiig fovea on forelictid ; cyes slightly promi- nent. Rostrunr about as lonm ns Iiead, vcry littlc Curved, sides subpnrallcl at bast, spica? lialf with a slight quadran- gulnr dilntntion ; uppcr stltfncc impunctate, tho sides faintly punctured ; antcniin: ferrugiiious, insorted well in front of niiddlc. Prothorax a littlo longcr than broad, sides slightly rounded, broadest behind middle, RPCX distinctly constricted, base gently lisiiiunte; up cr surfnce depressed, with a broad axid filled throughout mitli largc corifluerit piincturcs, exccpt ccntral impression, padun P ly dilated from near npcs to basc Mr. G. A. K. 1Inrsliall on tlrc 400 for a smooth ccntrnl lino of varinblc width ; discal nrcn im- p\inctnte, tlic sides closcly niid strongly liuiicturod ; colour black, glabrous. El!/tm subtriiiicntc at b:isc niid scarccly broader than thorns, slioiildcra sliglitly proniiucnt, the sides very grndunlly nsrro~od froin base to npcs ; upper surfacc dclircssed, wit11 iiiric complcte strongly piiicturcd strin., ivliiclr nre deep and crcnato-puiictatc on disk, but vcry slinllo\\~ l:iiernlly, tlic intervals plaiic but 1i:irrow and ir~i~)~ii~ct; colour ferriiginous browi, wit11 the suture and apes broadly, the laternl margins narrowly black. Legs black, siiiiiing, objolctcly punctured, tarsi 1iiccou2. j y p KATAL : Durban, '\'crirlani. TYPE iii tho Stockliolrii lluseum. A i A comnioii spccies occurring in soiiie iiunibcrs nncler the rotting bark of sevcral soft-wood trcea, atid cspccinlly tlic Kafir Loom (Erjthrizaa cqfrtr). Also rccordcd from tho Congo by Faust. South-11 fricnii Species of Co-- asOllllS. 40 I South-11 fricnii Species of Co-- asOllllS. 9. Cosso~iirs chirhiensis, sp. 11 tong. 5-52, Int. 13-1; iiini. tong. 5-52, Int. 13-1; iiini. tong. 5-52, Int. 13-1; iiini. g , ; IIctrd subcoiiicnl, iinpuiictatc on vcrtcs ; forclicnd finely puiictiircd :id with n dccp clongnte fovcn ; eye3 promincnt. lrostrvri~i scnrccly loiigcr tlinii Iie:id, sinuiito 1;rtcrnlly in tlic iiiiddlc, Lroirdly nnd snbqu:dlan; ul:irly dilated at apcs ; upiicr suil;ice closely niid fiiiely puiictiircd, nliiiost plniic npiciilly ; ntitclir1:c ferrtiginoiis, iiiscrtecl quite close to : I ~ C S . I'rollroIcts broiitlcr tlinn long, apes trtiricntc, niiicli iinrromcr 11i:rii bnsc, which is Lisiiiiinto ; s i d c ~ subpiirallrl to about ~~iiddlc, tlicn gradually iinrrowed to apes, wlicre tlicro is a sliglit constric~ion ; upper surfacc nliiiost pliint, vcry finely IIUIICI urcd on tlrc disk, but coarsclj so towards tlic sides, with :L slinllow loiigitudiii;il impression close to apex roiitniiiiiig nil ill-dcfiiicd ccntrul cnriiin, with n fcw lnrgc puiicturcs on c:rcli sidc 0 1 it, and n iiiucli dccpcr tr:ipezoidnl iiiil~rcssioii :it biisc, wliich is riigoscly punctured aiid contains n distinct Iinrrow cnriiio, the trso iru ircssioiis bciiig scpnrntcd Ly n narrow Xljtra sclmntely roundccl at Insc, bronder tlinii tlic pro- tliorns nt sliouldcru, which arc rouiidctily proiiiinciit, sid03 subp:irallcl to Icyoiid iiiidrllc ; iippcr siirfacc ])loiic, with tcn coiiiplcte, deep, niid stroiigly pictureti strix, tlic iiitervnls Lciiig iinrrow, coiivcs, niid iiiipuiictatc ; colour uiiiforiii tcstnccous bronn, a littlc dnrkcr tliaii tlie protliorns. Legs testnccous, bnrc, niitl finely puiicturcd. smooth spacc; co I oiir uiiiforiii light tcstnceoiis brown. y p S.K. ~lASl10NALAh.D : Ciiiriilda Forest. TYPE in tho British Uuscurn. illiis species as also C ztnizil r i lliis species, as also C. ztniziln, I captured flying in the daytime along shady pntlis in tlic detisc forest. 10. Cossoiius cloriyatiilus, Fn'nb. Cttrcrrlio rlo~~gdrzlu~~ Fnb. Eut. Sjst., Su pl. p. 1GS. CUSSOIIUS donplidifb, Doh. ScliCn. Gcn. &rc. is, p. 1022 (1837). 8. C'ossonrrs irwih, sp. 11. 111 Long. 32-4, Int. 1-11 mni. g , liead siiI~coiiical, vcrtes impunctntc; forellend clo3cly and finely punctimcl, but nitllout any cciitral fovea ; epca proiiii- nent. Roshini longer tlinri Iiend, subcylindrical nt bnso, distinctly tIioiigli iiot broadly t1il:itcd in thc apical Iialf ; iipper surface conyes, sliiiliiig black, with extrcniely fino puiictiintion ; antcrinn: iiiscrted iiear apcr, fcrrugiiious. Pro- t A o m z scarcely loiigcr t1i:iii broad, apcs truiicxte, base bi- sinuate, sides subliarnllcl to about iiiiddle, thence rapidly nnrrowed to npcx, wlierc tlicre is x slight constriction ;. upper surface pl:ineJ wi tIi n broad, dccp, triangular iriiprcssioii es- teridiiig froiii tlic apical constriction to base, tlic iiiipreesion being riigosely puiictiircd and containing x broad iiiipunctnte cariiia tliroughout ; the portions of tlie disk adjoining tile central impression are quite smooth nnd inipiinctnte, but the sides nrc very conrscly punctured ; shining black, bare. Eipu separately rounded at basc, a trifle broader tliaii pro- tliornx nt sllouldera, wliich nro eloping, sides subparallel to toyoiid niiddlc ; upper surface plnno, subdopresscd, witli nine deep strongly punctured strix, tlic tenth strin being sub- liuiiierd nnd mucli abbreviated, the intervals narrow, cnriiiatc, and inipunctnte. Legs piceous black, shining, barc, impuiic- tote ; tarsi fcrruginous. ; g S.E. ~IASIIONALAXD : Chirinda Forcst. TYPE in the British lhseum. 10. Cossoiius cloriyatiilus, Fn'nb. Cttrcrrlio rlo~~gdrzlu~~ Fnb. Eut. Sjst., Su pl. p. 1GS. CUSSOIIUS donplidifb, Doh. ScliCn. Gcn. &rc. is, p. 1022 (1837). Loiig. 3;-6:! lnt. 1-2 iim. Loiig. 3;-6:! lnt. 1-2 iim. IlmZ subcuiiical, piceous, sliining, inipuiictnto on vertex j forclicad witli fine scnttered puiictures ; eyes slightly promi- nent. It'ostrum stout, n little shorter tlraii. Iiead, broad and flatteiied; sides pnrallel in ;lie basal linlf niid with n sliglit y.a~rniigulnr dilntntiori nt apes, Llnck or ferrugiiious, s iiiiing, deeply puiicturrd, mid with n broad ceiitrnl furrow, ivliicli nsccnds the foreliead, but is nbbrevinted niiteriorly ; nntennm stout, ferrugiiious. Prothorax longcr tlinli broad, apes truncate, niucli iiarroiver tliau base, whicli is subbisinuate, Mr. G. A. K. Mnrslrall on the 402 sides gently rounded, broadest about middle, rapidly narrowing towards npex, wlicro tliero is n slight constriction ; upper surface almost plane, deeply an3 closely punctured, w t h a sliglitly raised sniooth ccntrril line and a faint longitudinal impression 0x1 encli side of it ; colour entirely black or fcrru- ginous, with the sides mid under snrfnce sometimes blnckish. Hyfra subtruncate at base, a little broader than the pro- thorax at slioulders, wliicli are subrectangular, sides pnriillel to hcjond middle; upper surface nlinost p!ane, with deep arid strongly p ~ t u r c d stritc, thc intervals narrow arid impunc- tate; colour cntirely black or ferrugirious, the suture and lateral margins being more or less infuscate. Legs short, ratlier elender, with faint scattered punctuatiori, black or ferruginons, with tlic knees rntlier darker. g , CAPE OF GOOD IlorE (teste Dolieman) ; S.E. lInsrrowA- LAND : Cliirindn Forest. I am not aware of the esistcnce of FrrbriciusJs type of this species ; but tlrc spccimons upon which Bolicinan founded his description are in tlre Stockliolm bIuseutn. p Bolieman also records tho insect from India and llanritius. Witlr the exception of C. abscissus, Boli., wliicli differs widely in other rcspects, this is tlie only South-African species wirli a furrow on tho uppcr surfacc of the roslruol. 11. Cossouus glabricollis, Boli. COSSOI~IU glnlricollk, Doh. op. cit. ir. p. 1034 (1838). Loeg. 4 , lat. 15 mm. g , Uectd conical, sliiniiig, impunctato, with a niinuto fovea on forohead and slifilitly coiistricted bcliiiid tho cyes, wliiclr are subdcpresscd. Rostrwir longcr tliari h a d , curved, suhcylin- drical a t basr, sliglirly nnd quadrangularly dilated apically ; smooth, shirring, and almost irnpuirctate ; atitennic compnra- tivcly long, ferruKinoua, inserted it: front of middle. Ply+ thorarz broadest nt base, grndudly Iiiirro\d till clojc to apex, where tlierc is n slight coiiatrictioii, npex truncate, bas0 bi- sinuate, posterior angles with n niiiiutc Eliarp prcjectiori ; upper surface plnne, sn~ootlr, sliiiiing black, impulictate, except for two irrcgular ceiitriil roivs of fine punctures (some- times partly obsolete). EZ’fru separately routided at base, s trifle broader tlinn protliorar nt sliouldcr~, wliicli are roundedly prominent, sides parallel to beyond middle ; upper surface dcprcsscd, aih niue complctc fino sl~allo~v stria? con- taining regular rows of sninll distant punctures, wliich aro faint near the suture and beconic gradually strouger Iatcrnlly, Soiith-Africun Species of Coasonua. 4 03 tllc iiitervals brond, plane, nnd impiinctate ; colour sliiniiig black. Legs black, shining, inipunctate; tarvi ferrugiiious. g NATAL : Alnlvern. l'we in tlie Stockliolni bluseum. This c10r.s not nppenr to Ic a coinnioii insect, and I liave t:lkeii it only uiider tlie bark of rotten Kalir booms (Erjlhrirra cn$rCl). 13. Cossori IN ivi 112 erif u s , Fa11 r. Cosioriits irr~nrcrifirs, F&r. (Efr. Vet.-Ali. F5rlr. 1871, p. 435. Cosioriits irr~nrcrifirs, F&r. (Efr. Vet.-Ali. F5rlr. 1871, p. 435. g , Ueud shortly conicnl, iiii~~i~iictato ; forcliend wit11 a central fovrn ; cycs sliglitly prrliiiiiicnt. Ii'ostrriw iicnrly twice ns long as Iicatl, stout, ~inrallel-sided, slightly ciirved, pcinctntc on tlie sidcs, but glabrous dorsally ; aiitciiiix stout, fcrru- giiious. .P~.otlrorns :iboiit ns loiig as broad, nlicx truiiwte, iiiiicli iin~ro~vcr tlinn Lnsc, wliicli is bisinuntc, sidcs subpnrallel froiii Lnsc to bcyoiid niiddlc, iinrrored nntcriorly, mid with n fiiint constriction at apes ; upper siirt'ncc slightly coiives, witli decp subrciiiotc ~~iiiicttintio~i and :i incdi:in basal iniprcs- sioii ; tlic cciitrnl cnriiia ecnrcely noticcnblc, exccpt in the Inanl iiii~~rc~sioii; colour Llnck, bnrc, sliiniiig. ELytrn a littlc brondrr tlinii protlioras at tlic slioulderj, wliicli are roundedly poiiiiiieiit, sides pnrallcl to bcyoiid iiiiddlc ; iippcr siirt'ilca sliglitly coiivcs, with tcri coinpletc dcep mid stroiigly Iiunc- turcd striz, the iiitsvals coiivcs aiid iuipuiictnto ; colour Llnck, ghibroiis. Legs Llncli, inil~uiictate, tnrji t'crrugitious. oaded by [University of Calgary] at 16:17 01 February 2015 , g g , CnFF1:AI:ra (fcslc Falrxlls). (f TYPE iii tlie S~ocklioliii Mimuin. 12. Cossonus cornplanatus, sp, n. Long. 5, lnt. I 4 nim. g , i h d elongate, subquadrate, constricted near h e , with vcly fine scattered puiictuntioti nrid n minute fovan oil forc- Iiend ; e p s EOtneWlifit prominent. Rostruiri rat Iicr shorter t1i:iri tlie liead, strongly and qundrnngulnrly dilated from niiddlc to npcr, upper surfncc nlniost plane nntl with the piwetiiation rntlicr stronger nnd closer than on tlic liond; antcriiim ferniginous, inserted close to npcs. Z'rothornx 3 rriHe loiigcr tlinn brond, npcr truncate and rnuch narrower than bnsr, which is bisinuatc, sides slightly roundcd, broadest bcliind middlc, gradually narrowed anteriorly, and with R constrictioii at nlies ; upper surfiice plaiic, niiicli depressed, with fiiie scattered punctuation, Iinviiig an imliunctnte central line, on cncli side of wliicli tlicre is iicnr tlic Lasc n very faint longitudinal impression containing rather deeper punctures, posterior angles with a minute slinrp projcction ; colour black, sliiniiig. E/IJ~I-U strongly depressed, jointly roundcd a t base, n little broader tlinn protliorax at tlia shoulders, which are rouiidrdly promincnt, sides pnrdlal to far beyond middlc; uplicr surfaco plane, with ten complctc dcop stria contniniiig rows of distinct closely-set puncturcs, tlio intervals narrow, plane, and inlpunctntc ; colour piceous black, shining. Legs black, bnro, finely puactulntc : tarsi piccous. g , , NATAL : Malvern. TTI Ei B i i h g , y p p NATAL : Malvern. TTI E TTI-E in tlie British Xuseuin. I l i l TTI-E in tlie British Xuseuin. I Iiavc sccii only a singlc esiimple of this very distinct specico, wliicli I cnpturcd flying nt sundown in August 1897. Its ertreniely depressed form will nt oiice distiiiguisli it from all other South-African specirs, its nearost ally in this respect bring ylaabricollis, Boll. Tliesc two spccics are tlie oiily ones in wliicti 1110 protliorax is smootli and without any distinct carinn or other sculpturing. Froin glnlricollis tlic present spccies may be further distingiiislied by its much shorter arid broador rostrum, diffcrciitly shaped tread, and by the decper and cloEer striatioil of tlic clytra. 404 018 trlle Soiith-Afiicnn Spies of Co;aonus. 14. Cussoriirs scrolicullis, sp. 11. Long. &.la, lilt. 1; 111111. iii l iiii Long. &.la, lilt. 1; 111111. g Ucatf coiiicnl, vcrtrs iiii~iiiiictntc j forcliccrd pniictulatc aiid with a cciitriil fovea ; cyrs subdeprcsscd. Ilustriiiri Ioiigcr tlinii Iicnd, curved, subcyliiidricnl, mid iiot dilated at npcs ; purictuntioii distiiict at bnsc, but bccoiiiiiig much tiricr apic.tlly ; aiiteiiiin: iiisertcd at iiiiddle, short, stout, fcrrugiiious. I+o- t h o r ~ z as loiig ns Lro:id, rips truiicatc niid much iinrrowcr tliaii Lnsc, wliicli is stroiigly bisiiitinte, sides bnt little rouidcd, slightly constrictcd behind apes, broadest about middle ; upper surface soiiicwliat coiives, clojcly and very coarsely puicturcd tlirougliout, niid with a decp triiingcilnr central impression, Iroadciiitig froiii apes tn bnso, tho iin - pression itself rugosely putictured and coiit;iiiiiiig a couiplete smooth cnrinn. Ehpa sepni-;itely rouiidcd :it bnse, a IittIo broader tlinii prothorax at shoulders, which arc obtiiscly prominent, sidcs pnriillcl to boyond iuiddlc ; upper surfiico iiiodciiitely couyes, with tcii dccp very strongly punctured striz, which nrc linrdly sliallower lnternlly (except tlie tenth, whicli is faint), the intervals liorrowJ sulcariiiate, airtl inipuuctate. Lcp piceouj, shining, tiircly puiictulatu ; t:irai fcrruginous. 405 IIr. L. J. Colc on Tcti-Iegged Pjciiogonids. IIr. L. J. Colc on Tcti-Iegged Pjciiogonids. NATAL : Vcrulnni ; POI:T[TGUESE E. AFRICA : Beirn (P. A. SAe]’l””‘d) - ] ) TYPE in tlic l3ritisli JIuscuai. In nddition to thc c!i:imctcra rnenticincd in the kcy, this spccics may be distiiiguislicd from it3 only near ally, C. iru- tncritus, Filir., Ly tlic following poiiits:-tlie sliortcr rostrum ; the closcr punctuation of tlie thorns, which is often loiigitu- dinally scrabicuhte ; tliu brondcr arid niore dccply ~iuriett~rc striac on tli.0 eljtra; and tlic nnrrowcr subcnritiatc iutorvds. j ; This sptcics was found urider tlic lmli of rotten Etip!iorbin trces. LI~’.-Ten-trygett Pycmgouids, with R-.rirarAs on the Clussijicatioii 01’ Ihc l’yciivgortida. By LEOS J. COLE. Iw a rccciit pnlicr publislicd in this Jouriinl Ly ‘1’. V. IIodpon (1904) Biologist to tlic National Antarctic Espcdition, np1icnrs n dcscriptiou of n most intcrcstiiig I’gciiogonid takcii during tlic stay of tlic Iliscovcry’ in wintcr-quarters iii McJlurdo Bay. It cliffcrs from tLc ordinary nicrnbcrs of thc group in poss~!ssing fivc pairs iustcncl of tlic usiinl riutubcr of four pairs oE mlkiiig or “ aiiiLulatorj ” lcgs. In othcr rcs~iccts it is very closc to tlic ~~-cll-kno~sii geuas ~Vyniphott IIorIgsoii coiisidcrs that this rcmnrliallc nuinid sliould be rcgnrdecl as rcprcscntitig n uc\s gcuiio as wcll qs a ncw spccics, ant1 lins proposccl for it tlic unrnc t’eiituiiynlpho~ aritarcticum. Tlint it is not n fortuitous or “frcnli ” variation is pretty wcll sliown by tlic fact that in all twcnty-cight iridiridunls m r c taken, Loth nialcs m i d fcmalcs. Tlicy ~ ~ r fouiid inllabitiiig watcr froin 13 to 125 fatlioius in tlcptli. g p IIorlgson Lelicscs that rr tlic prcscncc of n fifth pair of legs [is] a clinractcr wliicli scpnratcs it from all l’ycnogonids Iiitlicrto k i i o ~ n ~ ’ ; hit as cnrly ns 1837 Eights (1837) p b - lislictf a descriptioii of n l’ycnogonid with a fifth pair of \raking-lcgs from the South Slictlnncl Jslnuds, and gnvc to it tiic unme Decoloportrt airstmlis--a fact whicli sccuis 1ieyL.r to hare coiiic to tlic attcution of workcrs ou tlic group since that timc. Xiglits g i n s a wry good dcscriptiou mid illus- tratioii of tlic spccics-~nucli bcttcr tliau thc avcrngc at tIlat carly clntc,-tliougli tlic figilrcs do not seem to agrcc ill ;ill rlctails with tlic dcscriptioii. Ilc stntcs tlint tlic entire aiiirml was of n Lriglit scarlet c~luur, nud SO figures it; nii(1
https://openalex.org/W4395100679	https://stia-saidperintah.e-journal.id/ppj/article/download/137/94	Indonesian	null	Evaluasi Electronic Government Dalam Meningkatkan Kinerja Pelayanan Publik	Public Policy	2,023	cc-by-sa	3,972	Evaluasi Electronic Government Dalam Meningkatkan Kinerja Pelayanan Publik Evaluasi Electronic Government Dalam Meningkatkan Kinerja Pelayanan Publik Shofy Yorinda Febiyanti1) Arimurti Kriswibowo2) 1,2Universitas Pembangunan Nasional “Veteran” Jawa Timur shofyyorinda@gmail.com Abstract The implementation of online customer complaint handling at PDAM Surya Sembada Kota Surabaya is still not optimal and needs attention considering the importance of public services and PDAM is the only company engaged in the supply of clean water. This study aims to evaluate the implementation of e-government in PDAM Surya Sembada Kota Surabaya in terms of public services. The e-government management of PDAM Surya Sembada Kota Surabaya still has inaccuracies in handling online services, infrastructure indicators found that there is overlapping complaint data entering the system, and the application still does not have a customer service monitoring feature and the application is only available on Android. on Standard Operasional Prosedure (SOP) policy indicators it is necessary to carry out restandardization which is oriented towards e- government innovation. In terms of infrastructure indicators, it is necessary to evaluate the use of the company's internal system. In addition to application indicators, there is a need for innovation to add monitoring features for customers. LPPM STIA Said Perintah Volume 4, No. 2, September 2023 https://stia-saidperintah.e-journal.id/ppj Received; 2023-03-25 Accepted; 2023-05-03 Published; 2023-05-07 Keywords ;; Public Services, E-Government, Performance Keywords ;; Public Services, E-Government, Performance Public Policy Vol. 4, No. 2, September 2023 \|302 Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Pendahuluan Kemajuan teknologi informasi dan komunikasi (TIK) terus berlangsung setiap tahun. Berkembangnya TIK telah merambah banyak sektor, termasuk sektor publik yang salah satunya ditandai dengan adanya pengembangan TIK pada sektor publik oleh pemerintah yang dikenal dengan e-government (Didi Susianto, Eka Ridhawati dan Sucipto, 2022; Heriyanto, 2022). Berkat administrasi elektronik, semua lembaga negara, perusahaan, warga negara dan pihak berkepentingan lainnya dapat memanfaatkan layanan dan informasi resmi kapan saja. Pada tahun 2018, regulasi terkait e-government dikonsolidasikan dengan adanya PERPRES No. 95 Tahun 2018 tentang Sistem Pemerintahan Dasar Elektronik (SPBE). Menurut peraturan presiden mengenai SPBE, baik instansi pemerintah pusat maupun daerah menggunakan aplikasi SPBE dengan prinsip efisiensi, integrasi, kontinuitas, efisiensi, akuntabilitas, interoperabilitas dan keamanan. (Setneg, 2018). Pengaplikasian e-government pada pelayanan masyarakat yang telah disediakan oleh pemerintah daerah harus mengarah pada pelayanan masyarakat yang lebih baik, lebih cepat dan lebih efisien dan mempromosikan penerapan prinsip manajemen publik yang baik. Salah satu bidang penerapan e-government adalah penyediaan air bersih melalui Perusahaan Daerah Air Mainum (PDAM). PDAM Surya Sembada Kota Surabaya adalah perusahaan kelola daerah yang bertanggung jawab menyediaan air satu-satunya untuk masyarakat Kota Surabaya. Dalam memenuhi kebutuhan air minum, hal ini juga harus dilaksanakan dengan mengutamakan kualitas pelayanan pelanggan. (Syafirah, 2021; Astuti, 2014). Data Jumlah Pelanggan dan Pengaduan Pelanggan Data Jumlah Pelanggan dan Pengaduan Pelanggan PDAM Surya Sembada Kota Surabaya Tahun Jumlah Pelanggan Jumlah Pengaduan Presentase (%) 2018 562.381 209.129 37,19 2019 574.173 250.311 43,59 2020 585.604 302.872 51,72 2021 595.869 256.779 43,09 2022 (per-Oktober) 604.215 204.632 33,87 Sumber; PDAM Surya Sembada Kota Surabaya, (2022) PDAM Surya Sembada Kota Surabaya Berdasarkan tabel Data Jumlah Pelanggan dan Pengaduan Pelanggan mengindikasikan bahwa mulai tahun 2018 hingga tahun 2022 menunjukan bahwa pelanggan PDAM Surya Sembada Kota Surabaya terus mengalami peningkatan dari Public Policy Vol. 4, No. 2, September 2023 \|303 tahun sebelumnya. Namun, pada angka jumlah pengaduan pelanggan mengalami keadaan fluktuatif. Hal ini dapat dilihat pada tahun 2018 sampai pada tahun 2020 mengalami kenaikan yang signifikan. Namun, pada tahun 2021 angka presentase mengalami penurunan. Salah satu solusi yang ditempuh PDAM Surya Sembada Kota Surabaya dalam upaya untuk memenuhi tanggung jawab yakni memberikan pelayanan administrasi dan teknis kepada pelanggan/masyarakat adalah menerapkan prinsip e-government melalui penyediaan pelayanan pengaduan online dalam bentuk aplikasi dan website. Pendahuluan Salah satu solusi yang ditempuh PDAM Surya Sembada Kota Surabaya dalam upaya untuk memenuhi tanggung jawab yakni memberikan pelayanan administrasi dan teknis kepada pelanggan/masyarakat adalah menerapkan prinsip e-government melalui penyediaan pelayanan pengaduan online dalam bentuk aplikasi dan website. Domain pengaduan online yang dimiliki oleh PDAM Surya Sembada Kota Surabaya dapat diakses pada Website www.pdam-sby.go.id juga telah tersedia di gawai berbasis Android melalui Google Play Store. Pada era keterbukaan informasi layanan publik saat ini, fasilitas pengaduan online yang disediakan oleh Website dan Aplikasi PDAM Surya Sembada Surabaya menjadi sarana sistem informasi yang utama. Namun berdasarkan hasil observasi pada kondisi eksisting masih terdapat pengaduan pelanggan mengenai keluhan pemakaian pelayanan e-government yang belum optimal serta masih terdapat kendala log in aplikasi. Hal ini dibuktikan dalam ulasan pelanggan sebagai berikut; “Tempat saya daerah Gubeng mati air sudah 3 kali dalam 2 bulan terakhir. Kirim keluhan ke aplikasi dan ke website tidak ada respon, aplikasi tiap mau log in ke layanan juga selalu gagal. Tolong pelayanan online direview dan diperbaiki segera.” Kata Febri Gisella pada ulasan google PDAM Surya Sembada Kota Surabaya, (10/21) - (Diakses Pada Kamis 13 Oktober 2022, 11.49 WIB). Dalam proses pengaduan keluhan pelanggan, PDAM Surya Sembada Kota Surabaya telah memiliki Standar Operasional Prosedur (SOP). Berdasarkan Dokumen Internal Perusahaan SOP-PPG-03 Tahun 2019, standar operasional tersebut ditujukan sebagai petunjuk dalam menangani setiap pengaduan pelanggan, sehingga dapat diselesaikan secara cepat, tepat dan kepuasan pelanggan dapat tercapai. Berdasarkan hasil observasi secara langsung pada kondisi faktual dalam penerapan standar pelayanan penanganan pengaduan pelanggan ditemukan bahwa pelaksanaaannya belum selaras dengan acuan waktu yang telah diatur oleh perusahaan. Hal tersebut dapat dibuktikan dari komentar masyarakat pada ulasan google: “Website dan aplikasi tidak berfungsi. Pelayanan susah untuk dihubungi, teknisi pelayanan dijanjikan besok namun sampai minggu depan pun belum Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Public Policy Vol. 4, No. 2, September 2023 \|304 ada realisasi”. Kata Tyo Utama pada ulasan google PDAM Surya Sembada Kota Surabaya, (08/22) - (Diakses Pada Kamis 13 Oktober 2022, 12.06 WIB). Kondisi ini menandakan bahwa implementasi penanganan pengaduan keluhan pelanggan PDAM masih belum maksimal dan perlu menjadi perhatian mengingat pentingnya pelayanan publik serta PDAM merupakan perusahaan satu-satunya yang bergerak pada bidang pengadaan air bersih. Tujuan dari penelitian ini adalah guna mengevaluasi seberapa efektif PDAM Surya Sembada Kota Surabaya telah menerapkan e-government dalam kaitannya dengan pelayanan publik. Pendahuluan Teknik evaluasi penelitian ini menggunakan Pemeringkatan e-government Indonesia (PeGI) dengan tujuan dapat mempercepat perkembangan dan penerapan TIK di lembaga-lembaga pemerintahan Indonesia melalui evaluasi secara utuh dan menyeluruh. Penilaian Pemeringkatan E- government Indonesia (PeGI) di PDAM Surya Sembada Kota Surabaya menggunakan 5 indikator yaitu Kebijakan (Policy), Kelembagaan (Institution), Infrastruktur (Infrastructure), Aplikasi (Application) dan Perencanaan (Planning) (E- Government Department, Ditjen APTIKA, KEMKOMINFO, Republik Indonesia, 2012). Setiap indikator PeGI dijelaskan oleh beberapa atribut atau kriteria (Fitriansyah, Budiarto, dan Santoso, 2013). Pengevaluasian kualitas pelayanan berbasis elektronik menggunakan indikator serta atribut PeGI di PDAM Suryo Sembada Kota Surabaya disesuaikan dengan Bentuk Peraturan Birokrasi Menteri Pendayagunaan Aparatur Negara No. 38 Tahun 2012 pada Instruksi Penilaian Kinerja Satuan Kerja Administrasi Publik. Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur “evaluasi kebijakan publik” saling berkaitan; masing-masing mengacu pada proses penilaian hasil kebijakan dan program dengan menggunakan skala nilai tertentu. “evaluasi kebijakan publik” saling berkaitan; masing-masing mengacu pada proses penilaian hasil kebijakan dan program dengan menggunakan skala nilai tertentu. Pengaduan Menurut Setyarini, (2022) pengaduan adalah pernyataan publik yang timbul dari ketidakpuasan publik terhadap suatu penyedia produk atau layanan masyarakat. Pelanggan mengeluh ketika dia merasa bahwa keluhan tersebut telah dijawab secara positif dan tidak menghabiskan waktu maupun biaya. Hal ini membuat masyarakat enggan untuk mengadu karena tidak praktisnya pengaduan tersebut. Menurut Simanjuntak seperti yang dikutip oleh Saadah, M. A., (2023) bahwa pengaduan atau keluhan adalah pernyataan ketidaksenangan terhadap layanan, tindakan atau kurangnya respon yang dilaksanakan oleh penyedia layanan atau personel yang memengaruhi dan dirasakan oleh pengguna layanan, dalam bentuk apa pun (tertulis, lisan atau melalui bahasa tubuh). Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Tim Evaluasi Evaluasi merupakan salah satu langkah terpenting dalam proses pelayanan publik. Tujuan dari proses evaluasi ini adalah untuk menilai efektivitas dan efisiensi pelayanan publik agar dapat dipertanggungjawabkan kepada pemilih. Evaluasi merupakan kegiatan yang menilai tingkat kinerja suatu pelayanan publik. Menurut Khothimah dan Hertati, (2021) bahwa dalam keseluruhan proses analisis kebijakan publik, evaluasi merupakan tahapan krusial yang tidak hanya berlangsung diakhir tetapi juga pada proses pembuatan kebijakan yang sebenarnya. Efektivitas dan efisiensi implementasi kebijakan publik dinilai melalui evaluasi kebijakan. Pengertian Public Policy Vol. 4, No. 2, September 2023 \|305 Pemeringkatan e-Government Indonesia (PeGI) PeGI adalah kerangka kerja kegiatan Direktorat Jenderal Aplikasi dan Informatika (APTIKA), Kementerian Komunikasi dan Informatika (KEMKOMINFO) untuk mengetahui peta keadaan penggunaan teknologi informasi dan komunikasi pada organisasi sektor publik. Pemeringkatan ini diharapkan dapat mempercepat perkembangan dan penerapan TIK di lembaga-lembaga pemerintahan Indonesia. Evaluasi PeGI dilakukan menjadi sumber untuk kemajuan dan penggunaan TIK di lembaga pemerintahan, untuk merangsang penggunaan TIK dengan melakukan evaluasi secara menyeluruh, tidak memihak dan obyektif serta untuk mengumpulkan data tentang penggunaan TIK dikalangan pemerintahan dalam skala nasional. Terdapat lima komponen e-government yang masuk dalam kerangka Peringkatan e-government Indonesia (PeGI) antara lain; PeGI adalah kerangka kerja kegiatan Direktorat Jenderal Aplikasi dan Informatika (APTIKA), Kementerian Komunikasi dan Informatika (KEMKOMINFO) untuk mengetahui peta keadaan penggunaan teknologi informasi dan komunikasi pada organisasi sektor publik. Pemeringkatan ini diharapkan dapat mempercepat perkembangan dan penerapan TIK di lembaga-lembaga pemerintahan Indonesia. 1. Kebijakan, komponen kebijkan berkaitan dengan produk hukum dan resmi yang berfungsi sebagai petunjuk dan arahan untuk mempromosikan penggunaan Teknologi Informasi dan Komunikasi (TIK). 1. Kebijakan, komponen kebijkan berkaitan dengan produk hukum dan resmi yang berfungsi sebagai petunjuk dan arahan untuk mempromosikan penggunaan Teknologi Informasi dan Komunikasi (TIK). 2. Kelembagaan, aspek kelembagaan berkaitan dengan ada atau tidaknya suatu badan yang bertugas mengembangkan dan memanfaatkan TIK. 3. Infrastruktur, aspek infrastruktur berkaitan dengan penggunaan TIK, pembangunan infrastruktur, dan fasilitas pendukung. Public Policy Vol. 4, No. 2, September 2023 \|306 4. Aplikasi, aspek aplikasi berkaitan dengan aksesibilitas dan penggunaan perangkat lunak aplikasi alat yang mendukung operasi umum mendasar dari sistem layanan sektor publik. 5. Perencanaan, aspek perencanaan berkaitan dengan rencana induk pengembangan TIK dan proses perencanaan TIK. Metode Penelitian Penelitian ini menerapkan metodologi penelitian analisis deskriptif dengan menggunakan pendekatan kualitatif guna mendapatkan wawasan yang komprehensif serta mendalam terhadap analisis pemeringkatan e-government Indonesia dalam upaya meningkatkan kinerja pelayanan publik di PDAM Surya Sembada Kota Surabaya. Ungkapan konsep tersebut jelas bahwa yang diinginkan adalah informasi berupa uraian dan analisis permasalahan dengan menggunakan pemeringkatan e-government Indonesia (PeGI). Analisis pendekatan ini merujuk pada Instruksi Presiden Nomor 3 Tahun 2003 tentang Kebijakan dan Strategi Nasional Pengembangan E-Government (https://peraturan.bpk.go.id/). Data kajian ini bersumber data asli atau utama, observasi partisipan, wawancara lengkap dan menyeluruh kepada key informan yakni Manajer Pelayanan dan Pengaduan Pelanggan PDAM Surya Sembada Kota Surabaya serta pelanggan PDAM Surya Sembada Kota Surabaya digunakan sebagai strategi pengumpulan data. Metode analisis data menggunakan model analisis Miles dan Huberman. Pembahasan Hasil Penelitian Bagian ini memaparkan hasil penelitian yang berupa temuan-temuan di lapangan berdasarkan hasil pengamatan penulis terhadap peristiwa dan kejadian yang berkaitan dengan fokus penelitian. Melihat dan mengetahui tingkat keberhasilan e-government PDAM Surya Sembada Kota Surabaya, penulis melakukan penelitian dengan kerangka kerja pemeringkatan e-government Indonesia (PeGI) antara lain: Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur 1. Kebijakan 2, September 2023 \|308 Namun hasil temuan dilapagan dalam penerapan SOP tersebut masih membuktikan bahwa pelanggan masih mengeluhkan ketidaktepatan penanganan pelayanan online sesuai dengan acuan waktu yang telah ditetapkan oleh perusahaan. Seperti yang disampaikan oleh salah satu pelanggan “ES” berdomisili di Kebonsari Surabaya, sebagai berikut: Namun hasil temuan dilapagan dalam penerapan SOP tersebut masih membuktikan bahwa pelanggan masih mengeluhkan ketidaktepatan penanganan pelayanan online sesuai dengan acuan waktu yang telah ditetapkan oleh perusahaan. Seperti yang disampaikan oleh salah satu pelanggan “ES” berdomisili di Kebonsari Surabaya, sebagai berikut: “Saya sudah coba lapor ke petugas PDAM pakai aplikasi tapi setiap lapor jawabannya beda-beda, sudah dijanjikan petugas datang sampai 10 hari ini tidak ada. Seperti tidak ada SOP tetap.” (Hasil wawancara, 21 Maret 2023). 1. Kebijakan Kajian indikator kebijakan bertujuan guna untuk memastikan penerapan aturan kebijakan terkait teknologi informasi yang telah diberlakukan, seperti undang- undang, surat dinas dan barang legal lainnya yang mendorong penggunaan Kajian indikator kebijakan bertujuan guna untuk memastikan penerapan aturan kebijakan terkait teknologi informasi yang telah diberlakukan, seperti undang- undang, surat dinas dan barang legal lainnya yang mendorong penggunaan Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Public Policy Vol. 4, No. 2, September 2023 \|307 teknologi di lingkungan PDAM Surya Sembada Kota Surabaya. Peraturan Walikota Surabaya Nomor 29 Tahun 2011 tentang Pengelolaan dan Penyediaan Air Minum Bagi Perusahaan Daerah Air Minum Surya Sembada mengatur aspek kebijakan PDAM Surya Sembada Kota Surabaya. Berikut ini adalah fokus utama yang dipusatkan pada PDAM Surya Sembada Kota Surabaya untuk menentukan bagaimana undang-undang kebijakan yang berkaitan dengan layanan teknologi informasi online diterapkan antara lain; a) Koordinasi kegiatan dan anggaran Berdasarkan hasil temuan dilapangan dalam indikator penerapan peraturan kebijakan berkaitan dengan teknologi informasi pelayanan pengaduan online pelanggan telah diatur dengan baik. Hal tersebut dapat dibuktikan pada suatu kebijakan teknologi Strategic Plan yang tertuang dalam Surat Keputusan Direksi Nomor 55 Tahun 2016 serta tak terlepas dalam Peraturan Perusahaan Nomor 13 Tahun 2017. Penjelasan tersebut didukung oleh pernyataan informan kunci yakni Manajer Pelayanan dan Pengaduan Pelanggan PDAM Surya Sembada Kota Surabaya; “Jadi berbicara mengenai kebijakan pelayanan pengaduan online khususnya perencanaan kebijakan kami sudah membuat sebuah aturan, yang dimana aturan tersebut akan diputuskan dan di tanda tangani oleh para jajaran direksi. Selanjutnya juga ada masukan kebijakan dari stakeholder Pemerintah Kota Surabaya. Direksi disini menindaklanjuti input tersebut untuk diimplementasikan pada perusahaan” (Hasil wawancara, 02 Desember 2022). b) Standar Operasional Prosedure (SOP) pengadaan, perencanaan, pemanfaatan dan pengendalian. PDAM Surya Sembada Kota Surabaya memiliki SOP yang diatur dalam dokumen perusahaan SOP-PPG-03 Revisi 01, 2020. Prosedur pengaduan pelanggan ini bertujuan sebagai petunjuk dalam menangani setiap pengaduan pelanggan, sehingga diharapkan setiap pengaduan dapat diselesaikan secara cepat, tepat dan kepuasan pelanggan dapat tercapai. Keberhasilan penyelenggaraan ini juga disampaikan oleh informan kunci (Manajer Pelayanan dan Pengaduan Pelanggan) bahwa; “Kalau berbicara mengenai SOP pengaduan online sudah berjalan dengan baik, dan mengenai hambatan pengaplikasian SOP itu tidak ada ya, karena dari perancangan SOP itu harus dipatenkan sehingga sudah melewati standarisasi oleh pihak Peneliti dan Pengembangan.” (Hasil wawancara, 02 Desember 2022). Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Public Policy Vol. 4, No. Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Tim 3. Infrastruktur 3. Infrastruktur Kemudahan dan keterjangkauan akses informasi serta layanan pengaduan bagi masyarakat didukung dengan adanya penyediaan akses infrastruktur informasi yang bervariasi yang meliputi layanan Call Center, SMS, Website, Facebook, Twitter, Instragram dan Aplikasi. Idealnya jika terdapat variasi opsi penyediaan akses informasi dan penyampaian pengaduan pelanggan, maka dalam hal pelayanan dan respon ketepatan penyelesaian pengaduan oleh perusahaan juga akan semakin besar. Berdasarkan hasil penelitian di lapangan pada indikator infrastruktur PDAM Surya Sembada Kota Surabaya telah dilaksanakan sesuai dengan teknologi informasi Strategic Plan yang ada. Namun dalam hal implementasi dengan banyaknya variasi penyediaan akses penyampaian pengaduan dapat diketahui bahwa pelanggan menyampaikan pengaduan lebih dari satu kali serta melalui berbagai akses informasi, sehingga menyebabkan overlapping pengaduan yang masuk dan kecakupan dalam menyelesaian pengaduan online belum optimal. Dapat dibuktikan pada grafik penyelesaian pengaduan pelanggan berikut ini: Penyelesaian Pengaduan 2020-2022 No. Tahun Jumlah Pengaduan Pelanggan Jumlah Penyelesaian Pengaduan Presentase 1. 2020 302.872 132.414 44% 2. 2021 256.779 120.165 47% 3. 2022 (per-November) 204.632 117.404 57% Sumber; PDAM Surya Sembada Kota Surabaya, (2022) Penyelesaian Pengaduan 2020-2022 Sistem Informasi dapat dibantu supervisor dan staf yang formasi dan jabatannya sesuai kebutuhan dan ditetapkan dalam peraturan perusahaan. Sistem Informasi dapat dibantu supervisor dan staf yang formasi dan jabatannya sesuai kebutuhan dan ditetapkan dalam peraturan perusahaan. Sistem Informasi dapat dibantu supervisor dan staf yang formasi dan jabatannya sesuai kebutuhan dan ditetapkan dalam peraturan perusahaan. Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur 2. Kelembagaan Evaluasi aspek indikator kelembagaan bertujuan guna untuk penetapan keberadaan instansi yang berwenang dan bertanggungjawab atas penggunaan dan pemanfaatan teknologi informasi secara berkelanjutan. Evaluasi kelembagaan ini menyangkut; Evaluasi aspek indikator kelembagaan bertujuan guna untuk penetapan keberadaan instansi yang berwenang dan bertanggungjawab atas penggunaan dan pemanfaatan teknologi informasi secara berkelanjutan. Evaluasi kelembagaan ini menyangkut; a) Organisasi Fungsional Organisasi fungsional PDAM Surya Sembada Kota Surabaya telah dibentuk dengan baik serta memiliki legalitas. Sebagai bentuk komitmen perusahaan terhadap pengembangan teknologi informasi, PDAM Surya Sembada Kota Surabaya telah menyusun informasi elektronik Strategic Plan yang ditetapkan melalui Surat Keputusan Direksi Nomor 55 Tahun 2016 Tentang Penunjukan Pemimpin Proyek Tahun Anggaran 2016 di Bagian Teknologi Sistem Informasi (TSI). Hal tersebut didukung oleh pernyataan informan kunci (Manajer Pelayanan dan Pengaduan Pelanggan) sebagai berikut; “Sebagaimana yang sudah ada dalam peraturan perusahaan ya, memang pengelolaan TI itu yang menjadi tanggung jawab bagian Teknologi Sistem Informasi atau TSI itu. Tugas pokok yang satu diantaranya mengelola dan mengendalikan pengembangan teknologi dan sistem informasi perusahaan sesuai perkembangan bisnis yang ada.” (Hasil wawancara, 02 Desember 2022). a) Organisasi Fungsional Organisasi fungsional PDAM Surya Sembada Kota Surabaya telah dibentuk dengan baik serta memiliki legalitas. Sebagai bentuk komitmen perusahaan terhadap pengembangan teknologi informasi, PDAM Surya Sembada Kota Surabaya telah menyusun informasi elektronik Strategic Plan yang ditetapkan melalui Surat Keputusan Direksi Nomor 55 Tahun 2016 Tentang Penunjukan Pemimpin Proyek Tahun Anggaran 2016 di Bagian Teknologi Sistem Informasi (TSI). Hal tersebut didukung oleh pernyataan informan kunci (Manajer Pelayanan dan Pengaduan Pelanggan) sebagai berikut; “Sebagaimana yang sudah ada dalam peraturan perusahaan ya, memang pengelolaan TI itu yang menjadi tanggung jawab bagian Teknologi Sistem Informasi atau TSI itu. Tugas pokok yang satu diantaranya mengelola dan mengendalikan pengembangan teknologi dan sistem informasi perusahaan sesuai perkembangan bisnis yang ada.” (Hasil wawancara, 02 Desember 2022). b) Lembaga Khusus yang Menangani Teknologi Informasi Dalam sub-indikator ini PDAM Surya Sembada Kota Surabaya memiliki Bagian TSI yang dipimpin oleh seorang Manajer Teknologi Informasi yang memiliki tanggung jawab kepada Manajer Senior Kelola Sistem Informasi dan Aset Properti. Dalam melaksanakan tugas pokok dan fungsinya Manajer Teknologi Public Policy Vol. 4, No. 2, September 2023 \|309 4. Aplikasi 4. Aplikasi Hasil pengamatan di lapangan dalam indikator aplikasi PDAM Surya Sembada Kota Surabaya dapat diakses melalui website www.pdam-sby.go.id serta aplikasi PDAM Surabaya telah diperbaruhi menjadi Customer Information System (CIS) yang dapat diunduh pada gawai berbasis android. Melalui Bagian Teknologi Informasi (TSI) telah melakukan pengembangan aplikasi berbasis teknologi informasi terkait sistem pelayanan kepada masyarakat, pengendalian proses dan pengembangan sumber daya manusia. PDAM Surya Sembada Kota Surabaya telah melakukan resertifikasi ISO 27001:2013 untuk mempertahankan sertifikasi ISO 27001 yang telah didapatkan sejak tahun 2019. Hasil temuan dan informasi dapat diketahui bahwa dalam aspek aplikasi pelayanan online belum memiliki fitur monitoring progres pelayanan yang dapat dipantau oleh pelanggan serta aplikasi belum tersedia pada pengguna IOS/Play Store. Hal ini selaras dengan yang disampaikan oleh Manajer Pelayanan dan Pengaduan Pelanggan sebagai berikut; “Aplikasi atau teknologi informasi kami sudah berstandar ISO, jadi dari sini mungkin bisa menjadi jaminan bagi pelanggan bahwa data yang telah diserahkan pada kami akan terjamin keamanannya.” (Hasil wawancara, 02 Desember 2022). “Aplikasi atau teknologi informasi kami sudah berstandar ISO, jadi dari sini mungkin bisa menjadi jaminan bagi pelanggan bahwa data yang telah diserahkan pada kami akan terjamin keamanannya.” (Hasil wawancara, 02 Desember 2022). No. Sumber; PDAM Surya Sembada Kota Surabaya, (2022) Hasil wawancara oleh salah satu pelanggan “GSK” berdomisili di Wonocolo Surabaya menjadi pendukung bahwa masih terdapat kendala dalam implementasi pelayanan online di PDAM Surya Sembada Kota Surabaya sebagai berikut; “Pengaduan online PDAM itu saya tau, memang cara pakainya gampang dan jelas. Datangnya respon petugas ini yang lama sekali. Saya beberapa kali mengadu lewat aplikasi dan WhatsApp tetap tidak ada respon. Menghubungi call center juga lama untuk terhubung. Kalau saya milih dating langsung ke CS saja lebih cepat.” (Hasil wawancara, 08 Maret 2023). “Pengaduan online PDAM itu saya tau, memang cara pakainya gampang dan jelas. Datangnya respon petugas ini yang lama sekali. Saya beberapa kali mengadu lewat aplikasi dan WhatsApp tetap tidak ada respon. Menghubungi call center juga lama untuk terhubung. Kalau saya milih dating langsung ke CS saja lebih cepat.” (Hasil wawancara, 08 Maret 2023). Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Public Policy Vol. 4, No. 2, September 2023 \|310 Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Penutup Pengaplikasian e-government untuk pelayanan publik di PDAM Surya Sembada Kota Surabaya masih belum optimal. Namun demikian, pihak PDAM Surya Sembada Kota Surabaya terus berupaya untuk memperbaiki e-government untuk mengoptimalkan kinerja pelayanan publiknya. Situs e-government yang dapat diakses untuk mengetahui perkembangan PDAM Surya Sembada Kota Surabaya adalah dengan mengakses website www.pdam-sby.go.id juga dapat diakses melalui aplikasi Customer Information System (CIS). Kondisi saat ini bahwa dalam pengelolaan e-government untuk pelayanan publik masih belum optimal. Hal ini ditandai dengan masih didapati ketidaktepatan penanganan pelayanan online sesuai dengan acuan waktu yang diatur oleh perusahaan. Hal lainnya bahwa indikator infrastruktur masih belum optimal karena terdapat overlapping data pengaduan yang masuk pada sistem serta dalam indikator aplikasi masih belum memiliki fitur monitoring pelayanan bagi pelanggan dan ketersediaan aplikasi hanya dapat diakses pengguna android mobile. Dalam upaya meningkatkan penggunaan e-government pada rangka pengoptimalan kinerja pelayanan publik di PDAM Surya Sembada Kota Surabaya, disarankan pada indikator kebijakan SOP perlu dilakukan standarisasi ulang yang berorientasi dengan inovasi e-government guna untuk menentukan arah perbaikan kinerja sesuai dengan kebutuhan perusahaan. Pada indikator infrastruktur perlu adanya evaluasi penggunaan sistem internal perusahaan agar meminimalisir terjadinya overlapping data. Selanjutnya indikator aplikasi juga perlu dilakukan inovasi penambahan fitur monitoring bagi pelanggan dan PDAM Surya Sembada Kota Surabaya perlu menambah jangkauan pelanggan lebih luas melalui ketersediaan aplikasi berbasis IOS/App Store. 5. Perencanaan Berdasarkan hasil penelitian di lapangan, indikator perencanaan PDAM Surya Sembada Kota Surabaya sudah optimal, terlihat pada tata kelola teknologi informasi mendorong perusahaan untuk memiliki seperangkat kebijakan dan prosedur sistem informasi dalam bentuk tata kelola informasi perusahaan, rencana induk teknologi informasi (MPTI), informasi pedoman keamanan dan pengaturan pemanfaatan jaringan, email dan hak akses internet didalam perusahaan. Evaluasi peningkatan perencanaan kualitas layanan melalui pemanfaatan teknologi informasi selalu dilakukan sebagai satu upaya preventif dalam menghadapi resiko operasional yang berkaitan dengan penyusunan dokumen kebijakan internal yang berkaitan dengan e-government. Hal tersebut didukung oleh pernyataan Manajer Pelayanan dan Pengaduan Pelanggan sebagai berikut; Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Public Policy Vol. 4, No. 2, September 2023 \|311 “Tentang teknologi informasi kami melakukan perencanaan evaluasi yang dimana yang berkontribusi disini itu ranah Divisi Teknologi Sistem Informasi (TSI). Untuk perencanaan atau evaluasi pemeliharaan kita melakukan rutin dalam kurun waktu setahun sekali.” (Hasil wawancara, 02 Desember 2022). Astuti, N. (2014). Penyediaan Air Bersih oleh Perusahaan Daerah Air Minum (PDAM) Kota Sangatta Kabupaten Kutai Timur. 3 (2), 678–689. Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Daftar Pustaka Astuti, N. (2014). Penyediaan Air Bersih oleh Perusahaan Daerah Air Minum (PDAM) Kota Sangatta Kabupaten Kutai Timur. 3 (2), 678–689. Shofi Yorinda Febiyanti & Arimurti Kriswibowo – Univeristas Pembangunan Nasional “Veteran” Jawa Timur Public Policy Vol. 4, No. 2, September 2023 \|312 Didi Susianto, Eka Ridhawati dan Sucipto, (2022) Implementasi E-Government Berbasis Android untuk Meningkatkan pelayanan Publik, Jurnal Al Azhar Indonesia Seri Sains dan Teknologi, Vol. 7, No. 3, September 2022, http://dx.doi.org/10.36722/sst.v7i3.1143. Fitriansyah, A., Budiarto, H., & Santoso, J. (2013). Metode Pemeringkatan E- Government Indonesia (PeGI) untuk Audit Tata Kelola Teknologi Informasi. In Seminar Nasional Sistem Informasi Indonesia, https://is.its.ac.id/pubs/oajis/index.php/home/event/SESINDO. Heriyanto, (2022) Urgensi Penerapan E-Government dalam Pelayanan Publik, Musamus Journal of Public Adiministration, Vol. 4, No. 2, 2022, https://doi.org/10.35724/mjpa.v4i2.4128. Instruksi Presiden Nomor 3 Tahun 2003, Retrieved from https://peraturan.bpk.go.id/Home/Details/147277/inpres-no-3-tahun-2003. Instruksi Presiden Nomor 3 Tahun 2003, Retrieved from https://peraturan.bpk.go.id/Home/Details/147277/inpres-no-3-tahun-2003. Khothimah, K., & Hertati, D. (2021). Evaluasi Kebijakan Program Bantuan Sosial Tunai. Jurnal Governansi, 99-110, https://ojs.unida.ac.id/JGS/article/view/4230. Saadah, M. A. (2023). Responsivitas dan Transparansi Pemerintah Kota Yogyakarta dalam Menangani Keluhan. PUBLIKA: Jurnal Ilmu Administrasi Publik, 9(1), 99- 114, https://journal.uir.ac.id/index.php/JIAP/article/view/12719. Sekretariat Negara, Peraturan Presiden Republik Indonesia Nomor 95 Tahun 2018 Tentang Sistem Pemerintah Berbasis Elektronik, Retrieved from https://peraturan.bpk.go.id/Home/Details/147277/inpres-no-3-tahun-2003. Setyarini, W. A. (2022). Survei Kepuasan Masyarakat Terhadap Pelayanan Pengaduan Masyarakat Lapor Hendi Tahun 2021. Jurnal Riptek, 16(2), 90-96, https://doi.org/10.35475/riptek.v16i2.157. Setyarini, W. A. (2022). Survei Kepuasan Masyarakat Terhadap Pelayanan Pengaduan Masyarakat Lapor Hendi Tahun 2021. Jurnal Riptek, 16(2), 90-96, https://doi.org/10.35475/riptek.v16i2.157. Syafirah, L., & Nawangsari, E. R. (2021). Efektivitas Pelayanan Pengaduan di Perusahaan Daerah Air Minum (PDAM) Giri Tirta Kabupaten Gresik. Syntax Literate ; Jurnal Ilmiah Indonesia, 6(2), 928. doi: 10.36418/syntax- literate.v6i2.2170, https://doi.org/10.36418/syntax-literate.v6i2.2170.
https://openalex.org/W2100666401	https://ojs.unisa.edu.au/index.php/IJEI/article/download/672/499	English	null	The customer isn't always right: Limitations of 'customer service' approaches to education or why Higher Ed is not Burger King	International journal for educational integrity	2,010	cc-by	6,299	Abstract The increasingly popular trend of conceptualising education in terms of “customer service” is, in some ways, attractive. It encourages educators to think in terms of meeting students’ needs and to develop innovative ways to deliver their “product.” In other ways, however, it fails to convey the essential collaborative, participatory, reciprocal relationship that is central to effective teaching and learning. With respect to academic integrity, the customer service model also obscures students’ roles and responsibilities. In this paper, we identify some of the ways this model provides an inappropriate metaphor for understanding the project of teaching and learning (i.e., education) and argue that, when embraced uncritically, the model has the potential both to undermine education and at the same time derail efforts to develop and sustain a culture of integrity. After identifying this model’s shortcomings, we suggest ways to develop and promote a more robust model in which faculty and students work together toward a shared purpose while recognising and embracing their interlocking responsibilities. Teddi Fishman The International Centre for Academic Integrity, Clemson University (USA) TFISHMA@clemson.edu Teddi Fishman The International Centre for Academic Integrity, Clemson University (USA) TFISHMA@clemson.edu Keywords: customer service, integrity, reciprocal roles and responsibilities, teaching and learning. This is a revised version of a paper presented at the 4th Asia Pacific Conference on Educational Integrity: Creating an Inclusive Approach, University of Wollongong, NSW, Australia, 2009. International Journal for Educational Integrity Daniel E Wueste Rutland Institute for Ethics, Clemson University (USA) ERNEST@clemson.edu A re-examination Sometimes it is helpful to think in a new way, to see things differently, because, inter alia, things that were not seen or appreciated previously come into view, perhaps revealing new possibilities/approaches that hold great promise for success in an undertaking. In a complex multi-faceted undertaking, however, one has to be careful not to adopt an explanatory schema uncritically; what works very well with one facet of the operation – in the sense of revealing new possibilities and approaches, for example, may be unhelpful or counterproductive in another. It is important to recognise the limitations inherent in each approach – the ways in which the analogy (in this case, the analogy of customer service to teaching and learning) falls short. Failing to do so can limit the potential usefulness of the approach, and frustrate the operation as the new way of thinking meets with resistance from practitioners better positioned to see and anticipate problems than administrators or managers who work some distance from the trenches. There is another reason for care in the choice of how to see and talk about things. How we conceptualise an undertaking can shape our choices and actions. We can see the need for caution in the business arena, where people have been encouraged to think of business as war, a money making machine, a race, or a game; to believe that Machiavelli and Attila the Hun are good managerial role models; and to see all business people as entrepreneurs, for example. Discussing these ways of thinking about business, each of which must be rejected, because (first) they misrepresent business, Robert Solomon puts the key point forcefully: How we talk reflects how we think, and how we think affects how we act and the nature of the organizations and institutions we create for ourselves. If we talk like brutes and we think like brutes, we will act like brutes and build organizations suitable only for brutes. To be sure, even in such organizations, some people will prosper, but life for most of them will be nasty, brutish and short. (Solomon, 1989, p. 12) The key point bears repetition. There is a connection between the way we talk and the way we think; the way we think influences our actions, including those involved in the creation, maintenance, functioning and success of the institutions we work in. Customer service as a conceptual model for education There are many reasons why customer service has become increasingly popular as a way to conceptualise higher education. Indeed, some features that recommend it are compelling: We should strive to be responsive to student needs in much the same way that businesses try to anticipate and meet the demands of the market. We must compete for the best students in a way that is similar to businesses competing for customers. Accordingly, we have indeed come to recognise the benefits of advertising, marketing ourselves, and inspiring “brand loyalty”. Additionally, whether we like it or not, because of decreasing public funding, higher education is increasingly subject to market forces. In fact, for public universities at least, the ability to carry out the mission often calls for entrepreneurial activity aimed at making universities self-sustaining financially. So too, we know that we must respond swiftly and intelligently to trends in The International Journal for Educational Integrity is available online at: http://www.ojs.unisa.edu.au/journals/index.php/IJEI/ © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 3 order to remain on the cutting edge and be able to offer the latest and greatest in courses, facilities, and approaches. At the same time, however, we must be cognisant of the ways in which what we do in higher education differs from commerce. Similarities can be pressed too hard, and when they are, what looked to be sheer gain can end up being an unbalanced and infelicitous mix. Customer service The customer service model, which has its roots in the total quality movement that started with William Edwards Deming (Lomas, 2007, p. 31) has been aggressively promoted in higher education. The idea that has been pressed is that an embrace of this model is imperative, “given the fact that in order to be effective organizations must be customer-driven,” (Maguad, 2007, p. 334) or, alternatively, the model must be embraced because “even the best organization in the world will be ineffective if the focus on ‘customers’ is lost” (Grant & Anderson, 2002, p. 23). Resisters are told that properly understood the model is “generally applicable to all kinds of organizations”; moreover, “[e]veryone of us is a customer. Everyone of us serves customers” (Maguad, 2007, p. 334). Those of us who resist thinking of our students as customers are told to “wake up and smell the coffee,” and stop thinking that there is something special about higher education (Lomas, 2007, p. 33). Managerial principles applied in commercial service organisations and other public-sector service organisations, such as health trusts, hospitals. and local councils, are just as relevant in a higher education context. Managerialism is universal and, as Ritzer (1996) suggests, consumers in universities require the same standardisation, reliability and predictability as they do when purchasing a burger meal or dealing with their bank. (Lomas, 2007, p. 33) Interestingly, the extraordinary and tortured efforts to salvage the notion that students are customers reveal much of what’s wrong with pressing the idea in the academy, or more precisely, in the arena of teaching and learning. The extent to which the definition of “customers” must be stretched to include students illustrates the significant differences in the two groups. Part of the confusion stems from a conflation of educational and business functions within universities. Clearly, there are customer service functions within institutions. Entities such as student housing (analogous to other real estate), food services (analogous to restaurants), and even some functions of bursar’s offices (analogous to banks) may, or should be run with an eye toward customer service. When it comes to the education of students, however, the goals, functions, practices and relationships are different from what one finds in commercial undertakings. A re-examination Indeed, this point is presupposed when ideas such as those Solomon rigorously and decisively critiques are put forward. Trouble is, these ways of thinking about business (that Solomon debunks) are, in fact, pernicious. Our point is softer than Solomon’s, since, as we shall see, the customer service model has some felicitous applications in colleges and universities. The same can not be said of the suggestion that Attila the Hun is a good managerial role model. Still, like Solomon, we have two main points: (1) the way we are asked to think about higher education does not accurately represent the relationship at the heart of the enterprise of teaching and learning, that is, the relationship between teacher and student, and (2) because of this it frustrates the maintenance, functioning and success of the enterprise and, not incidentally, our efforts to promote academic integrity. We will argue, therefore, that we should not use customer service terms to conceptualise education because, as Kenneth Burke points out, “the nature of our terms affect[s] the nature of our observations” (Burke, 1989, p. 115). Choosing a set of terms, therefore, is significant, because in choosing, one highlights some aspects of the thing described and obscures others. “[By] its very nature as a terminology, it must be a selection of reality” (Burke, 1989, p. 166). Put more simply, the way we talk about what we do matters in part because it affects how we think about it. ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 4 Customer service Moreover, the delivery of knowledge does not entail a change of ownership – instead, it is an extension of ownership; the educator still “owns” the knowledge that has been given to the student – it has simply been shared; the student’s acquisition of knowledge does not diminish in any way the educator’s supply. One aspect of “return business,” of course, is customer satisfaction. This is a commonplace familiar to most people from the old saying that “the customer is always right”. Where market forces operate, the buyer is right in this sense: it is not in the interest of the seller to question or criticise the customer’s views since doing so would quite likely thwart his effort to make a sale (Lomas, 2007, p. 35). In commercial transactions, then, and in the ordinary understanding of the term, customers are the ultimate arbiters of quality, which is, of course, the linchpin of customer satisfaction. It is evident that advocates of the customer service model in higher education recognise the obvious problem here in thinking of students as customers, yet they insist on using the word “customer”. For example, Maguad writes, “The term customer can be defined as the recipient or beneficiary of the outputs of work efforts or the purchaser of products and services” (2007, p. 334). That this definition would have us calling those persons whose wartime deaths are called collateral damage, as well as the victims of the terrorist attacks on September 11, 2001 customers does not slow him down. Without missing a beat he writes, “Students are appropriately viewed as customers in the sense that they are beneficiaries with needs that should be satisfied. This view does not imply that they are the ultimate arbiters of quality in the commercial sense” (Maguad, 2007, p. 335). Later in the same article he lists several other ways in which students, though properly seen as customers, “differ from your typical business customers” (Maguad, 2007, p. 339). They are, he says, “admit[ted] selectively based on certain academic standards and requirements,” they are sometimes “prevent[ed] ...from purchasing ...[higher education] products and services,” and “often do not totally pay for the full cost of their tuition and fees”. Further, once admitted students “are continually tested and graded”; moreover, they “must maintain their good academic standing in order to be able to take more advanced courses and complete their programs of study” (Maguad, 2007, p. Customer service Commercial enterprises provide goods or services in a way that allows them to “make a profit, i.e., to obtain some value in excess of what [it] had before the exchange which is sufficiently flexible that it can be put to uses other than the immediate satisfaction [of the] needs and desires” of the provider (Camenisch, 1981, p. 62). With an eye to achieving this two-fold purpose a commercial enterprise will set itself to attracting customers who will buy – and continue to buy – its product or service. In the case of a product, there is a transfer or exchange of ownership and the provider’s holdings of the product that has been exchanged are diminished; the business needs to make more widgets. It’s fair to say that the educational enterprise is supposed to deliver something – knowledge – which will then be “owned” by the student. But while commercial enterprises can (and often do) measure success in terms of repeat business, in education one measure of success is that the student “gets” the “product” the first time and need not come back a second time; repeating a course represents failure – a bad thing – rather than “brand loyalty” – a good thing. The point can be seen from the other side as well: A brewery, for example, wants its customers to keep buying beer. That customers would learn to brew beer for themselves and thus reduce their reliance on the brewery runs contrary to the business model. Further, the transfer of knowledge that occurs in education does not diminish the provider’s holdings and prompt the provider to produce more. To be sure, educators are interested in and 5 ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 strive to increase knowledge, but what’s going on here is quite unlike the situation in business where, for example, having delivered widgets to many customers, more will have to be made in order to have a supply adequate to meet demand. Professional practice The practice of clinical psychology is another example of a profession where cooperative engagement is required to bring about the desired outcome. Here, in order to be successful, a client must work with a therapist to bring about a desired result. Although the professional guides the process, it is the client who must do the work and the client’s progress (not the work of the professional) is the ultimate measure of success. It is worth noting, in this connection, that in the practice of clinical psychology a professional must sometimes reject a request made by the client/consumer because, in the therapist’s professional judgment, the client’s needs would not be well served; so too in education, where the client/consumer does not always have the requisite knowledge to know what is needed. Much the same thing could be said of physicians, attorneys, and architects, i.e., professionals. As sociologist Everett C. Hughes notes, “‘[p]rofession’ originally meant the act or fact of professing. It has come to mean: ‘The skilled occupation that one professes to be skilled in and to follow”. The implications are significant. For the present purpose, this is key: because they profess, Hughes continues, professionals ask to be trusted. The rule with professional practice, he says, is credat emptor. The reason for this is that, in general, because they lack the expertise of the professional they have come to, nonprofessionals are not in a position to evaluate their judgments, advice, and performance; in short, they must trust them. This rule, credat emptor (i.e., buyer give credence to, trust) distinguishes the professional relation from the relation of vendor- vendee found in markets where, as is well known, the rule is caveat emptor (buyer beware) (Hughes, 1965, pp. 1–3). All of this suggests what we believe to be quite true, namely, that looking to professional practice (Wueste, 1994, pp. 4–16) for help in understanding the educational enterprise holds considerably more promise than looking to business practice. Lastly but perhaps most significantly, the relationship between student and teacher is very different from that of vendor and vendee, i.e., customer. In the case of the latter, the relationship exists to facilitate a transaction or transactions. Customer service Knowledge and truth are not market commodities in the way that pork bellies and petroleum are; recall the earlier point about how delivery does not diminish the supply. Our professional roles and our institutional charters both dictate that we recognise educational needs and attempt to meet them. demand is not enough to justify a change in course. Put another way, whereas consumer markets can respond to wants, even wants that are arbitrary or manufactured, educators are obliged to meet needs, even when those needs are unrecognised by those who have them. Knowledge and truth are not market commodities in the way that pork bellies and petroleum are; recall the earlier point about how delivery does not diminish the supply. Our professional roles and our institutional charters both dictate that we recognise educational needs and attempt to meet them. The third and perhaps the most obvious way that education differs from commercial enterprises has to do with their practices, and more specifically, who engages in them. In commercial exchanges, the terms of the exchange usually specify that the customer gives something (usually money) in exchange for goods or services which the purveyor is then obliged to provide. It is an exchange so familiar that we take it for granted – I pay, you provide. In education, however, although payment is made the “product” can never be successfully “delivered” unless the student participates actively. An education can not be had unless the person being educated is engaged in the process. Lessons can be given, but learning remains the responsibility of the learner. The practice of education is, then, necessarily cooperative; it is not a simple exchange of services for pay. It is more like what most people would readily recognise as professional practice than it is like business practice, the customer service model’s suggestion to the contrary notwithstanding. Customer service 339). Would we be mistaken to suggest that by this account students are customers only in a Pickwickian sense? Perhaps it would be more politic to say it is a highly theoretical sense. Either way, the problem is the same. Those who hear the term take it not in a Pickwickian or theoretical sense, but in its ordinary sense, i.e., the sense in which the term is most commonly used. More than one student has said, during the advising meetings in which they choose courses to fulfill university requirements, that they don’t understand why they are required to take courses that they have no interest in. “I’m paying for the courses – why can’t I choose the ones that I want?”. When seen through the lens of a customer, this seems a perfectly reasonable expectation whereas when seen through the lens of a student, the expectation that curricular choices are exclusively theirs seems far less apt. To return to Burke, we find that the use of particular terms directs our attention. In this case, we suggest that the use of customer service terminology often misdirects. A second way that the educational enterprise differs significantly from commerce emerges when we consider the relationship between functions and constraints. While the market is free to respond to trends, whims, and fancy, and to deliver whatever product seems attractive at the time, the raison d’etre of education is much more narrowly defined. We have one primary function, which although responsive to the greater world, and flexible in some measure, remains relatively constant: we educate. It will never (and should never) be the case that the secondary services we provide (food, housing, transportation, banking, and athletic spectacle/entertainment, for example) become primary, regardless of how popular or financially profitable they become. To be sure, education is not immune to market forces. However, the action- reaction nexus in education is not the same as in business. In education, mere market 6 ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 demand is not enough to justify a change in course. Put another way, whereas consumer markets can respond to wants, even wants that are arbitrary or manufactured, educators are obliged to meet needs, even when those needs are unrecognised by those who have them. Teacher – Learner The relationship between teacher and learner is also a dyadic relation: xTy “x teaches y” (alternatively yLx “y learns from x”). The upshot of the relation is not, however, mere exchange: The relation creates or produces something (education) rather than transferring something. There is another difference between this relation and that of vendor and customer: In this case the expectations which, if thwarted, lead to complaints of wrongdoing – that one party has been wronged – are found on both sides. This is a rich reciprocal relation. It’s here that one finds the moral nub of academic integrity; it’s intrinsic to the enterprise of education, the heart of which is the relationship between teacher and learner. One seeming complication is that in contemporary higher education students work with and rely on their peers in making the most of collaborative learning environments and opportunities. Yet, in a very real sense, in these learning situations they, i.e., the students, are in it together and something akin to the relationship between teacher and learner obtains among all those involved in the teaching and learning experience creating a nexus of (potentially) beneficial reciprocal relations. Nothing like this occurs with customers; customers aren’t “in it together”; indeed in some cases (think Christmas toy shopping or online auctions) they are competing with one another. Still, the customer model does direct us to things that need attention. For instance, the model reveals how important it is for teachers to be aware of the needs and abilities of students (learning styles and so on too). That’s part of its attractiveness, perhaps even its intuitive appeal. Trouble is, it also (wrongly) positions students as passive rather than active participants in the process. In a customer-service model, there is no expectation that they will be contributing partners in what is in fact a necessarily participatory process (of teaching and learning). This model, as it is commonly understood, dictates that apart from wanting what the other seems positioned to provide, and paying to effect a transfer of it, there’s nothing for the student to do. If we are guided by these understandings, we face two very unpalatable consequences: (1) success in the educative enterprise becomes less likely, and (2) the interactive reciprocity of the enterprise becomes obscured, thereby diminishing the significance (or the salience) of academic integrity. Vendor – Vendee, i.e., Customer Vendor – Vendee, i.e., Customer The relationship between vendor and vendee is a dyadic relation: xSy “x sells to y” (alternatively yBx “y buys from x”). The relation is a simple one: a transaction/ exchange in which a good or service is exchanged for money. The relation is not entirely balanced as the obligation to perform rests almost completely with the vendor. Apart from a failure to pay, it would be unusual, at least, if the vendor were to claim that he had been wronged by the customer. The seller needs to attend to the needs of the customer in a way that the customer does not and needn’t match – it’s a sparse rather than a rich reciprocal relation. Professional practice The vendor hopes to maintain the relationship as long as possible in order to continue to amass transactions (and therefore continue to amass profits.) In the former case, the goal is focused upon the improvement of the student, and the goal is similar to that of a 7 ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 parent – to reach a point where though continued interactions may be welcome indeed, they are unnecessary. Continued interaction is then by choice rather than necessity. Teacher – Learner As we have seen, there are several reasons for rejecting the language and practices of customer service in education, but perhaps the most important is this: In most customer service environments, the responsibility of customers is limited to articulating their needs and paying for the goods or services that are produced in response. Even though there are a number of businesses that refer to their customers as “partners,” there are very few for whom the responsibility for what is produced is truly a shared concern. ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 8 An alternative model A good place to start in developing an alternative to the customer service model is the idea, discussed briefly above, that there is much to be gained in looking to professional practice. In fact, starting here promises forward progress on two fronts: (1) in our understanding of the educational enterprise, and (2) in seeing new and promising ways to promote academic integrity. By returning to the idea that education is, in fact, a professional rather than customer service concern, we will focus attention more properly on the practices, essential goals, functions, and relationships that are most central to teaching and learning. Practices comprise norms that prescribe or proscribe certain acts as well as norms that specify roles and functions; the accountability of those responsible for discharging these functions is largely, though not completely, a matter of compliance with these norms. Legal philosopher Lon Fuller puts the point succinctly; practices include both “a vocabulary of deeds and a basic grammar” (Fuller, 1981, pp. 243–244). The norms of a practice constitute a set of internal principles that, in fact, make the practice possible, in the sense of creating the capacity to achieve its purpose(s). Fuller’s idea about the norms internal to a practice, which we are adapting here, emerges in a story he tells about a hapless King Rex who fails as a lawmaker in eight distinguishable ways. The lesson we learn, according to Fuller, is that lawmakers will fail to make law unless they comply with principles internal to the legal enterprise. The eight principles of the legal enterprise require laws to be 1) general, 2) promulgated, 3) typically prospective, not retroactive, 4) clear – that is, readily intelligible. Laws must also be such that the acts they require are 5) neither incompatible nor 6) impossible to perform. Moreover, 7) laws must not be changed too frequently, and finally, 8) there must be a congruence between the rules as declared and the rules as administered. As Fuller has it, these eight principles constitute the morality that “makes law possible”. Put another way, these norms are key elements in an explanation of how lawmaking is possible, in particular, of how the legal enterprise can achieve the purpose of making behaviors normatively non-optional, in a word, obligatory (Fuller, 1969, p. 39). Fuller can speak for himself: Certainly there can be no rational ground for asserting that a man can have a moral obligation to obey a legal rule that does not exist, or is kept secret from him, or that came into existence only after he had acted, or was unintelligible, or was contradicted by another rule of the same system, or commanded the impossible, or changed every minute. It may not be impossible for a man to obey a rule that is disregarded by those charged with its administration, but at some point obedience becomes futile – as futile, in fact, as casting a vote that will never be counted. As the sociologist Simmel has observed, there is a kind of reciprocity between government and the citizen with respect to the observance of rules. (Fuller, 1969, p. 39) The “principle of reciprocity” is a key element in Fuller’s account of law’s capacity to obligate. Indeed, as Fuller has it, “the notion of reciprocity [is] implicit in the very notion of duty – at least in the case of every duty that runs toward society or toward another responsible human being” (Fuller, 1969, p. 21). Thus, a complete rupture of the “bond of reciprocity” between government and citizen leaves nothing “on which to ground the citizen’s duty to observe the rules” (Fuller, 1969, pp. 39–40). Fuller maintains, rightly we believe, that the principle of reciprocity “has roots…in our professions [and] in our practices” (Fuller, 1969, p. 21). In some cases, he says, “it is obvious to those affected by it; [in others] it traces a more subtle and obscure course through the institutions and practices of society” (Fuller, 1969, p. 22). ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 9 Returning to the idea that norms internal to a practice explain its ability to achieve its purpose(s), it may be helpful to consider a simpler case than lawmaking. The practice of casting ballots involves a settled and stable expectation that a vote cast for candidate A will be counted as a vote for candidate A. The principle here, that votes should (must) be faithfully counted, is intrinsic to the practice of voting or elections. Fuller can speak for himself: Both the meaning of a voter’s participation in an election and the successful functioning of the practice depend on compliance with it (Fuller, 1981, p. 92). Again, the key idea of the approach we advocate in developing an alternative to the customer service model is the idea that the capacity of a practice or enterprise to achieve its purpose depends on compliance with a set of internal principles. Fuller puts the idea another way, and as we shall see, it resonates deeply with our suggestion that by returning to the idea that education is, in fact, a professional rather than customer service concern and focusing attention on the essential practices, goals, functions, and relationships that are most central to teaching and learning we will be able to see new and promising ways to promote academic integrity. What he says is this: an enterprise such as legislation or election, for example, “[has] an integrity of [its] own which must be respected if [it is] to be effective at all” (Fuller, 1981, p. 91). Let’s apply this idea to the academy and ask what is required if the educational enterprise of higher education is to achieve its purpose(s): please take a moment to think about this and then write down what you have come up with. Please do this before reading further. Really.... Here’s a provisional response to the question “What is required if the educational enterprise of higher education is to achieve its purpose(s)?”:  honest reports and representations  transparency/openness rather than secretiveness  trust, which is the foundation of reasonable reliance  accountability, on an individual as well as institutional level  recognition of limits  respect (but not deference) for established authority and innovation  a mechanism for attribution and citation that creates an accurate “trail” of ideas and findings; this is necessary for several purposes including  giving credit where credit is due  knowing where an investigator/writer/thinker made a wrong turn  knowing where to go to find out more – to continue and enhance the inquiry h i i ht i id (i t ll t l t )  honoring rights in ideas (intellectual property)  a sense of community and common purpose that can keep the competitive drive of individuals on a short leash. Fuller can speak for himself:  a sense of community and common purpose that can keep the competitive drive of individuals on a short leash. What we have here are ideas/norms implicit in the educational enterprise that are essential elements in the collection of things necessary for successful teaching and learning. It’s not surprising that they might just as well be referred to as elements of academic integrity in the Fullerian sense that the academy “[has] an integrity of [its] own which must be respected if [it is] to be effective at all”. Moreover, the reciprocity implicit in them seems to confirm the idea, stressed earlier, that the success of the educational enterprise depends on the efforts of those being “served” at least as much as it depends on the efforts of those providing the “service”. The suggestion that we will make in closing (since making the full dress case is beyond the scope of our project here) is that seeing the principles of academic integrity in this way should make it easier for students to (1) understand why those of us who have made our 10 ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 careers in the academy expect, in fact, demand compliance with them, and (2) come to “own” them and see them as constraints one accepts voluntarily when one becomes a participant in the enterprise of higher education. careers in the academy expect, in fact, demand compliance with them, and (2) come to “own” them and see them as constraints one accepts voluntarily when one becomes a participant in the enterprise of higher education. About the authors Daniel E. Wueste is director of the Robert J. Rutland Institute for Ethics at Clemson University (www.clemson.edu/ethics). He did his graduate work in philosophy at the University of Wisconsin-Milwaukee (M.A., 1979) and Washington University in St. Louis (Ph.D., 1985). His research and writing focus on issues in three areas: legal philosophy, social and political philosophy, and professional ethics. Wueste’s work has appeared in various journals including Cornell Law Review, Harvard Journal of Law and Public Policy, the Canadian Journal of Law and Jurisprudence, Teaching Ethics, and Dimensions in Critical Care Nursing. He is the author of the chapter on professional ethics in The Biomedical Engineering Handbook, 2nd edition and the chapter on biomedical ethics in the Encyclopedia of Biomaterials and Biomedical Engineering. In 2009 Wueste was reelected president of the Society for Ethics Across the Curriculum. Teddi Fishman is Director of the International Center for Academic Integrity (http:// www.academicintegrity.org/). She earned M. A. degrees in both Professional Communication and English from Clemson University in 1995 and 1996 respectively before completing a Ph.D. in Rhetoric and Composition at Purdue University in 2002. Teddi’s research is interdisciplinary and addresses such diverse topics as the rhetoric of police writing, identity in online environments, and the teaching of multi-media literacy. Her work has been published in a number of edited collections as well as in online and print journals including Kairos and The Journal of Managerial Accounting. One of the core beliefs that unites her eclectic and seemingly disparate collection of professional endeavors and achievements is the belief that personal and systemic integrity in education, communication, and media are essential for the maintenance of a successful, sustainable, and just society. Conclusion We believe that an alternative to the customer service model should speak straightforwardly rather than in a Pickwickian fashion. The model we prefer relies on the mores of professional practice, highlights the reciprocal dimensions of teaching and learning, and articulates the principles that are central to the enterprise of education. ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 ternational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 Wueste, D. E. (1994). Introduction. In D. E. Wueste (Ed.), Professional ethics and social responsibility (p. 1). Lanham, Maryland: Rowman and Littlefield. References Burke, K. (1989). Symbols and society. Chicago: University of Chicago Press. Camenisch, P. (1981). Business ethics: On getting to the heart of the matter. Business and Professional Ethics Journal, 1, 59–62. Camenisch, P. (1981). Business ethics: On getting to the he and Professional Ethics Journal, 1, 59–62. Fuller, L. L. (1969). The morality of law (Rev. ed.). New Haven and London: Yale University Press. Fuller, L. L. (1981). In Winston K. I. (Ed.), The principles of social order. Durham, North Carolina: Duke University Press. Grant, G. B., & Anderson, G. (2002). Customer relationship management, A vision for higher education. In Katz, Richard N. and Associates (Ed.), Web portals and higher education technologies to make IT personal () Jossey-Bass Inc. Hughes, E. C. (1965). Professions. In K. S. Lynn, & & the editors of Daedalus. (Eds.), The professions in America (pp. 1–2,3). Boston: Houghton Mifflin Company. Lomas, L. (2007). Are students customers? Perceptions of academic staff. Quality in Higher Education, 13(1), 31–33, 45. 11 ernational Journal for Educational Integrity Vol. 2 No. 2 December 2006 pp. xx-xx ISSN 1833-2595 © International Journal for Educational Integrity Vol. 6 No. 1 July, 2010 pp. 3–12 ISSN 1833-2595 Maguad, B. A. (2007). Identifying the needs of customers in higher education. Education, 127(3), 332–343. Retrieved from http://search.ebscohost.com/ login.aspx?direct=true&db=aph&AN=24928359&site=ehost-live Solomon, R. (1989). A better way to think about business. New York: Oxford University Press. Wueste, D. E. (1994). Introduction. In D. E. Wueste (Ed.), Professional ethics and social responsibility (p. 1). Lanham, Maryland: Rowman and Littlefield. Maguad, B. A. (2007). Identifying the needs of customers in higher education. Education, 127(3), 332–343. Retrieved from http://search.ebscohost.com/ login.aspx?direct=true&db=aph&AN=24928359&site=ehost-live Solomon, R. (1989). A better way to think about business. New York: Oxford University Press. Wueste, D. E. (1994). Introduction. In D. E. Wueste (Ed.), Professional ethics and social responsibility (p. 1). Lanham, Maryland: Rowman and Littlefield. g p p Solomon, R. (1989). A better way to think about business. New York: Oxford University Press. Wueste, D. E. (1994). Introduction. In D. E. Wueste (Ed.), Professional ethics and social responsibility (p. 1). Lanham, Maryland: Rowman and Littlefield. 12
https://openalex.org/W3203395581	https://www.nature.com/articles/s41598-021-98660-y.pdf	English	null	Information flow in the rat thalamo-cortical system: spontaneous vs. stimulus-evoked activities	Scientific reports	2,021	cc-by	11,359	Information flow in the rat thalamo‑cortical system: spontaneous vs. stimulus‑evoked activities Kotaro Ishizu1, Tomoyo I. Shiramatsu1, Rie Hitsuyu1, Masafumi Oizumi2, Naotsugu Tsuchiya3,4,5 & Hirokazu Takahashi1* Kotaro Ishizu1, Tomoyo I. Shiramatsu1, Rie Hitsuyu1, Masafumi Oizumi2, Naotsugu Tsuchiya3,4,5 & Hirokazu Takahashi1* The interaction between the thalamus and sensory cortex plays critical roles in sensory processing. Previous studies have revealed pathway-specific synaptic properties of thalamo-cortical connections. However, few studies to date have investigated how each pathway routes moment-to-moment information. Here, we simultaneously recorded neural activity in the auditory thalamus (or ventral division of the medial geniculate body; MGv) and primary auditory cortex (A1) with a laminar resolution in anesthetized rats. Transfer entropy (TE) was used as an information theoretic measure to operationalize “information flow”. Our analyses confirmed that communication between the thalamus and cortex was strengthened during presentation of auditory stimuli. In the resting state, thalamo-cortical communications almost disappeared, whereas intracortical communications were strengthened. The predominant source of information was the MGv at the onset of stimulus presentation and layer 5 during spontaneous activity. In turn, MGv was the major recipient of information from layer 6. TE suggested that a small but significant population of MGv-to-A1 pairs was “information-bearing,” whereas A1-to-MGv pairs typically exhibiting small effects played modulatory roles. These results highlight the capability of TE analyses to unlock novel avenues for bridging the gap between well-established anatomical knowledge of canonical microcircuits and physiological correlates via the concept of dynamic information flow. The interaction between the thalamus and cortex is thought to play critical roles in sensory processing1,2. Ana- tomically, the middle cortical layer is the predominant recipient of thalamocortical projections, whereas the deep cortical layer is the source of cortico-thalamic projections3–9. This general structural pattern is observed across different thalamo-cortical systems and mammalian species, and is thus considered a canonical microcircuit in the thalamo-cortical system. These hodological motifs suggest that feedforward pathways originate principally from the supragranular layer (L2/3) and terminate in the granular layer (L4), whereas feedback pathways originate from the infragranular layers (L5/6) and avoid terminating in L410–16. Information flow within these anatomi- cal circuits is thought to be dynamic, with moment-to-moment variation in active pathways17–21. For example, communication between the thalamus and cortex is expected to be strengthened during stimulus presentation, whereas communication within the cortex is likely to be strengthened in the resting state in the absence of overt sensory processing. www.nature.com/scientificreports www.nature.com/scientificreports Methods i l Animals. This study was performed in strict accordance with the “Guiding Principles for the Care and Use of Animals in the Field of Physiological Science” published by the Japanese Physiological Society, and the recom- mendations in the ARRIVE guidelines (https://arriveguidelines.org/). The experimental protocol was approved by the Committee on the Ethics of Animal Experiments at the Research Center for Advanced Science and Tech- nology, University of Tokyo (Permit Number: RAC 130107). All surgeries were performed under isoflurane anesthesia. All efforts were made to minimize animal suffering. Following the experiments, animals were eutha- nized with an overdose of pentobarbital sodium (160 mg/kg, i. p.).h p g g p Four male Wistar rats were used in this study (11–13 weeks old; body weight, 290–330 g). The protocols for animal preparation and neural recordings have been described elsewhere20,31,37. Briefly, the rats were anesthetized with isoflurane and air at a concentration of 3% for induction and 1% for maintenance during the surgery and experiments. Animals were held in place with a custom-made head-holding device. Atropine sulfate (0.1 mg/ kg) was administered pre- and post-surgery to reduce the viscosity of bronchial secretions. A skin incision was made at the start of surgery under local anesthesia using lidocaine (0.3–0.5 mL). A needle electrode was subcu- taneously inserted into the right forepaw and used as the ground. A small craniotomy was performed close to bregma in order to embed a 0.5 mm-thick integrated circuit socket as a reference electrode, with electrical contact to the dura mater. The right temporal muscle, cranium, and dura overlying the auditory cortex were surgically removed. The exposed cortical surface was perfused with saline to prevent desiccation. Cisternal cerebrospinal fluid drainage was performed to minimize cerebral edema. The right eardrum, ipsilateral to the exposed cortex, was ruptured and waxed to ensure unilateral sound inputs from the ear contralateral to the exposed cortex. Respiratory rate, heart rate, and hind-paw withdrawal reflexes were monitored throughout the surgery to ensure maintenance of stable and sufficient anesthesia. For acoustic stimulation, a speaker (Technics EAS-10TH800, Matsushita Electric Industrial Co. Ltd., Japan) was positioned 10 cm from the left ear (contralateral to the exposed cortex). Test stimuli were calibrated at the pinna with a 0.25-inch microphone (4939, Brüel & Kjær, Denmark) and spectrum analyzer (CF-5210, Ono Sokki Co., Ltd., Japan). Stimulus levels were presented in dB SPL (sound pressure level in decibels with respect to 20 μPa). Electrophysiology. www.nature.com/scientificreports/ termed driver or modulator, respectively). Class 1 inputs express ionotropic glutamate receptors and constitute the main information-bearing pathway, whereas Class 2 projections express metabotropic receptors and modu- late the transmission of Class 1 inputs22–24. In the auditory system, Class 1 constitutes the main pathway from the ventral division of the medial geniculate body (MGv) to L4–L6 in the primary auditory cortex (A1). Class 2 projections are observed from the MGv to L2/3 and from L5/6 to the MGv25,26. Within the cortex, Class 1 and Class 2 are likely intermingled27–30. However, this synapse-based pathway characterization has yet to be validated by physiological neural recordings paired with information theoretical analyses, which will enable dynamic and quantitative determination of the nature of information flow. l To characterize the electrophysiological responses in the auditory thalamo-cortical system, we previously designed a microelectrode array that enabled simultaneous neural measurements in the MGv and every layer in A131. In the present study, we used transfer entropy (TE) to characterize pathway-specific information flow in the MGv-A1 system32–36. TE is a metric based on information theory that statistically quantifies the directed influence between two sets of time-series data.l l Here, we test our hypotheses (i) that TE analyses are able to reveal that information flow during spontaneous activity is distinct to that during stimulus-driven activity, (ii) that TE is able to capture feedforward/feedback flow during stimulus-evoked activity, which is consistent with well-established canonical microcircuits in the thalamo-cortical system, and (iii) that the TEs in MGv-to-A1 direction vary widely because of intermingled pro- jections of Class 1 and Class 2, while A1-to-MGv transmission is commonly characterized as small TEs of Class 2. Information flow in the rat thalamo‑cortical system: spontaneous vs. stimulus‑evoked activities Nevertheless, these differences have yet to be characterized comprehensively in the thalamo- cortical sensory system, especially at the level of neuronal spiking. y y , p y p g Beyond layer-based categorization, further subdivisions of thalamo-cortical pathways have been proposed based on synaptic properties, which may delineate specific roles in information transmission. For example, glu- tamatergic pathways in the thalamo-cortical system can be classified into either Class 1 or Class 2 (previously 1Department of Mechano‑Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 7‑3‑1 Hongo, Bunkyo‑ku, Tokyo 113‑8656, Japan. 2Department of General Systems Studies, Graduate School of Arts and Science, The University of Tokyo, 3‑8‑1 Komaba, Meguro‑ku, Tokyo 153‑0041, Japan. 3School of Psychological Sciences and Turner Institute for Brain and Mental Health, Monash University, Melbourne, VIC, Australia. 4Advanced Telecommunications Research Computational Neuroscience Laboratories, 2‑2‑2 Hikaridai, Seika‑cho, Soraku‑gun, Kyoto 619‑0288, Japan. 5Center for Information and Neural Networks (CiNet), National Institute of Information and Communications Technology (NICT), Suita, Osaka 565‑0871, Japan. email: takahashi@i.u-tokyo.ac.jp 1Department of Mechano‑Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 7‑3‑1 Hongo, Bunkyo‑ku, Tokyo 113‑8656, Japan. 2Department of General Systems Studies, Graduate School of Arts and Science, The University of Tokyo, 3‑8‑1 Komaba, Meguro‑ku, Tokyo 153‑0041, Japan. 3School of Psychological Sciences and Turner Institute for Brain and Mental Health, Monash University, Melbourne, VIC, Australia. 4Advanced Telecommunications Research Computational Neuroscience Laboratories, 2‑2‑2 Hikaridai, Seika‑cho, Soraku‑gun, Kyoto 619‑0288, Japan. 5Center for Information and Neural Networks (CiNet), National Institute of Information and Communications Technology (NICT), Suita, Osaka 565‑0871, Japan. email: takahashi@i.u-tokyo.ac.jp \| https://doi.org/10.1038/s41598-021-98660-y Scientific Reports \| (2021) 11:19252 www.nature.com/scientificreports/ Methods i l We used a surface microelectrode array and depth electrode array (NeuroNexus, Ann Arbor, MI, USA) to simultaneously measure neural activity in the auditory cortex and thalamus, as previously described28 (Fig. 1a). The surface microelectrode array comprising a 10 × 7 grid within 4 × 3 mm2 mapped local field potentials (LFPs) in the right temporal cortex to identify the location of the primary auditory cortex (A1)37. The depth microelectrode array was then inserted perpendicular to the cortical surface in A1. The array com- prised three shanks (6 mm in length), each of which constituted 15 distal recording sites for MGv and 17 proxi- mal sites for A1. The array simultaneously measured multi-unit activity (MUA) and LFPs from the MGv and A1. The diameter of recording sites was 30 μm. The center-to-center inter-electrode distance was 120 μm. The most distal site was placed 100 μm from the tip of the shank, and the distance between the most proximal site in the MGv and the most distal site in A1 was 1200 μm. Each electrode was composed of iridium oxide and coated with platinum black.i p Neural signals were amplified with a gain of 1000 (Cerebus Data Acquisition System; Cyberkinetics Inc. Salt Lake City, UT, USA) software. The digital filter bandpass was 0.3–500 Hz for LFP and 250–7500 Hz for MUA. The sampling rates for LFPs and MUA were 1000 Hz and 30 kHz, respectively. Multi-unit spikes were detected online from MUA by threshold-crossing (− 5.65 times root mean square of MUA).i y g Spontaneous activity was first characterized as MUA in a silent environment for 5 min. Auditory-evoked activity was then characterized in response to clicks and tone bursts. Clicks were presented at a rate of 1 Hz. Tone bursts were used to characterize the characteristic frequency (CF) at each recording site. CF was determined as the frequency at which test tones evoked MUA with the lowest intensity or the largest response at 20 dB SPL (the minimum intensity used in this study). Test frequencies ranged from 1.6 to 64 kHz with an increment of 1/3 octaves and intensities from 20 to 80 dB SPL with an increment of 10 dB. Each test tone was repeated 20 times Scientific Reports \| (2021) 11:19252 \| https://doi.org/10.1038/s41598-021-98660-y www.nature.com/scientificreports/ www.nature.com/scientificreports/ i Figure 1. Electrophysiological measurements of the auditory thalamo-cortical system. (a) Experimental setup. Methods i l (a) Experimental setup. A custom-designed microelectrode array was used to simultaneously measure multi-unit activity (MUA) and local field potentials (LFPs) from the ventral division of the medial geniculate body (MGv) in the thalamus and primary auditory cortex (A1) of anesthetized rats (i). The array was composed of three shanks (6 mm in length) (ii), each of which comprised 15 recording sites for the MGv at the distal edge and 17 recording sites for A1 on the proximal side (iii). (b) Identification of cortical layers. For LFPs in response to a click sound (i), current source density analysis (CSD) was performed (ii). (c) Click-evoked MUA in the MGv (bottom) and A1 (top). Raster plots for 5 s (i) and discharge rates within a given bin (ii) at 32 recording sites in a representative shank are depicted. The longitudinal axis corresponding to the depth of recording sites as depicted in inset (a-iii). (d) Spontaneous MUA in the MGv (bottom) and A1 (top). Data in (c) and (d) were obtained from the same rat. Red lines indicated burst periods in (c-i) and (d-i). in a pseudorandom order with an inter-tone interval of 600 ms. Recording sites at which CF was identified were defined as either MGv or A1, whereas those at which CF was not identified were excluded from further analyses. For the grand average of 240-trial click-evoked LFPs from the depth array, one-dimensional current source density (CSD) analysis (Fig. 1b) was conducted, as described previously28,38,39. Briefly, twice the potential at a given depth (V0) was subtracted from the sum of the potentials at the upper and lower adjacent sites of a given depth (Vu and Vl), and then divided by the square of the distance (Δx) between the recording sites (120 μm): (Vu + Vl −2V0)/x2. Each layer was defined based on the CSD results as follows: L4 was first defined as the site with the earliest sink and adjacent sites as sinks and no source. L2/3 was defined as sites above L4 with sinks, followed by short sources. L5 was defined as two successive sites with sources below L4. Weak sinks were identified in deeper sites, of which the second deeper site was defined as L6. Transfer entropy. TEs of either thalamo-cortical, intracortical, or cortico-thalamic projections were derived from MUA data of either spontaneous activity or click-evoked activity in a pairwise manner. Methods i l A custom-designed microelectrode array was used to simultaneously measure multi-unit activity (MUA) and local field potentials (LFPs) from the ventral division of the medial geniculate body (MGv) in the thalamus and primary auditory cortex (A1) of anesthetized rats (i). The array was composed of three shanks (6 mm in length) (ii), each of which comprised 15 recording sites for the MGv at the distal edge and 17 recording sites for A1 on the proximal side (iii). (b) Identification of cortical layers. For LFPs in response to a click sound (i), current source density analysis (CSD) was performed (ii). (c) Click-evoked MUA in the MGv (bottom) and A1 (top). Raster plots for 5 s (i) and discharge rates within a given bin (ii) at 32 recording sites in a representative shank are depicted. The longitudinal axis corresponding to the depth of recording sites as depicted in inset (a-iii). (d) Spontaneous MUA in the MGv (bottom) and A1 (top). Data in (c) and (d) were obtained from the same rat. Red lines indicated burst periods in (c-i) and (d-i). igure 1. Electrophysiological measurements of the auditory thalamo-cortical system. (a) Experimental setup. A d d l d d l l l ( A) d Figure 1. Electrophysiological measurements of the auditory thalamo-cortical system. (a) Experimental setup. A custom-designed microelectrode array was used to simultaneously measure multi-unit activity (MUA) and local field potentials (LFPs) from the ventral division of the medial geniculate body (MGv) in the thalamus and primary auditory cortex (A1) of anesthetized rats (i). The array was composed of three shanks (6 mm in length) (ii), each of which comprised 15 recording sites for the MGv at the distal edge and 17 recording sites for A1 on the proximal side (iii). (b) Identification of cortical layers. For LFPs in response to a click sound (i), current source density analysis (CSD) was performed (ii). (c) Click-evoked MUA in the MGv (bottom) and A1 (top). Raster plots for 5 s (i) and discharge rates within a given bin (ii) at 32 recording sites in a representative shank are depicted. The longitudinal axis corresponding to the depth of recording sites as depicted in inset (a-iii). (d) Spontaneous MUA in the MGv (bottom) and A1 (top). Data in (c) and (d) were obtained from the same rat. Red lines indicated burst periods in (c-i) and (d-i). Figure 1. Electrophysiological measurements of the auditory thalamo-cortical system. Methods i l TE was estimated from MUA data binarized with a bin of 1 ms (Fig. 2a). Bins with spikes were labeled as 1; those without Scientific Reports \| (2021) 11:19252 \| https://doi.org/10.1038/s41598-021-98660-y www.nature.com/scientificreports/ Figure 2. Estimation of transfer entropy from spike train data. (a) Schematic of transfer entropy (TE) from electrodes Y to X. Binned spike train data sets were generated from MUA data with 1-ms bins. The joint probability p Xt+lag, Xt+lag−d, Yt was estimated within a certain sampling window. See text for details. (b) Sampling window for TE analyses. (i) Long window of 10 s. TE was computed for neural activity with and without stimulus presentation (long-window TEstim and long-window TEspon, respectively). (ii) Short window of 15 ms. The time course of short-window TE was examined using moving window analyses. Figure 2. Estimation of transfer entropy from spike train data. (a) Schematic of transfer entropy (TE) from electrodes Y to X. Binned spike train data sets were generated from MUA data with 1-ms bins. The joint probability p Xt+lag, Xt+lag−d, Yt was estimated within a certain sampling window. See text for details. (b) Sampling window for TE analyses. (i) Long window of 10 s. TE was computed for neural activity with and without stimulus presentation (long-window TEstim and long-window TEspon, respectively). (ii) Short window of 15 ms. The time course of short-window TE was examined using moving window analyses. spikes were labeled as 0. None of the bins contained two or more spikes. The TE of Y to X or TEY→X was defined as follows: (1) TEY→X = H Xfuture\|Xpast −H Xfuture\|Xpast, Ypast (1) where H(A\|B) represents the conditional entropy in information theory, which indicates the unpredictability of A when information on B is known. TEY→X estimates how spikes at electrode Y ( Ypast ) improve the predic- tion of spikes at electrode X ( Xfuture ), beyond the prediction based on past data of X ( Xpast ). Here, TEY→X was calculated as follows: (2) TEY→X(t, lag) = Xt+lag X t + lag −d Yt p Xt+lag, Xt+lag−d, Yt log2 p Xt+lag\|Xt+lag−d, Yt p Xt+lag\|Xt+lag−d , (2) where t, lag, and d represent the time, transfer lag, and delay, respectively, between the future and past. Yt repre- sents the past state of electrode Y ( Ypast ). Xt+lag and Xt+lag−d represent the future and past states, respectively, of electrode X ( Xfuture and Xpast ). (ii) Short-window TE Short-window TE was computed using 15-ms windows to characterize information transmission in the thalamo-cortical system during the time window surrounding stimulus onset. The time course of information transmission for short-window TE was investigated using moving window analysis. Short-window TE was computed using 15-ms windows to characterize information transmission in the thalamo-cortical system during the time window surrounding stimulus onset. The time course of information transmission for short-window TE was investigated using moving window analysis. g g g y For trial i (= 1, …, 240), in response to a click delivered at time si, spike trains within 15-ms post-stimulus latency were used to derive short-window TE. Based on short-window TE at stimulus onset (short-window TEonset), we first identified significant information transmission and the optimal transfer lag, Lagopt, for a given electrode pair. For [si + 1, si + 15] := {si + 1 ≤t ≤si + 15} , the joint probability, p Xt+lag, Xt+lag−d, Yt , was obtained to derive TEY→X at a given lag (= 1, …, 30) according to Eq. (2). Short-window TEonset was ultimately defined as the median across 240 trials for each lag. The optimal transfer lag, Lagopt, was determined as the transfer lag that maximized the short-window TEonset. g We next characterized the time-course of short-window TE, i.e., how TE evolved over time in the thalamo- cortical system during the time window surrounding stimulus onset. We computed the short-window TE for T − 15+Lagopt 2 , T + 15+Lagopt 2 := T − 15+Lagopt 2 ≤t ≤T + 15+Lagopt 2 , where T ranged from si -10 to si + 40 and the lag was the optimal value in the short-window TEonset. When t was not an integer, t was rounded off to the nearest integer. The time course of short-window TE was ultimately defined as the median across 240 trials for each T. The earliest T when TE > 0 after bias correction (see the next section) was defined as the onset latency of information transmission. Statistical analyses for identification of significant information transfer. To identify electrode pairs with significant information transfer, we compared the above TEs derived from experimental data with those derived from shuffled data (TEshuffled). To generate the shuffled data, we randomly shuffled the inter-spike intervals (ISIs) of Xt and Yt without changing the ISI distribution. Methods i l The past data of X were obtained from d bins before a given time point of (t + lag), which were optimized as follows, assuming that Xt depends predominantly on past Xt-d: (3) d = argminH Xt\|Xt−d . . . (3) According to Eq. (1), we quantified TEY→X for given electrode pairs with either a short window (15 ms) or long window (10 s) (Fig. 2b). i) Long-window TE with and without stimuli (long-window TEstim and long-window TEspon, respectively) Long-window TE was derived using 10-s windows to assess if information transmission differed depend- ing on the state of the thalamo-cortical system (i.e., during sensory processing vs. resting state). Long-window TEstim was derived from MUA over a continuous period of 240 s, during which clicks were presented every second. Long-window TEspon was derived from a separate 240-s time period of data during which no stimulus was delivered. Ten sets of 10-s p Xt+lag, Xt+lag−d, Yt and spike trains were randomly selected to derive the joint probability, p Xt+lag, Xt+lag−d, Yt . Based on Eq. (1), 10 sets of TE were then estimated in the transfer lag rang- ing between 1 and 30 ms. Long-window TEs were ultimately defined as the median across 10 sets for each lag. https://doi.org/10.1038/s41598-021-98660-y Scientific Reports \| (2021) 11:19252 \| www.nature.com/scientificreports/ Figure 3. Identification of significant information transfer. We shuffled the raw spike data to generate 100 datasets without changing inter-spike interval (ISI) distribution (a). Transfer entropy (TE) values were derived from either raw spike data (red line; TEraw) or shuffled data (blue dots; TEshuffled) at every transfer delay (b). Significant information transfer was defined as TEraw exceeding the top 5% of 100 TEshuffled (black dotted lines). An electrode pair was defined as having functional connectivity if significant information transfer was observed within a time window of 5 ms or more. Figure 3. Identification of significant information transfer. We shuffled the raw spike data to generate 100 datasets without changing inter-spike interval (ISI) distribution (a). Transfer entropy (TE) values were derived from either raw spike data (red line; TEraw) or shuffled data (blue dots; TEshuffled) at every transfer delay (b). Significant information transfer was defined as TEraw exceeding the top 5% of 100 TEshuffled (black dotted lines). An electrode pair was defined as having functional connectivity if significant information transfer was observed within a time window of 5 ms or more. (ii) Short-window TE Shuffling disrupted the temporal structure underlying functional connectivity between Xt and Yt.i To assess statistical significance of information transfer, we estimated p-values as the rank order of empirically identified TE values among the null distributions arising from 100 TEshuffled. For example, if the empirical TE was larger than the top 5% of 100 sets of TEshuffled, we regarded the p-value to be less than 0.0538. We corrected for multiple comparisons across transfer lags (1–30 ms) using the false discovery rate (FDR) method39. Further, we defined a pair of functionally connected electrodes as those with significant information transfer within a time window of 5 ms or more (Fig. 3). g When quantifying the amount of information transfer, we considered the degree of positive bias caused by a limited amount of sample data. Theoretically, TEshuffled must become 0 because shuffling should disrupt any causality between X and Y. However, the actual TEshuffled was larger than 0 due to biases, which were removed by subtracting the median TEshuffled from the TE. When TE was smaller than TEshuffled, no information transfer was assumed (i.e., TE = 0). Normalized TE (nTE). Mean firing rates of evoked activity was substantially higher than those of spontane- ous activity (Fig. 1c,d). To eliminate the bias due to differences in mean firing rate, we introduced the nTE. This normalization was necessary when comparing TEs derived from evoked and spontaneous states with different probability densities as follows: https://doi.org/10.1038/s41598-021-98660-y Scientific Reports \| (2021) 11:19252 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ nTEY→X = H Xfuture\|Xpast −H Xfuture\|Xpast, Ypast H Xfuture\|Xpast = TEY→X H Xt+lag\|Xt+lag−d Practically, the bias of nTE was corrected as (4) nTEY→X = TEY→X −TEshuﬄed Y→X H Xt+lag\|Xt+lag−d ∈[0, 1] . . . (4) nformation transmission in a given pathway. We characterized the information transmission in each athway as the average of the peaks of nTE among pairs with significant information transfer: nformation transmission in a given pathway. We characterized the information transmission in each athway as the average of the peaks of nTE among pairs with significant information transfer: (5) Average of nTE peaks = 1 n (peak of nTE) × n Npathway . . . (5) where n is the number of pairs with significant information transfer, and Npathway is the number of possible pairs of electrodes. Role of a given region in information transmission. (ii) Short-window TE Based on the average of the nTE peaks defined above, we quantified whether each region (X) served as either a receiver ( RX ) or a sender ( SX ). The metrics SX and RX were defined as the summation of the average of the nTE peaks as follows: (6) SX = iaverage of nTE peaks X →regioni . . . (6) (7) RX = iaverage of nTE peaks regioni →X . . . (7) regioni : one of (MGv, L2/3, L4, L5, and L6) with the exception of X where the average of nTE peaks(pathway) is the average of nTE peaks in a given pathway, as defined in Eq. (5). We then characterized each region X using the SR ratio: regioni : one of (MGv, L2/3, L4, L5, and L6) with the exception of Xi regioni : one of (MGv, L2/3, L4, L5, and L6) with the exception of X where the average of nTE peaks(pathway) is the average of nTE peaks in a given pathway, as defined in Eq. (5). We then characterized each region X using the SR ratio: where the average of nTE peaks(pathway) is the average of nTE peaks in a given pathway, as defined in Eq. (5) We then characterized each region X using the SR ratio: (8) SR ratio = SX −RX SX + RX ∈[−1, 1] . . . (8) A positive SR ratio indicated that region X served as a sender, whereas a negative SR ratio indicated that region X served as a receiver. Burst detection. Under isoflurane anesthesia, cortical LFPs exhibited an alternating pattern of high amplitude bursts and suppressed activity, which might influence the information flow and transmission in the thalamo-cortical system. The burst activities were easily detectable because they simultaneously appeared in all recording sites over the auditory cortex21,40. For the burst detection, the variances of LFP over time per electrode were measured with every 100-ms window at each time point. If the variance exceeded a threshold over 100 ms in > 25 (out of 96) electrodes, we regarded the analyzed period as burst. The burst threshold was identified heu- ristically. Results In the four rats tested, 96 sites in the MGv and 138 sites in A1 exhibited tone-evoked MUA, which exhibited definable auditory responses and a CF. Among these sites, we simultaneously measured click-evoked and spon- taneous MUA in the MGv and A1 (Fig. 1c,d). Under isoflurane anesthesia, burst activities were often observed as barrages of MUAs in A1 (red lines in Fig. 1c,d), which might influence the information flow. To address this concern, we first measured the burst period in our data. As a result, burst periods did not depend on stimulus presentation significantly (mean ± SD: 25.3 ± 5.4 s/min in spontaneous activities vs. 30.2 ± 5.8 s/min during stimu- lus presentation; t-test, P = 0.2), and hence, bursts were likely to have little effects to characterize the difference between TEspon and TEstim. p We derived long-window TE and short-window TE between all possible pairs among available sites. Sig- nificant information transfer was identified in 11,483 pairs (98% of all possible pairs) in long-window TEstim, 3964 pairs (36%) in long-window TEspon, and 5246 pairs (45%) in short-window TEonset. These significant pairs were further characterized as follows. Among these pairs, 2430, 107, and 1578 pairs (83%, 4%, and 54% of pairs, respectively) transmitted information from the MGv to A1; 2005, 108, and 743 pairs (69%, 4%, and 25% of pairs, respectively) transmitted information from A1 to MGv; and 2671, 2548, and 1405 pairs (75%, 71%, and 40% of pairs, respectively) transmitted information from A1 to A1 in long-window TEstim, long-window TEspon, and short-window TEonset, respectively. Supplementary Fig. S1 summarizes the distribution of CF at test sites in A1 and MGv, and the difference of CF (delta CF) among pairs with significant TE. Because each shank of the microelectrode array penetrated a single tonotopic column in A1, the CFs in A1 were more narrowly distributed than those in MGv. As the figure shows, the majority of delta CF was confined within ± 1 octave. To test if pairs with significant TE have the matched CF, we performed the following. Using bootstrap analysis, we constructed surrogate delta CF distributions by randomly sampling MGv and A1 sites by resampling from the real distribution. We found this distribution did not differ from the real distribution. This suggests that this Scientific Reports \| (2021) 11:19252 \| https://doi.org/10.1038/s41598-021-98660-y .com/scientificreports/ Figure 4. Long-window TE differentiates information flow with and without stimulus presentation. Results Long-window TE differentiates information flow with and without stimulus presentation. (a) Normalized TE (nTE) as a function of transfer lag: (i) nTE during stimulus presentation (long-window TEstim) and (ii) nTE during spontaneous activity (long-window TEspon). Average and standard error across subjects are presented. Colors indicate pathways: red, MGv-to-A1; blue, A1-to-MGv; green, A1-to-A1 pathways. (b) Information flow in a given pathway. Recording sites in A1 were classified into Layers 2/3, 4, 5, and 6. TEstim and TEspon were characterized in four thalamo-cortical pathways, four cortico-thalamic pathways, and 16 intracortical pathways between cortical nodes (L2/3, L4, L5, and L6) and nodes in the MGv. Information flow in each pathway was quantified as the average of nTE peaks (see text for details). The filled symbols indicate that information flow was significantly larger than that in the opposite pathway (one-sided Student’s t-test, P < 0.05). (c) Schematic diagram of information flow. Significant directional influences in (b) are depicted. (d) Sender/ receiver (SR) ratio at each region. SR ratios were derived for TEstim and TEspon. Regions with a positive SR ratio served as senders, whereas those with a negative ratio served as a receiver. Figure 4. Long-window TE differentiates information flow with and without stimulus presentation. (a) Normalized TE (nTE) as a function of transfer lag: (i) nTE during stimulus presentation (long-window TEstim) and (ii) nTE during spontaneous activity (long-window TEspon). Average and standard error across subjects are presented. Colors indicate pathways: red, MGv-to-A1; blue, A1-to-MGv; green, A1-to-A1 pathways. (b) Information flow in a given pathway. Recording sites in A1 were classified into Layers 2/3, 4, 5, and 6. TEstim and TEspon were characterized in four thalamo-cortical pathways, four cortico-thalamic pathways, and 16 intracortical pathways between cortical nodes (L2/3, L4, L5, and L6) and nodes in the MGv. Information flow in each pathway was quantified as the average of nTE peaks (see text for details). The filled symbols indicate that information flow was significantly larger than that in the opposite pathway (one-sided Student’s t-test, P < 0.05). (c) Schematic diagram of information flow. Significant directional influences in (b) are depicted. (d) Sender/ receiver (SR) ratio at each region. SR ratios were derived for TEstim and TEspon. Regions with a positive SR ratio served as senders, whereas those with a negative ratio served as a receiver. relatively narrow range of delta CF was caused by the sampling bias of CF at test sites. Results (a) Normalized TE (nTE) as a function of transfer lag: (i) nTE during stimulus presentation (long-window TEstim) and (ii) nTE during spontaneous activity (long-window TEspon). Average and standard error across subjects are presented. Colors indicate pathways: red, MGv-to-A1; blue, A1-to-MGv; green, A1-to-A1 pathways. (b) Information flow in a given pathway. Recording sites in A1 were classified into Layers 2/3, 4, 5, and 6. TEstim and TEspon were characterized in four thalamo-cortical pathways, four cortico-thalamic pathways, and 16 intracortical pathways between cortical nodes (L2/3, L4, L5, and L6) and nodes in the MGv. Information flow in each pathway was quantified as the average of nTE peaks (see text for details). The filled symbols indicate that information flow was significantly larger than that in the opposite pathway (one-sided Student’s t-test, P < 0.05). (c) Schematic diagram of information flow. Significant directional influences in (b) are depicted. (d) Sender/ receiver (SR) ratio at each region. SR ratios were derived for TEstim and TEspon. Regions with a positive SR ratio served as senders, whereas those with a negative ratio served as a receiver. www.nature.com/scientificreports/ Figure 4. Long-window TE differentiates information flow with and without stimulus presentation. (a) Normalized TE (nTE) as a function of transfer lag: (i) nTE during stimulus presentation (long-window TEstim) and (ii) nTE during spontaneous activity (long-window TEspon). Average and standard error across subjects are presented. Colors indicate pathways: red, MGv-to-A1; blue, A1-to-MGv; green, A1-to-A1 pathways. (b) Information flow in a given pathway. Recording sites in A1 were classified into Layers 2/3, 4, 5, and 6. TEstim and TEspon were characterized in four thalamo-cortical pathways, four cortico-thalamic pathways, and 16 intracortical pathways between cortical nodes (L2/3, L4, L5, and L6) and nodes in the MGv. Information flow in each pathway was quantified as the average of nTE peaks (see text for details). The filled symbols indicate that information flow was significantly larger than that in the opposite pathway (one-sided Student’s t-test, P < 0.05). (c) Schematic diagram of information flow. Significant directional influences in (b) are depicted. (d) Sender/ receiver (SR) ratio at each region. SR ratios were derived for TEstim and TEspon. Regions with a positive SR ratio served as senders whereas those with a negative ratio served as a receiver Figure 4. Long-window TE differentiates information flow with and without stimulus presentation. (a) l d ( ) f f f l ( ) d l (l d Figure 4. www.nature.com/scientificreports/ 4d, which characterized the role of each region as either a sender or receiver based on the SR ratio as defined by Eq. (8). Short‑window TE. Based on the above analysis, we conjectured that sender/receiver characteristics may change as a function of time during the temporal window surrounding stimulus onset, and that feedforward and feedback information transmission may be temporally segregated. For each significant electrode pair in the short-window TEonset, the time course of short-window TE was derived using moving window analyses (Fig. 2b). We first compared information transmission in the feedforward (i.e., 1578 pairs from MGv to A1; 54% of all possible pairs) and feedback direction (i.e., 743 pairs from A1 and MGv; 25% of all possible pairs). For both the average of all test pairs (Fig. 5a) and individual test pairs (Fig. 5b), we observed that feedforward informa- tion transmission was larger in amount and earlier in latency compared to feedback information transmission. g y To test whether feedforward pathways (Fig. 5b-i) were activated earlier than feedback pathways (Fig. 5b-ii), we quantified the onset latency of (i) feedforward and (ii) feedback information transmission in each layer-specific pathway (Fig. 5c). L4 received feedforward information with the earliest onset from MGv, whereas L2/3 received information with the latest onset (Fig. 5c-i; Kruskal–Wallis followed by Tukey–Kramer test, P < 0.05 for all pairs). In contrast, feedback information transmission from A1 to MGv was initiated in L6 (Fig. 5c-ii). These properties of feedforward information transmission were consistent with previous physiological findings10–16, underscoring the major benefits of the estimates of feedback transmission in our analyses.fi ji y To dissect the differences between feedforward and feedback pathways in more detail, we quantified feed- forward and feedback information transmission in each layer-specific pathway (Fig. 5d). L4 received the larg- est influence from MGv (Fig. 5d-i; Kruskal–Wallis followed by Tukey–Kramer test, L4 vs. L2/3 (P < 10−6), L5 (P < 10−4), and L6 (P < 10−8)). In contrast, L2/3 exerted the least influence on MGv in the feedback pathway (P < 10−8) (Fig. 5d-ii). These results are consistent with well-established anatomical projections10–16. Notably, information transmission in the feedforward direction contained several outliers which exerted extremely large influences. In contrast, information transmission in the feedback direction was more normally distributed. www.nature.com/scientificreports/ way from MGv to A1 and intracortical pathway was larger than that in the feedback pathway from A1 to MGv. TEspon (Fig. 4a-ii) indicated that the forward transmission of information from MGv to A1 almost disappeared during spontaneous activity compared to TEstim. These results support the notion that stimulus-driven infor- mation flow is distinct to spontaneous information flow and that each pathway possesses different information flow properties.f way from MGv to A1 and intracortical pathway was larger than that in the feedback pathway from A1 to MGv. TEspon (Fig. 4a-ii) indicated that the forward transmission of information from MGv to A1 almost disappeared during spontaneous activity compared to TEstim. These results support the notion that stimulus-driven infor- mation flow is distinct to spontaneous information flow and that each pathway possesses different information flow properties. l p p We further subdivided the cortical recording sites into four different layers (L2/3, L4, L5, and L6) according to the CSD analysis (Fig. 1b) and characterized information flow as the averages of nTE peaks in 24 pathways (Fig. 4b), as defined in Eq. (5). To evaluate the consistency of this measure across subjects, we compared infor- mation flow patterns of 24 pathways across subjects and quantified the correlation coefficients of the patterns between subjects in a pairwise manner. The correlation coefficients across subjects for TEstim and TEspon were 0.665 ± 0.185 and 0.954 ± 0.013 (average ± SD), respectively (t-test: TEstim, P < 0.05 in 5 out of 6 pairs of test rats; TEspon, P < 10−11 for all pairs; see Supplementary Fig. S2a and S2b). The moderately high correlation coef- ficient in the presence of stimuli (0.665) and high correlation coefficient during spontaneous activity (0.954) verified the ability of our analyses to capture general patterns of information flow.li i y y gl We next investigated the directionality of information flow. Based on TEstim and TEspon, the filled symbols in Fig. 4c-i,c-ii indicate that information flow was significantly larger than that in the opposite direction (one- sided Student’s t-test; P < 0.05). These pathways are highlighted in the schematic diagram of the thalamocortical system in Fig. 4c. Neural activity originating from L5 spread to other cortical layers and terminated in L2/3 in both TEstim and TEspon. In the presence of stimulation, additional activity originated from the MGv and L4. These trends of information flow were supported by data presented in Fig. Results In sum, we interpret this to mean that significant TE pairs did not have matched CF more often than unmatched CF, at least in our data of click-evoked MUAs. Long‑window TE. For long-window TE, we compared information flow with and without stimulus inputs. The nTE of (i) long-window TEstim and (ii) long-window TEspon as a function of transfer lag is presented in Fig. 4a. TEstim decayed abruptly at a transfer lag of approximately 15 ms in the MGv-to-A1 (red) direction, whereas it decayed smoothly in the A1-to-MGv (blue) direction (Fig. 4a-i). TEstim in the feedforward path- https://doi.org/10.1038/s41598-021-98660-y Scientific Reports \| (2021) 11:19252 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ This observation is consistent with the notion that a subpopulation of projections in the feedforward pathway is information-bearing, i.e., Class 1 projection, and that feedback pathways play a modulatory role through Class 2 projection22–30. p j Similar to the analysis for long-window TE, we characterized information transmission based on short- window TE in 24 pathways (Fig. 5e). The correlation coefficient of information transmission patterns of the 24 pathways across subjects was 0.629 ± 0.148 (P < 0.05 for all test pairs; see Supplementary Fig. S2c). The filled symbols in Fig. 5e indicate that information transmission was significantly larger than that in the opposite direction (one-sided Student’s t-test; P < 0.05). These pathways are highlighted in the schematic diagram of the thalamocortical system in Fig. 5f. Neural activity originated from MGv, L4, and L5, and terminated in L2/3. As depicted in Fig. 5g, MGv served as a sender and L2/3 as a receiver, whereas L4 and L5 served as relay stations with inward and outward transmission of similar magnitude. The difference between long-window TEstim (Fig. 4c-i) and short-window TE (Fig. 5f), i.e., L5 not always serving as a sender, indicated that the MGv was the origin of stimulus-driven information transmission in the thalamo-cortical system. y Lastly, Supplementary Fig. S3 characterized how the information transmission between MGv and A1 depended on the tonotopic axis, i.e., whether CF of a test pair matched (delta CF ≤ 1/3 oct) or unmatched (delta CF > 1). In the feedforward pathways, i.e., from MGv to A1, the amount of transmission did not depend on the tonotopic axis (Fig. S3a). In terms of onset latency, L4 and L5 in A1 tended to receive information from MGv earlier than other layers (Fig. S3b; P < 0.05), consistent with the direct tonotopic projection from MGv to L4 and L531. In the feedback pathways, L6 tended to send more information to MGv in pairs with unmatched CFs than in those with matched CFs (Fig. S3c; P < 0.05) although no difference was found in latency (Fig. S3d). Such feedback connectivity might play crucial roles in regulating gain and selectivity along the tonotopic axes in the thalamocortical system41. https://doi.org/10.1038/s41598-021-98660-y Scientific Reports \| (2021) 11:19252 \| www.nature.com/scientificreports/ Figure 5. Short-window TE to characterize feedforward and feedback information transmission. www.nature.com/scientificreports/ (a) The average time-course of short-window TE between MGv and A1: red, feedforward thalamo-cortical pathway (i.e., from MGv to A1); blue, feedback cortico-thalamic pathway (i.e., from A1 to MGv). The average and standard deviation (solid and dotted lines) are provided for available pairs. (b) Time-course of TE for individual test pairs. (i) Feedforward pathway. (ii) Feedback pathway. Cortical nodes were classified into Layers 2/3, 4, 5, and 6 (L2/3, L4, L5, and L6). (c) Onset latency of nTE transfer. (d) nTE at the peak in the time-course analyses. For each box, the central mark indicates the median (across all available/significant pairs and animals), and edges indicate the 25th and 75th percentiles. The whiskers extend to the most extreme data points that were not considered outliers, which exceeded the 75th percentile or were less than the 25th percentile by 1.5 times the inter-quartile range. Large outliers, i.e., nodes that exerted extremely large influence, were more frequently observed in the feedforward direction than in the feedback direction. (e) Information transmission in a given pathway. (f) Schematic diagram of information transmission. (g) Sender/receiver (SR) ratio at each region. For (e–g), see conventions in Fig. 4c–e. Figure 5. Short-window TE to characterize feedforward and feedback information transmission. (a) The average time-course of short-window TE between MGv and A1: red, feedforward thalamo-cortical pathway (i.e., from MGv to A1); blue, feedback cortico-thalamic pathway (i.e., from A1 to MGv). The average and standard deviation (solid and dotted lines) are provided for available pairs. (b) Time-course of TE for individual test pairs. (i) Feedforward pathway. (ii) Feedback pathway. Cortical nodes were classified into Layers 2/3, 4, 5, and 6 (L2/3, L4, L5, and L6). (c) Onset latency of nTE transfer. (d) nTE at the peak in the time-course analyses. For each box, the central mark indicates the median (across all available/significant pairs and animals), and edges indicate the 25th and 75th percentiles. The whiskers extend to the most extreme data points that were not considered outliers, which exceeded the 75th percentile or were less than the 25th percentile by 1.5 times the inter-quartile range. Large outliers, i.e., nodes that exerted extremely large influence, were more frequently observed in the feedforward direction than in the feedback direction. (e) Information transmission in a given pathway. (f) Schematic diagram of information transmission. (g) Sender/receiver (SR) ratio at each region. For (e–g), see conventions in Fig. 4c–e. Discussion h d In this study, we performed TE analysis to characterize information flow in the thalamo-cortical pathway between the MGv and A1 in anesthetized rats. We simultaneously recorded MUA in both the MGv and A1 (Fig. 1), and estimated TE using two different sampling windows (Fig. 2). We employed long-window TE to compare informa- tion flow with and without stimulus presentation, and short-window TE to scrutinize feedforward and feedback transmission around the time of stimulus onset. TE analyses were applicable to MUA data to characterize such bidirectional information flows, while traditional cross-correlation approach42,43 can be in principle applied to MUA data. However, in such a case, we cannot unambiguously interpret which MUA caused the other (as MUA represents spikes of many neurons). Further, traditional cross correlation applied to single-unit activities is also difficult to interpret when the firing rate is very low. Our analyses were consistent with well-established neuroanatomical literature and demonstrated that information flow was dynamic, with moment-to-moment variability in active pathways depending on the mode of stimulus processing.l Long-window TEspon indicated that thalamo-cortical information flow almost disappeared and intracortical communication became dominant during spontaneous activity. Short-window TE revealed that the MGv acted as a sender to L4 and L5 in response to a click stimulus. The time-course of short-window TE demonstrated that feedforward thalamo-cortical information flow preceded feedback cortico-thalamic information flow. L4 received the greatest influence and earliest latency from the MGv in the feedforward direction. These results are in accordance with well-characterized anatomical structures and canonical microcircuits in the thalamo-cortical system, thus confirming the validity of our analyses3–15. However, this structure of information flow was not revealed by long-window TEstim, in which L4 and L5 were not predominant receivers from MGv, and instead, L2/3 was the predominant receiver. This discrepancy between short-window and long-window TE was caused by the temporal resolution of the analyses. The 10-s window of long-window TEstim was able to capture some thalamo-cortical information flows, i.e., MGv-to-L2/3, but was too long to reveal the precise pathways, i.e., MGv-to-L4/5, which were active only within 50 ms following the stimulus onset.fl Our analyses revealed several differences in aspects of information flow between the MGv and A1. First, the information transfer window was approximately 15 ms for MGv-to-A1 transmission, which was narrower than that in the A1-to-MGv direction (Fig. 4a-i). This order of transfer lags for information transmission to A1 is consistent with previous findings44. Discussion h d Furthermore, false-positive TE may have been obtained for a pair of nodes which both receive projections from a common origin33,34,63. For example, a proportion of information flow between L4 and L5 may have been false positives, because both L4 and L5 receive dense projections from the MGv3,4,7,8,11–13,15,26,64–66. This false positive information flow may be more frequently observed in the L4-to-L5 direction than in the opposite direction because click- evoked responses occur earlier in L4 than in L5. To overcome these limitations, conditional mutual information methods such as momentary TE67 should be employed to estimate direct causality by conditioning out the effects of possible common drivers. Other alternatives to reduce the effects of common drivers exist68–71. Nevertheless, these techniques share the issue of estimation of neural interactions when the number of nodes in the analysis is large. Given that we have 96 electrode sites, there are currently no known techniques to address the problem of combinatorial explosion. Therefore, we believe that the pairwise TE is a well-balanced solution at present to give reasonable validation from the anatomical and physiological viewpoints. Our pairwise estimation of information flow has several limitations, which may complicate the interpreta- tion of our results. Although monosynaptic connections in L5 were previously identified in 0.25% of test pairs43, significant information flow was observed for 25–49% of possible test pairs in our analyses, suggesting that TE based on MUA was a measure of polysynaptic connections, rather than monosynaptic connectivity. Furthermore, false-positive TE may have been obtained for a pair of nodes which both receive projections from a common origin33,34,63. For example, a proportion of information flow between L4 and L5 may have been false positives, because both L4 and L5 receive dense projections from the MGv3,4,7,8,11–13,15,26,64–66. This false positive information l origin , , . For example, a proportion of information flow between L4 and L5 may have been false positives, because both L4 and L5 receive dense projections from the MGv3,4,7,8,11–13,15,26,64–66. This false positive information flow may be more frequently observed in the L4-to-L5 direction than in the opposite direction because click- evoked responses occur earlier in L4 than in L5. To overcome these limitations, conditional mutual information methods such as momentary TE67 should be employed to estimate direct causality by conditioning out the effects of possible common drivers. Other alternatives to reduce the effects of common drivers exist68–71. Discussion h d Such transfer lags are substantially longer than the cortical synaptic delay of 2 ms43,45 and are therefore likely generated within abundant recurrent connections in A1, but not in MGv11–13,46,47. Further, the order of the time window for integration is reminiscent of a cycle of gamma-band oscillations, which are generated via the interaction between pyramidal and inhibitory interneurons48–50 and subserve information integration51–55. Second, the influence of A1-to-MGv nodes was typically small (Fig. 5d-ii), whereas the influence of MGv-to-A1 nodes varied considerably (Fig. 5d-i). Furthermore, the outliers in Fig. 5d-i imply that a small proportion of MGv-to-A1 information transmission was significantly higher than the average value, indicative of high efficiency in driving post-synaptic neurons. These information-bearing nodes are likely to be classified as Class 1 projections, which express glutamatergic ionotropic receptors. In contrast, other nodes may be classified as Class 2 projections, which express metabotropic receptors22–30. The MGv may comprise more Class 1 pathways compared to A1, enabling transfer of external stimulus information (outliers in Fig. 5d-i vs. d-ii). Our analyses demonstrated that communication between the thalamus and cortex is strengthened during stimulus presentation, whereas communication within the cortex is strengthened during spontaneous activity. Furthermore, differences and similarities between long-window TEstim and long-window TEspon provide criti- cal insight into cortical computational processes. For example, our results in Fig. 4 are consistent with past reports in that the major source of information flow during spontaneous activity likely originated from L517,56. L5 is more likely to serve as the source of spontaneous activity in A1 because L5 exhibits more depolarized membrane potentials and higher firing rates compared to other layers47,57,58 with less inhibition59,60. During both spontaneous and evoked activity, L2/3 constituted more pathways for information inflow than for information outflow. These patterns of information flow suggest that L2/3 has a higher-dimensional space of representation compared to L4, corroborating the conceptual framework of sparse coding formation in L2/3 from high activity in L417,47,57,61,62. f f fl h l l h h l h g p p g g y Our pairwise estimation of information flow has several limitations, which may complicate the interpreta- tion of our results. Although monosynaptic connections in L5 were previously identified in 0.25% of test pairs43, significant information flow was observed for 25–49% of possible test pairs in our analyses, suggesting that TE based on MUA was a measure of polysynaptic connections, rather than monosynaptic connectivity. www.nature.com/scientificreports/ https://doi.org/10.1038/s41598-021-98660-y Scientific Reports \| (2021) 11:19252 \| www.nature.com/scientificreports/ Discussion h d Nevertheless, these techniques share the issue of estimation of neural interactions when the number of nodes in the analysis is large. Given that we have 96 electrode sites, there are currently no known techniques to address the problem of combinatorial explosion. Therefore, we believe that the pairwise TE is a well-balanced solution at present to give reasonable validation from the anatomical and physiological viewpoints. of possible common drivers. Other alternatives to reduce the effects of common drivers exist68 71. Nevertheless, these techniques share the issue of estimation of neural interactions when the number of nodes in the analysis is large. Given that we have 96 electrode sites, there are currently no known techniques to address the problem of combinatorial explosion. Therefore, we believe that the pairwise TE is a well-balanced solution at present to give reasonable validation from the anatomical and physiological viewpoints. In conclusion, we simultaneously measured MUA in the MGv and A1 in rats and harnessed TE analyses to characterize information flow in the auditory thalamo-cortical system. Long-window TE revealed that com- munication between MGv and A1 was strengthened during stimulus presentation, whereas thalamo-cortical communications almost disappeared and intracortical communications were strengthened during spontaneous activity. Short-window TE indicated that feedforward (thalamo-cortical) information transmission was followed Scientific Reports \| (2021) 11:19252 \| https://doi.org/10.1038/s41598-021-98660-y www.nature.com/scientificreports/ by feedback (cortico-thalamic) transmission at stimulus onset, and L4 exerted the largest influence with the earli- est latency from the MGv in the feedforward direction, corroborating anatomical reports on thalamo-cortical projections. Furthermore, consistent with the notion of Class 1 and Class 2 synaptic properties, a small but significant population of MGv-to-A1 pairs was information-bearing, whereas A1-to-MGv pairs that typically exhibited a small influence were likely to play modulatory roles. Our results highlight the capability of TE analy- ses to unlock novel avenues for bridging the gap between well-established anatomical knowledge of canonical microcircuits and physiological findings via the concept of dynamic information flow. 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https://openalex.org/W4360601955	https://digital.csic.es/bitstream/10261/337746/1/mnras_GMolpeceres_et_al_2023.pdf	English	null	Processing of hydroxylamine, NH2OH, an important prebiotic precursor, on interstellar ices	Monthly Notices of the Royal Astronomical Society	2,023	cc-by	16,693	A B S T R A C T Hydroxylamine, NH 2 OH, is one of the already detected interstellar molecules with the highest prebiotic potential. Yet, the abundance of this molecule found by astronomical observations is rather low for a relatively simple molecule, ∼10 −10 relative to H2. This seemingly low abundance can be rationalized by destruction routes operating on interstellar dust grains. In this work, we tested the viability of this hypothesis under several prisms, ﬁnding that the origin of a lower abundance of NH 2 OH can be explained by two chemical processes, one operating at low temperature (10 K) and the other at intermediate temperature (20 K). At low temperatures, enabling the hydrogen abstraction reaction HNO + H → NO + H 2 , even in small amounts, partially inhibits the formation of NH 2 OH through successive hydrogenation of NO, and reduces its abundance on the grains. We found that enabling a 15–30 per cent of binding sites for this reaction results in reductions of NH 2 OH abundance of approximately one to two orders of magnitude. At warmer temperatures (20 K, in our study), the reaction NH 2 OH + H → HNOH + H 2 , which was found to be fast ( k ∼10 6 s −1 ) in this work, followed by further abstractions by adsorbates that are immobile at 10 K (O, N) are the main route of NH 2 OH destruction. Our results shed light on the abundance of hydroxylamine in space and pave the way to constraining the subsequent chemistry experienced by this molecule and its deri v ati ves in the interstellar prebiotic chemistry canvas. Key words: astrochemistry – molecular data – methods: numerical – ISM: molecules. (NH 2 CH 2 CH 2 OH) (Rivilla et al. 2021 ) a key molecule in the synthesis of phospholipids, and the molecule subject of this study, hydroxylamine NH 2 OH (Rivilla et al. 2020 ), which is assumed to be crucial in forming amino acids and RNA nucleotides (Sakurai & Yanagawa 1984 ; Snow et al. 2007 ; Becker et al. 2019 ; Xu et al. 2022 ). ( ) Published by Oxford University Press on behalf of Royal Astronomical Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://cr eativecommons.or g/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. MNRAS 521, 6061–6074 (2023) https://doi.org/10.1093/mnras/stad892 Advance Access publication 2023 March 23 MNRAS 521, 6061–6074 (2023) https://doi.org/10.1093/mnras/stad892 Advance Access publication 2023 March 23 https://doi.org/10.1093/mnras/stad892 ⋆ E-mail: molpeceres@astron.s.u-tokyo.ac.jp ⋆ E-mail: molpeceres@astron.s.u-tokyo.ac.jp signiﬁcant theoretical and experimental effort has been dedicated © 2023 The Author(s). Published by Oxford University Press on behalf of Royal Astronomical Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://cr eativecommons.or g/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Processing of hydroxylamine, NH 2 OH, an important prebiotic precursor, on interstellar ices Germ ´an Molpeceres , 1 ‹ V ´ıctor M. Ri villa , 2 K enji Furuya, 3 Johannes K ¨astn and Yuri Aikawa 1 ceres , 1 ‹ V ´ıctor M. Ri villa , 2 K enji Furuya, 3 Johannes K ¨astner , 4 Bel ´en Mat ´e 5 1 1 Department of Astronomy, Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan 2 Centro de Astrobiolog ´ıa (CSIC, INTA), Ctra. de Ajalvir, km. 4, Torrej ´on de Ardoz, E-28850 Madrid, Spain 3 National Astronomical Observatory of Japan, 181-8588 Tokyo, Japan 4 Institute for Theoretical Chemistry, University of Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany 5 Instituto de Estructura de la Materia (IEM-CSIC), Calle Serrano 113, E-28006 Madrid, Spain 1 Department of Astronomy, Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan 2 Centro de Astrobiolog ´ıa (CSIC, INTA), Ctra. de Ajalvir, km. 4, Torrej ´on de Ardoz, E-28850 Madrid, Spain 3 National Astronomical Observatory of Japan, 181-8588 Tokyo, Japan Accepted 2023 March 20. Received 2023 March 20; in original form 2023 January 12 1 Department of Astronomy, Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan 2 Centro de Astrobiolog ´ıa (CSIC, INTA), Ctra. de Ajalvir, km. 4, Torrej ´on de Ardoz, E-28850 Madrid, Spain 3 National Astronomical Observatory of Japan, 181-8588 Tokyo, Japan 4 Institute for Theoretical Chemistry, University of Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany 5 Instituto de Estructura de la Materia (IEM-CSIC), Calle Serrano 113, E-28006 Madrid, Spain 2.1.1 Reactivity The study of the reactivity of hydroxylamine uses density functional theory (DFT) in combination with high-level calculation to reﬁne the electronic energy. In particular, geometries and vibrational frequencies were obtained using the DSD-PBEP86-D3BJ double hybrid exchange and correlation functional (Grimme, Ehrlich & Goerigk 2011 ; Kozuch & Martin 2011 ), and the def2-TZVP basis set (Weigend & Ahlrichs 2005 ). The electronic energies were reﬁned utilizing DLPNO-CCSD(T) (domain-based local pair natural orbital coupled cluster) calculations using a two-point extrapolation to the basis set limit (CBS) (Helgaker et al. 1997 ; Zhong, Barnes & Petersson 2008 ; Neese & Valeev 2011 ; Guo et al. 2018 ). For the extrapolation, we used the energies obtained using with the cc-pVTZ, and cc-pVQZ basis sets (Woon & Dunning 1993 ). The level of theory used in the reactivity calculations can be therefore abbreviated as DLPNO-CCSD(T)/CBS//DSD-PBEP86-D3BJ/def2- TZVP. The frozen-core approximation was applied in all wavefunc- tion theory-based calculations. The DFT and DLPNO-CCSD(T) calculations include resolution of the identity techniques to speed up the calculations via the RIJK approximation for the Coulomb and Exchange integrals additional RI approximations for the correlation part of the exchange and correlation e v aluation. The deﬁciency of NH 2 OH could be due to NH 2 OH being liable for being chemically converted (destroyed) on the surface of in- terstellar dust grains through hydrogenations and other reactions (Garrod 2013 ; Garrod et al. 2022 ). In this study, we have explicitly addressed this scenario under a series of possible reactions, namely isomerization reactions (cis-trans, c-NH 2 OH/t-NH 2 OH), irreversible destructions to produce the highly stable H 2 O and NH 3 molecules, and reversible destructions through an H-addition and H-abstraction cycle (Molpeceres et al. 2022 ). We study these reactions using high- precision quantum chemical calculations. In particular, the reactions that we study are: We used two different structural models for all the reactions we present in Section 3 . In the ﬁrst place (Model A ), we used a gas- phase structural model, and then the derived rate constants were corrected to account for the ﬁxated rotational degrees of freedom by ﬁxing the rotational partition function on the surface to the unity. 2.1.1 Reactivity Such an approach, named the implicit surface approach, is helpful in many cases where the surface does not play an active role in the reaction (Molpeceres, Garc ´ıa de la Concepci ´on & Jim ´enez-Serra 2021b and Molpeceres & Rivilla 2022 show two recent examples of such approach). c −NH 2 OH ⇌t −NH 2 OH (1) NH 2 OH + H −→ NH 3 + OH (2) NH 2 OH + H −→ H 2 O + NH 2 (3) NH 2 OH + H −→ H 2 NO + H 2 (4) NH 2 OH + H −→ HNOH + H 2 (5) HNOH + H −→ NH 2 OH (6) H 2 NO + H −→ NH 2 OH . (7) (1) Hydroxylamine, as already pointed out in Wakelam et al. ( 2017a ), has a peculiar behaviour when interacting with a water surface because of its concomitant behaviour as an H-donor and H-acceptor in hydrogen bonds. Hence, we also used a model (Model B ) explicitly considering two water molecules but maintaining the ﬁxing of the rotational partition function to account for possible orientation effects stemming from the -NH 2 and -OH interaction with water. The initial conﬁguration for Model B is obtained from exploratory calculations of the interaction of NH 2 OH with a water dimer in its global minimum. A total of 25 initial positions, for NH 2 OH were tried, from where the conﬁguration with the lowest energy is used for further calculations. An example of the two different models for the adsorption of NH 2 OH is depicted in Fig. 1 . (6) (7) Later, we merge the results attained in this work with further works available in the literature (He et al. 2015 ; Nguyen et al. 2019 ) and complement our theoretical calculations with tailored astrochemical models. This article’s structure is as follows. In Section 2 , we present our computational framework for studying the candidate reactions for the destruction of NH 2 OH. In Section 3 , we give a complete picture of the destruction and reformation of NH 2 OH through isomerization and hydrogenation, in combination with previous works (Nguyen et al. 2019 ). 1 INTRODUCTION Understanding the origin of life is one of the so-called ‘Holy Grails of Chemistry’ (Burrows 2017 ). To take steps towards this ambitious goal, one of the most fundamental conundrums remains to establish the transition from purely abiotic compounds to prebiotic ones (i.e aminoacids, nucleotides, lipids, and sugars) (Plankensteiner et al. 2004 ; Chandru et al. 2016 ; Yadav, Kumar & Krishnamurthy 2020 ). Postulating routes of formation of prebiotic molecules is a crucial ﬁrst step for more advanced origin of life theories, such as the ‘RNA World Hypothesis’ (Gilbert 1986 ), one of the most accepted theories for the emergence of life, which proposes that ribonucleic acid (RNA) may have played a twofold role in primordial Earth, ﬁrst as a carrier of genetic material and second as a catalyst in the biosynthesis of proteins and the subsequent DN A/RN A/Proteins cycle. The presence of these prebiotic precursors in space is a fascinating subject that opens many branches, such as the follo wing. Ho w are these molecules synthesized under interstellar conditions? How do the y evolv e? Are the y the summit of comple xity e xpectable in the ISM? These questions require tailored, experimental, or theoretical studies to reveal these precursors’ physical and chemical nature under interstellar conditions. For NH 2 OH, several of these questions have been explicitly addressed. For example, literature shows that it is produced mostly through nitric oxide (NO) hydrogenation (Congiu et al. 2012 ; Fedoseev et al. 2012 , 2016 ; Nguyen et al. 2019 ) on the surface of dust grains. Alternative routes include non-energetic oxidation of ammonia (He et al. 2015 ), and other energetic routes, e.g. by cosmic ray irradiation of O 2 and NH3 ices (Tse ga w et al. 2017 ). Finally, the classical route for hydroxylamine is the radical recombination of NH 2 and OH (Nishi, Shinohara & Okuyama 1984 ). Concerning the evolution of hydroxylamine, a signiﬁcant theoretical and experimental effort has been dedicated Many of the prebiotic precursors do not present a complicated chemical structure and, consequentially, are susceptible to being detected in the interstellar medium (ISM). For example, recent astronomical surv e ys hav e found 1-2-ethenediol ((C 2 H 2 O) 2 (Rivilla et al. 2022 ) a key molecule in the synthesis of sugars, ethanolamine G. Molpeceres et al. 1 INTRODUCTION 6062 to determining its reactivity in the gas phase, primarily through its ionized (NH 2 OH + ) form (Snow et al. 2007 ; Largo et al. 2009 ; Barrientos et al. 2012 ; Redondo, Barrientos & Largo 2013 ; Sanz- Novo et al. 2019 ). reactions in the bigger astrochemical picture. Also, we reanalyse some of the previously thought formation reactions of NH 2 OH. Then we include reactions in the literature and our data into astrochemical models. Finally, we provide an overview and outlook in Section 5 . ) One key issue surrounding hydroxylamine pre v alence under in- terstellar conditions is its elusive nature. For a relatively simple molecule containing ﬁve atoms, with equally standard functional groups, e.g. amino −NH 2 and hydroxyl -OH, models predict total abundances in the range 10 −5 –10 −6 (Garrod 2013 ; He et al. 2015 ; Garrod et al. 2022 ). Ho we ver, the abundance of NH 2 OH in the ISM is surprisingly low, of 2.1 × 10 −10 to H 2 (Rivilla et al. 2020 ). Besides, NH 2 OH has only been detected towards our Galactic Centre, speciﬁcally in the molecular cloud G + 0.693-0.027, an astronomical environment characterized by rather unique conditions in terms of energetic input. For example, temperatures of the gas in the range between 50 and 150 K (Krieger et al. 2017 ; Zeng et al. 2018 ), dust temperatures of 15 K (Rodr ´ıguez-Fern ´andez et al. 2004 ), and most importantly, are subjected to mechanical shocks due to cloud– cloud collisions (Mart ´ın-Pintado et al. 1997 ) and a high cosmic-ray ionization rate ( ∼10 −15 –10 −14 s −1 ) (Goto et al. 2013 ; Zeng et al. 2018 ) that might alter the molecular inventory of G + 0.693-0.027. Hydroxylamine has not been detected in other regions, neither in cold environments (molecular clouds) nor warmer ones (hot-corinos and hot-cores) (Pulliam, McGuire & Remijan 2012 ; McGuire et al. 2015 ; Codella et al. 2018 ; Ligterink et al. 2018 ). The upper limits for Sgr B2 and IRAS 16293–2422 are 8 × 10 −12 and 3.1 × 10 −11 , respectively. 2.1.2 Binding energy calculations The discrete nature of the rate constants obtained by instanton theory, in contrast to the classical continuous counterparts, makes the calculation of these quantities and their convergence behaviour at low temperatures e xpensiv e and complicated. For all the reactions presented in Section 3 we indicate the lowest temperature of our calculation, assuming that the extrapolation to lower temperatures should yield very similar values granted by the asymptotic behaviour of tunnelling-dominated rate constants at low temperatures. The magnitude that controls the temperature at which we start to include quantum effects (e.g. tunnelling starts to play a signiﬁcant role) is the crosso v er temperature: T c = ℏ νi k B , (8) T c = ℏ νi k B , The electronic structure method we used for reactivity, presented in Section 2.1.1 , is too e xpensiv e to sample binding energies. For the binding energy calculations, we used the B97-3c method in the geometry optimizations (Brandenburg et al. 2018 ) with energy reﬁnement coming from the DLPNO version of the DSD-PBEP86- D3BJ. Hence, the method is abbreviated as DLPNO-DSD-PBEP86- D3BJ/def2-TZVP//B97-3c. It is worth mentioning that even though the level of theory for the binding energies is more approximated than the local coupled cluster methods used in the reactivity part of this work, using a double-hybrid DFT method for the electronic energies should ensure good treatment of the weak interatomic interactions in the adsorbate-ice system (Kozuch & Martin 2011 ). (8) where νi is the transition frequency at the transition state (e.g. imaginary frequency). Additionally, since our work aims to reﬁne astrochemical models, we have not included symmetry factors ( σ) in our rate constants. Even if in the gas phase, they may be present, on interstellar ice, the rotational symmetry is broken. Similar to the PES exploration, our instanton calculations employ a dual-level formalism (Meisner & K ¨astner 2018 ). Finally, we considered radical–radical reactions in the reformation of hydroxylamine through reactions ( 6 ) and ( 7 ). These two reactions are the only ones rele v ant to the formation of hydroxylamine after the reaction of NH 2 OH with an H atom; see Section 3.2 . They were simulated using a broken-symmetry formalism, ﬁrst converging the pre-reactant complex’s high-spin state, followed by downhill optimizations using the correct spin multiplicity. 2.1.2 Binding energy calculations Binding energies are critical in grain surface chemistry, interpreted as the attractive interaction between an adsorbate and a surface. To account for a diversity of binding sites stemming from the amorphous solid water (ASW) structure, we have sampled the distribution of binding energies of the primary intermediates of the reactions considered in this work (e.g. NH 2 OH, HNOH, and H 2 NO), and incorporated these values into the astrochemical model. The structural models for the ices are the same (H 2 O) 20 clusters that were used in Molpeceres et al. ( 2021b ). These clusters were constructed following a heating and cooling cycle of molecular dynamics simulations with an empirical force ﬁeld followed by a geometry optimization using an electronic structure method. Once the clusters are prepared, the abo v e-mentioned molecules are situated at a distance given by a tolerance parameter plus the maximum { x , y , z} value of the cluster. The orientation of the molecules is randomized, and the system is allowed to relax via a geometry optimization. The binding energies are then obtained as the difference between the bimolecular system of cluster and adsorbate and the complex formed by the relaxed molecule on the cluster. We sampled 10 initial positions per cluster for ﬁve clusters (50 binding energies per considered molecule). More details on the protocol can be found in Molpeceres & K ¨astner ( 2021 ). For computational saving reasons and due to the considerable binding energy found for the adsorbates, we omitted ZPVE in calculating the binding energies, and we ﬁxed the Cartesian positions of all the water molecules 8 Å away from any of the initial positions of the adsorbate. While ZPVE effects are known to shift the binding energy distributions, the high binding energy from the electronic energy contribution is high enough to assume that this effect will be minor at 10–20 K. Figure 1. Structural models employed for the study of the reactivity of hydroxylamine. Left: implicit surface (Model A ). Right: two model water approach (Model B ). The donor–acceptor nature of NH 2 OH can be inferred from the ﬁgure. quantum effects) rate constants. These are later expanded by locating the instantons at different temperatures, e.g. performing a sequential cooling scheme. 2.1.2 Binding energy calculations This set of calculations requires a more e xtensiv e sampling, and therefore we reduced the basis set of the DSD-PBEP86-D3BJ calculations to def2-SVP (Weigend & Ahlrichs 2005 ). We initially sampled 150 starting positions for the downhill optimizations, from which we ensured that less than 10 per cent did not converge, a sometimes una v oidable outcome due to the complicated electronic structure of the system. All the calculations use the ORCA (v.5.0.2) software (Neese et al. 2020 ) interfaced with DL-FIND/CHEMSHELL (K ¨astner et al. 2009 ; Metz et al. 2014 ). The sampling of reaction outcomes for the radical–radical reactions follows the procedure presented in Section 2.1.2 for Model A , modiﬁed to account for the presence of water molecules in Model B (see Method section in Molpeceres et al. 2021b ). water molecules in Model B (see Method section in Molpeceres et al. 2021b ). 2021b ). 2.1.1 Reactivity Section 4 builds on the quantum chemical ﬁndings and provides a comprehensive analysis of the importance of these The formalism for studying the reactions is based on transition state theory (TST) incorporating quantum nuclear effects from semiclassical instanton theory (Langer 1967 ; Miller 1975 ; Coleman 1977 ; Rommel & K ¨astner 2011 ; Rommel, Goumans & K ¨astner 2011 ). Thus, for a given reaction, we determine the stationary points in the potential energy surface (PES) (reactant, transition states, and products) from where we extract the classical (e.g. not including MNRAS 521, 6061–6074 (2023) Hydroxylamine chemistry in the ISM Hydroxylamine chemistry in the ISM 6063 Figure 1. Structural models employed for the study of the reactivity of hydroxylamine. Left: implicit surface (Model A ). Right: two model water approach (Model B ). The donor–acceptor nature of NH 2 OH can be inferred from the ﬁgure. 3.1 Isomerism of hydroxylamine on ASW The ﬁrst reaction we looked at is the interconversion in the gas and on the grains of trans-NH 2 OH (t-NH 2 OH) and cis-NH 2 OH (c-NH 2 OH). This is because we e v aluated se veral hypotheses for the mismatch between observ ations (Ri villa et al. 2020 ) and models (Garrod 2013 ; Garrod et al. 2022 ), and one of them could be preferential destruction of a particular isomer on the grain or isomerization in the gas. So far, only t-NH 2 OH has been detected in the ISM (Rivilla et al. 2020 ). Both isomers are separated by ∼2300 K in the gas phase at the DLPNO-CCSD(T)/CBS//DSD-PBEP86-D3BJ/def2-TZVP level of theory. NH 2 OH can isomerize via two distinct molecular motions. MNRAS 521, 6061–6074 (2023) 6064 G. Molpeceres et al. than in the gas phase (Model A ). This is a consequence of the steric hindrance produced by the water molecules. Ho we ver, in both cases, cis → trans isomerization is a fast process in the gas and the surface. All rate constants for trans–cis isomerization are negligible at low temperatures, indicating that any t-NH 2 OH should not convert to c-NH 2 OH. Likewise, any c-NH 2 OH formed through chemical reactions (see, for example, Section 3.2.5 ) must isomerize quite fast under astrophysical conditions (e.g. below 150 K). Even the lowest rate constant for c −NH 2 OH → t −NH 2 OH is of the order of 10 −4 s −1 which is higher than the estimated accretion rate of H of 10 −5 s −1 (Wakelam et al. 2017b ), which translates in time-scales of 10 4 and 10 5 s −1 , respectively. Hence, in the following sections, any chemistry of NH 2 OH will imply t-NH 2 OH, assuming that abundances of c- NH 2 OH must be negligible. Finally, it is interesting to mention that NH 2 OH isomerization through the classical torsion barrier is faster for the whole considered temperature range than through nitrogen in- v ersion, ev en considering that tunnelling is more critical for the latter. This is due to the signiﬁcantly lower value of the torsional barrier. Table 1. 3.1 Isomerism of hydroxylamine on ASW Molecular mode (nitrogen inversion or torsion of HONH bond), reaction energies ( U R in K), acti v ation energies ( U a , in K) and transition frequencies ( νi in cm −1 ) for the c −NH 2 OH → t −NH 2 OH reaction. The direction of the reaction is chosen for the reaction to be exothermic. Structural model Mode U R ( K ) U a (K) νi (cm −1 ) Model A (cis → trans) Inversion −2026 3666 956 i Model B (cis → trans) Inversion −2291 5274 865 i Model A (cis → trans) Torsion −2026 1091 476 i Model B (cis → trans) Torsion −2291 1257 257 i First, via nitrogen inversion, e.g. simultaneous migration of the two hydrogen atoms for the NH 2 moiety. Second, via torsion of the HONH bond. While the former is potentially affected by quantum tunnelling, this effect on the latter is minor. We sampled both molecular motions for Model A and Model B . In this case, for Model A , we do not ﬁx the rotational partition function because we are interested in the gas-phase process of inter-converting trans and cis isomers. The energetic quantities describing the process are gathered in T able 1 . W e attribute an increase in the acti v ation energies between Model A and Model B to restraining motions on the surface. In the case of the isomerization via the torsion motion, this increase is not very pronounced ( ± 200 K). Still, for the inversion of nitrogen, the acti v ation energy change is ±1600 K. The reaction energies are similar both in Model A and Model B , indicating a similar binding of cis and trans isomers to the surface. Finally, for the imaginary transition mode, we observe minor differences between both mechanisms. Ho we ver, for the torsion mechanism, a reduction of merely 200 cm −1 in the transition frequencies signiﬁcantly affects the rate constants, as we observed for the OCSH radical on ice surfaces (Molpeceres et al. 2021b ). 3.2 Destruction of NH 2 OH Right: transition state for reaction ( 2 ) in Model B . Reaction Label Model U R (K) U a (K) NH 2 OH + H → NH 3 + OH ( 2 ) A −24 893 3723 ( 2 ) B −24 695 4903 NH 2 OH + H → H 2 O + NH 2 ( 3 ) A −29 762 6437 ( 3 ) B −24 877 6206 NH 2 OH + H → H 2 NO + H 2 ( 4 ) A −13 172 4206 ( 4 ) B −13 444 7439 NH 2 OH + H → HNOH + H 2 ( 5 ) A −9527 3409 ( 5 ) B −10 763 3218 Reaction Label Model U R (K) U a (K) ademic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Figure 4. Instanton rate constants for reaction ( 2 ) in Model A and Model B . The crosso v er temperature is 300 K for the former and 308 K for the latter. Figure 5. IRC calculations for reaction ( 2 ). The IRC path is neither corrected for ZPVE contributions nor DLPNO-CCSD(T) energies. Figure 3. Left: product of reaction ( 2 ) in Model B . As a reference, the OH–H distance in the water dimer is 1.94 Å at the DLPNO-CCSD(T)/CBS//DSD- PBEP86-D3BJ/def2-TZVP level of theory. Right: transition state for reaction ( 2 ) in Model B . is supposed to dominate at warm temperatures ( k = 1.6 × 10 −13 cm 3 s −1 at 300 K) according to the KIDA data base and references therein (Atkinson et al. 2004 ; Sander et al. 2011 ; Wakelam et al. 2012 ). It is important to note that reaction ( 9 ) is only tested for intermediate temperatures (230–450 K) and in the gas phase. Hence, low temperature surface studies are desirable. Therefore, reaction ( 2 ) would result in a total loss of hydroxylamine and a reduction of the abundance of this molecule in the ISM. Reaction ( 2 ) is exothermic both in Model A and Model B , with energies of reaction of U R , ModelA = −24 893 K and U R , ModelB = −24 695 K, e.g. a tiny inﬂuence of the addition of explicit water molecules to the reaction. 3.2 Destruction of NH 2 OH The high reaction energy can be explained by the strong interaction of the NH 3 –OH pair formed during the reaction. This is not the case for acti v ation energies, with values: U a , ModelA = 3723 K and U a , ModelB = 4903 K. While normally, and especially for weakly bound adsorbates, the ASW surface does not play a signiﬁcant role (Lamberts & K ¨astner 2017 ; Meisner, Lamberts & K ¨astner 2017 ; Molpeceres & Rivilla 2022 ), the case of hydroxylamine seems particular, owing to the donor– acceptor nature of the NH 2 OH −2H 2 O complex. The reaction’s increased exothermicity and higher acti v ation energy in Model B can be visualized in Fig. 3 . Contributing to the reaction exothermicity, a cooperative four-centre network is formed in the product, and contributing to the high acti v ation energy we ﬁnd a temporary break of a hydrogen bond to initiate the reaction. Figure 5. IRC calculations for reaction ( 2 ). The IRC path is neither corrected for ZPVE contributions nor DLPNO-CCSD(T) energies. less, this reaction is, in absolute terms, a reasonably fast reaction with reaction rate constants of the order of 10 −1 –10 0 s −1 , i.e. it can proceed in astronomical time-scales in the absence of competitive reactions. Ho we ver, we will show in Section 3.2.4 that this reaction channel is minor compared with the competitive H 2 abstraction reactions due to the increased inﬂuence of quantum tunnelling in the latter. From a chemical point of view, it is important to ascertain that reaction ( 2 ) proceeds in a single elementary step, meaning that no intermediate reaction that can be long-lived on an ASW surface is formed during the reaction. We conﬁrmed this behaviour using the transition state structure’s intrinsic reaction coordinate (IRC) calcu- lations. The results for the IRC are presented in Fig. 5 . Note that the path-length to products is notoriously considerable but leads without any additional barrier to the bimolecular products NH 3 and OH. Instanton rate constants for reaction ( 9 ) down to 50 K are presented in Fig. 4 , a temperature where the asymptotic regime for tunnelling is reached. 3.2 Destruction of NH 2 OH Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 20 Hydroxylamine chemistry in the ISM 6065 6065 Figure 4. Instanton rate constants for reaction ( 2 ) in Model A and Model B . The crosso v er temperature is 300 K for the former and 308 K for the latter. Table 2. Reaction energies, U R , and acti v ation energies, U a (in K, including ZPVE), for the reactions proceeding with an acti v ation barrier, and studied using quantum chemical methods in this work. Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 202 Reaction Label Model U R (K) U a (K) NH 2 OH + H → NH 3 + OH ( 2 ) A −24 893 3723 ( 2 ) B −24 695 4903 NH 2 OH + H → H 2 O + NH 2 ( 3 ) A −29 762 6437 ( 3 ) B −24 877 6206 NH 2 OH + H → H 2 NO + H 2 ( 4 ) A −13 172 4206 ( 4 ) B −13 444 7439 NH 2 OH + H → HNOH + H 2 ( 5 ) A −9527 3409 ( 5 ) B −10 763 3218 Figure 3. Left: product of reaction ( 2 ) in Model B . As a reference, the OH–H distance in the water dimer is 1.94 Å at the DLPNO-CCSD(T)/CBS//DSD- PBEP86-D3BJ/def2-TZVP level of theory. Right: transition state for reaction ( 2 ) in Model B . Reaction Label Model U R (K) U a (K) NH 2 OH + H → NH 3 + OH ( 2 ) A −24 893 3723 ( 2 ) B −24 695 4903 NH 2 OH + H → H 2 O + NH 2 ( 3 ) A −29 762 6437 ( 3 ) B −24 877 6206 NH 2 OH + H → H 2 NO + H 2 ( 4 ) A −13 172 4206 ( 4 ) B −13 444 7439 NH 2 OH + H → HNOH + H 2 ( 5 ) A −9527 3409 ( 5 ) B −10 763 3218 Figure 3. Left: product of reaction ( 2 ) in Model B . As a reference, the OH–H distance in the water dimer is 1.94 Å at the DLPNO-CCSD(T)/CBS//DSD- PBEP86-D3BJ/def2-TZVP level of theory. 3.2 Destruction of NH 2 OH In addition to the reactions presented in Nguyen et al. ( 2019 ) and He et al. ( 2015 ), whose implications are discussed in Section 4 , we have studied reactions ( 2 ) to ( 7 ) in Model A and Model B . The rationale behind the explicit study of these reactions is to determine viable NH 2 OH destruction routes to understand the abundances observed in Rivilla et al. ( 2020 ). A summary of the energetic descriptors for the reaction can be found in Table 2 for a quick o v erview of the results. At the same time, in the text, we deepen in the discussion of the reactions from a chemical point of view. The endothermicity of the trans → cis reaction, summed to the high acti v ation energies of the inversion reaction (more af fected by tunnelling), make that, for this particular reaction, we calculated the rate constants of reaction employing a TST corrected by a tunnelling factor obtained from the ﬁt of an asymmetric Eckart barrier. The rate constants are presented in Fig. 2 . From the trends, we observe that, in general, rate constants in Model B are lower 3.2.1 NH 2 OH + H → NH 3 + OH The ﬁrst of the destruction reactions that we studied is the addition of a hydrogen atom to the NH 2 moiety of hydroxylamine, leading to ammonia and the hydroxyl radical. Subsequently, the reaction NH 3 + OH −→ H 2 O + NH 2 (9) (9) Figure 2. Rate constants for isomerizations in NH 2 OH. Left panel – isomerization rate constants in the gas. Right panel – isomerization rate constants in the 2H 2 O explicit model (Model B ). Figure 2. Rate constants for isomerizations in NH 2 OH. Left panel – isomerization rate constants in the gas. Right panel – isomerization rate constants in the 2H 2 O explicit model (Model B ). MNRAS 521, 6061–6074 (2023) Hydroxylamine chemistry in the ISM 6065 Figure 4. Instanton rate constants for reaction ( 2 ) in Model A and Model B . The crosso v er temperature is 300 K for the former and 308 K for the latter. Figure 5. IRC calculations for reaction ( 2 ). The IRC path is neither corrected for ZPVE contributions nor DLPNO-CCSD(T) energies. 3.2 Destruction of NH 2 OH The reaction energies for reaction ( 3 ) are U R , ModelA = −29 762 K and U R , ModelB = −29 877 K, whereas acti v ation energies were found to be U a , ModelA = 6437 K and U a , ModelB = 6206 K. In this case, the effect of the inclusion of explicit water molecules in the model does not lead to signiﬁcant changes in the energetics of reaction. This is due to the transition state for the reaction being located separated from the H-bonding network of the NH 2 OH–H 2 O system. None the less, the acti v ation energies for the reaction are much higher for reaction ( 3 ) than for reaction ( 2 ). The instanton rate constants for reaction ( 3 ) are presented in Fig. 6 . From them, we determine that in the shallow binding sites deﬁned by Model A , reaction ( 3 ) is much slower than reaction ( 2 ). Ho we ver, it is interesting to observe that in Model B , the increased acti v ation energy of reaction ( 2 ), an effect not present in reaction ( 3 ), makes reactions ( 2 ) and ( 3 ) competitive. As we will show later, this stabilization is not ultimately rele v ant to the ISM chemistry of NH 2 OH, because H abstraction reactions dominate the chemistry. Finally, we also conﬁrmed that reaction ( 3 ) proceeds through a single elemental step, like reaction ( 2 ), using IRC calculations. The instanton rate constants for reaction ( 4 ) are presented in Fig. 7 . From the plot, we can see the immense effect of the different structural models on the rate constants. While in model A the reaction is the fastest of this study, in model B , we ﬁnd the slowest reaction. This ﬁnding is essentially the same provided in Nguyen et al. ( 2019 ) for the hydrogenation of NO, i.e. a critical rele v ance of the ice surface in the reaction. We note that for dual-level instantons in Model A , we observed that the rate constant at 50 K is ∼1.4 higher than at 80 K (not perceptible in the graph). 3.2 Destruction of NH 2 OH We note a decrease in the reaction rate constants of three orders of magnitude in the case of Model B , which is a better descriptor of deep binding sites, compared with Model A . None the MNRAS 521, 6061–6074 (2023) 6066 G. Molpeceres et al. Figure 7. Instanton rate constants for reaction ( 4 ) in Model A and Model B . The crosso v er temperature is 539 K for the former and 514 K for the latter. Figure 6. Instanton rate constants for reaction ( 3 ) in Model A and Model B . The crosso v er temperature is 406 K for the former and 399 K for the latter. Figure 7. Instanton rate constants for reaction ( 4 ) in Model A and M The crosso v er temperature is 539 K for the former and 514 K for the Figure 6. Instanton rate constants for reaction ( 3 ) in Model A and Model B . The crosso v er temperature is 406 K for the former and 399 K for the latter. Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Figure 7. Instanton rate constants for reaction ( 4 ) in Model A and Model B . The crosso v er temperature is 539 K for the former and 514 K for the latter. Figure 6. Instanton rate constants for reaction ( 3 ) in Model A and Model B The crosso v er temperature is 406 K for the former and 399 K for the latter. 3.2.2 NH 2 OH + H → H 2 O + NH 2 The second reaction we focus on in this work is reaction ( 3 ). Similar to the case found in Section 3.2.1 for NH 3 , once hydrogen adds to the OH moiety of NH 2 OH, it breaks it. Subsequent reactions to reform NH 2 OH, through for example the back-reaction: is exothermic but signiﬁcantly less than the H addition counter- parts (Reactions 2 and 3 ). The reaction energies are U R , ModelA = −13 172 K and U R , ModelB = −13 444 K. In the case of acti v ation energies, we found the highest gap between Model A and Model B for this reaction with U a , ModelA = 4206 K and U a , ModelB = 7439 K. 3.2 Destruction of NH 2 OH This fact is unsurprising considering that the OH moiety of NH 2 OH is interacting directly with a water molecule of the 2H 2 O cluster. Therefore, in Model B the hydrogen atom needs to o v ercome the combined effect of two processes, ﬁrst the breaking of a hydrogen bond and second the short-range barrier inherent to the reaction. H 2 O + NH 2 −→ NH 2 OH + H (10) (10) are endothermic (provided that the forward reaction is exothermic), proceed through high acti v ation barriers, or are not competitive with H abstraction reactions. The reaction energies for reaction ( 3 ) are U R , ModelA = −29 762 K and U R , ModelB = −29 877 K, whereas acti v ation energies were found to be U a , ModelA = 6437 K and U a , ModelB = 6206 K. In this case, the effect of the inclusion of explicit water molecules in the model does not lead to signiﬁcant changes in the energetics of reaction. This is due to the transition state for the reaction being located separated from the H-bonding network of the NH 2 OH–H 2 O system. None the less, the acti v ation energies for the reaction are much higher for reaction ( 3 ) than for reaction ( 2 ). The instanton rate constants for reaction ( 3 ) are presented in Fig. 6 . From them, we determine that in the shallow binding sites deﬁned by Model A , reaction ( 3 ) is much slower than reaction ( 2 ). Ho we ver, it is interesting to observe that in Model B , the increased acti v ation energy of reaction ( 2 ), an effect not present in reaction ( 3 ), makes reactions ( 2 ) and ( 3 ) competitive. As we will show later, this stabilization is not ultimately rele v ant to the ISM chemistry of NH 2 OH, because H abstraction reactions dominate the chemistry. Finally, we also conﬁrmed that reaction ( 3 ) proceeds through a single elemental step, like reaction ( 2 ), using IRC calculations. are endothermic (provided that the forward reaction is exothermic), proceed through high acti v ation barriers, or are not competitive with H abstraction reactions. 3.2 Destruction of NH 2 OH The reason for this unphysical effect can be found in slightly different pre-reactive complex geometries between our energy method DLPNO-CCSD(T) and our geometry method DSD-PBEP86-D3BJ/def2-TZVP. Ho we ver, this ef fect’s impact is minute in the o v erall conclusions. In Section 4.1.1 , we discuss the chemical implications of this ﬁnding and the current limitations of quantum chemical calculations to co v er the div ersity of chemically activ e sites on a real ASW surface. From a purely chemical perspective and for reaction ( 4 ) we must conclude that the most likely representation of the real situation on a surface must be closer to Model B than to Model A. Depending on the ice morphology, in the case of, for example, numerous H 2 O dangling bonds on the surface in porous ices (Bossa et al. 2015 ; Mat ´e, Satorre & Escribano 2021 ), the situation might be different, and it is hard to predict which model will predominantly describe the reaction. 3.2.3 NH 2 OH + H → H 2 NO + H 2 3.2.4 NH 2 OH + H → HNOH + H 2 For all three reactive trajectories and irrespective of the structural model, the reactions that we found are: HNOH + H −→ NH 2 OH (11) H 2 NO + H −→ NH 2 OH (12) H 2 NO + H −→ NH 3 O . (13) HNOH + H −→ NH 2 OH (11) H 2 NO + H −→ NH 2 OH (12) H 2 NO + H −→ NH 3 O . (13) (11) The instanton rate constants for this reaction are presented in Fig. 8 and support the energetic point of view presented abo v e. The y are similar both for Model A and Model B . It is important to note that in the gas phase, these rate constants are missing a factor 2 from the rotational symmetry number σ. In amorphous ices such as the ones these models aim for, such a de generac y must be broken. Our values for the acti v ation energies of reactions ( 3 ) and ( 4 ) vary with respect to the values in Nguyen et al. ( 2019 ) that reported acti v ation energies of ∼1500 K. While such deviations may stem from the computational method, they do not affect the main conclusion of this or their work, that is, NH 2 OH reacts with H via abstraction reactions, and mainly from the NH 2 moiety in the presence of strong H-bond networks. (13) H 2 NO + H −→ NH 3 O . For Model A and reactions ( 12 ) and ( 13 ), we found roughly a 40/60 per cent ratio in fa v our of reaction ( 13 ). The same reactions in Model B yield 45/55 per cent. For HNOH, reaction ( 11 ) dominates in 100 per cent of the reaction events, both for Models A and B . The zwitterion NH 3 O, ammonia oxide, was brieﬂy mentioned in Tse ga w et al. ( 2017 ), indicating that it is less stable than hydroxylamine, with an energy gap of around 1 eV ( ∼11 600 K, Wang et al. 2010 ). 3.2.4 NH 2 OH + H → HNOH + H 2 While the formation of NH 3 O in our simulations resembles a non-equilibrium situation and the actual relative energy between the structural isomers is not crucial in this context, we hypothesize that NH 3 O undergoes very fast isomerization on water ice, following, for example, a proton shuttle mechanism (Molpeceres et al. 2021a ; Perrero et al. 2022 ). In the following sections, we assume a rapid NH 3 O → NH 2 OH conversion, and we do not deepen in the interaction of NH 3 O with water ices. 3.2.4 NH 2 OH + H → HNOH + H 2 3.2.4 NH 2 OH + H → HNOH + H 2 3.2.4 NH 2 OH + H → HNOH + H 2 The ﬁnal reaction with acti v ation energy that we considered is represented in equation ( 5 ). This reaction is equi v alent to equation ( 4 ) except that the abstracted H comes from the NH 2 moiety that does not directly interact with the ice because, in interaction with water, the amine group acts as an H-bond acceptor. This ef fecti v ely leav es the H atoms free to react. The reaction energies are U R , ModelA = −9527 K and U R , ModelB = −10 763 K and the acti v ation energies are U a , ModelA = 3409 K and U a , ModelB = 3218 K. It is clear then that the major product of reaction of NH 2 OH + H must be HNOH + H 2 , both in Model A and in Model B. That is why, despite the reaction energies being lower than in the case of reaction ( 4 ), and H 2 NO being the thermodynamical product of the reaction, H 2 NO is not the most likely product of reaction at ISM conditions due to the inﬂuence of the water matrix in protecting the -OH moeity from abstraction. susceptible to reacting via radical–radical recombination. In the same vein as for the destruction reactions, we have sampled the reactions reforming NH 2 OH from H atoms, reactions ( 6 ) and ( 7 ). An example of the sampling for HNOH, Model B is depicted in Fig. 9 . Involving radical–radical recombinations, for this type of reaction we e v aluate the branching ratio of the reaction, which is the deﬁning quantity to input in astrochemical models. As we mentioned in Section 2.1.1 , we sampled 150 initial positions for each chemical model (Models A and B ). We must mention that in some of our other works (Miksch et al. 2021 ; Molpeceres et al. 2021b ) not all the geometry optimizations that we sampled ended in a reactive event due to the ﬂatness of the PES and the location of spurious minima. 3.2.3 NH 2 OH + H → H 2 NO + H 2 Reaction ( 4 ) involves the abstraction of H from an OH moiety, forming H 2 during the process. In the case of formic acid (HC(O)OH) (Molpeceres et al. 2022 ), we observed that abstracting hydrogen from this position is endothermic and proceeds through high acti v ation energy. For NH 2 OH we found that the abstraction in the OH moiety MNRAS 521, 6061–6074 (2023) Hydroxylamine chemistry in the ISM 6067 Figure 8. Instanton rate constants for reaction ( 5 ) in Model A and Model B . The crosso v er temperature is 420 K for the former and 416 K for the latter. Figure 9. Initial positions for the sampling of reaction outcomes in radical– radical recombinations (Reactions 6 and 7 ) for Model B . The image shows 77 initial structures of the 150 considered for this reaction. The white spheres represent initial poisions (one at a time) of the hydrogen atoms. Figure 9. Initial positions for the sampling of reaction outcomes in radical– radical recombinations (Reactions 6 and 7 ) for Model B . The image shows 77 initial structures of the 150 considered for this reaction. The white spheres represent initial poisions (one at a time) of the hydrogen atoms. Figure 8. Instanton rate constants for reaction ( 5 ) in Model A and Model B . The crosso v er temperature is 420 K for the former and 416 K for the latter. 3.3 NH 2 OH, HNOH, and H 2 NO binding energies Binding energies for NH 2 OH were calculated to include a reﬁned guess into astrochemical models. The moti v ation for updating these binding energies in the context of hydroxylamine destruction dis- cussion can be found in Wakelam et al. ( 2017a ) (to the best of our knowledge, the only study explicitly addressing NH 2 OH) where the authors state that they found NH 2 OH binding to be likely o v eresti- mated due to the computational protocol that they applied (that could not account for the donor–acceptor nature of the hydroxylamine molecule) with reported values between 6000 and 7400 K, depending on the theoretical model. Our results for the average binding energies using high-level quantum chemical calculations are presented in Table 3 , and the binding energy distributions can be found in Fig. 10 . From both the distributions and the average values, we conﬁrm that the assumption presented in Wakelam et al. ( 2017a ) was correct and that there was an o v erestimation of the NH 2 OH binding energies on water ice. Ho we v er, we do not e xpect this deviation to inﬂuence their study focused on the role of binding energies on chemical desorption since NH 2 OH remains less volatile than water. second 20 K, where we determined the importance of alternative NH 2 OH formation routes (e.g. ammonia oxidation). We used the chemical network presented in Garrod ( 2013 ), updated by the reactions presented in Section 3 , and in Congiu et al. ( 2012 ), Nguyen et al. ( 2019 ), and He et al. ( 2015 ). Further details of the speciﬁc chemical reactions are provided later in the text (Section 4.1 ). The molecular cloud physical model consists of a pseudo-time-dependent model whose physical conditions are presented in Table 4 and elemental abundances relative to hydrogen nuclei in Table 5 . In our model, grain-surface reactions use the modiﬁed rate approach of Garrod ( 2008 ), Model A. In e v aluating the rate of grain-surface reactions with acti v ation barrier, the diffusion-reaction competition is considered (Chang, Cuppen & Herbst 2007 ). As deﬁned abo v e, we assume that the diffusion energy of a given adsorbate is ∼0.4 its binding energy. 3.3 NH 2 OH, HNOH, and H 2 NO binding energies We set the exceptions in this factor for H 2 (0.5), H (0.5), and CO (0.3) based in our recent studies (Furuya, Oba & Shimonishi 2022a ; Furuya et al. 2022c ). In our model, chemistry on the grain takes place in the outermost four layers of the dust Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Element Initial abundance H 2 0.5 He 9 .8 × 10 −2 N 2 .5 × 10 −5 O 1 .8 × 10 −4 C + 7 .9 × 10 −5 S + 9 .1 × 10 −8 Si + 9 .7 × 10 −9 Fe + 2 .7 × 10 −9 Na + 2 .3 × 10 −9 Mg + 1 .1 × 10 −8 Cl + 2 .2 × 10 −10 P + 1 .0 × 10 −9 3.2.5 Hydro g enation of HNOH and H 2 NO Reactions ( 2 ) and ( 3 ) are irreversible in the context of the destruction of hydroxylamine by H atoms. As we discussed in Section 3.2.1 , there is not, to the best of our knowledge, a signiﬁcant production of NH 2 OH from OH + NH 3 or NH 2 + H 2 O (Reactions 9 and 10 ). Ho we ver, this is not the case for HNOH or H 2 NO, both radicals MNRAS 521, 6061–6074 (2023) 6068 G. Molpeceres et al. Table 4. Initial physical conditions utilized for modelling the evolution of NH 2 OH under molecular cloud conditions. Parameter Value n gas 2 × 10 4 cm −3 A v 5 mag ζ 1.3 × 10 −17 s −1 T g 10–20 K T d 10–20 K Table 5. Initial elemental abundances with respect to H nuclei. Taken from Aikawa & Herbst ( 1999 ). Element Initial abundance H 2 0.5 He 9 .8 × 10 −2 N 2 .5 × 10 −5 O 1 .8 × 10 −4 C + 7 .9 × 10 −5 S + 9 .1 × 10 −8 Si + 9 .7 × 10 −9 Fe + 2 .7 × 10 −9 Na + 2 .3 × 10 −9 Mg + 1 .1 × 10 −8 Cl + 2 .2 × 10 −10 P + 1 .0 × 10 −9 Table 4. Initial physical conditions utilized for modelling the evolution of NH 2 OH under molecular cloud conditions. Table 4. Initial physical conditions utilized for modelling the evolution of NH 2 OH under molecular cloud conditions. Table 3. Average binding energies (in K) of the main intermediates and NH 2 OH derived in this study. Molecule Binding energy (K) NH 2 OH 5382 HNOH 5536 H 2 NO 5925 It is important to mention that both in reactions ( 11 ) and ( 12 ) a mixture of cis-NH 2 OH and trans-NH 2 OH is formed, but in light of the results obtained in Section 3.1 , we conclude that the isomerization must take place in very short time-scales, as shown by the blue solid line (for gas) and blue dashed line (for surface isomerization) in Fig. 2 . Table 5. Initial elemental abundances with respect to H nuclei. Taken from Aikawa & Herbst ( 1999 ). Hydroxylamine chemistry in the ISM Hydroxylamine chemistry in the ISM 6069 Table 6. Surface reactions added to Garrod ( 2013 ) reaction network and employed in this work. Note that H 2 NO gas-phase chemistry was also included, and we considered it equi v alent (e.g. same reactions and rate constants) to the chemistry of HNOH (assuming different products). α, β, γ are the branching ratio of the reaction accounting for all possible reaction channels for a reaction, the acti v ation energy in K, and the barrier width in Å for the tunnelling rate constants. For a more in-depth description of the formalism, we refer to Ruaud, Wakelam & Hersant ( 2016 ), section 2.3. An α value of 0 means deacti v ating the reaction (see the text), and the rest of the parameters are not applicable (n/a in the table). Reaction α β γ Reference H + HNO → NO + H 2 0.00–0.33 i 0 0 .00 Nguyen et al. ( 2019 ) H + HNO → H 2 NO 0.50–0.33 i 734 1 .00 Nguyen et al. ( 2019 ) H + HNO → HNOH 0.50–0.33 i 8167 1 .00 Nguyen et al. ( 2019 ) HNO + HNO → N2O + H 2 O 1.00 0 0 Nguyen et al. ( 2019 ) HNO + NO → N2O + OH 1.00 0 0 Nguyen et al. ( 2019 ) H + H 2 NO → HNO + H 2 0.00 n/a n/a This work (Section 3.2.5 ) H + HNOH → HNO + H 2 0.00 n/a n/a This work (Section 3.2.5 ) H + H 2 NO → NH 2 OH ii 0.50 0 0 .00 Nguyen et al. ( 2019 ) & This work (Section 3.2.5 ) H + HNOH → NH 2 OH 0.50 0 0 .00 Nguyen et al. ( 2019 ) & This work (Section 3.2.5 ) O + NH 3 → NH 2 OH 1.00 1500 1 .00 He et al. ( 2015 ) & This work (Section 4.1.2 ) NH 2 + OH → NH 2 OH 1.00 0 0 .00 Zheng & Kaiser ( 2010 ) & Nishi et al. 4.1 Molecular cloud model Fig. 12 shows the abundances of the main NO-bearing compounds for the model, neglecting chemical desorption (bottom panel) and assuming a constant desorption probability (top panel). From the plots, we deduce that for this chemical model, NH 2 OH is the most abundant NO-bearing molecule in the ice, formed mainly by reaction ( 12 ). This also reﬂects in a high abundance of NH 2 OH in the gas phase, in the order of a few 10 −8 at the NH 2 OH peak, which is two orders of magnitude higher than the values reported in Rivilla et al. ( 2020 ). When we neglect chemical desorption, the abundance in the gas-phase declines to values around 10 −11 , which is lower than the observational value in G + 0.693 (Rivilla et al. 2020 ) by around a factor of 2. This observation is unsurprising considering the high binding energy of NH 2 OH that precludes chemical desorption. When chemical desorption is deacti v ated, most of the NH 2 OH gas abundance is triggered by photodesorption. When comparing with the upper limit of the abundance presented in Ligterink et al. ( 2018 ) of the IRAS16293-2422 hot corino, we observe that the current model predicts the opposite trend with respect the ratio n(NH 2 OH)/n(N 2 O) that in our models fa v ours hydroxylamine (gas + ice, since in hot corinos ices are sublimated) in contrast to the observations. The explanation for this discrepancy can be found in the alternative reactions in the hydroxylamine formation reaction network. In particular, the reaction During our chemical simulations, we found that the reaction rate of HNO + H → NO + H 2 is key for the evolution of NH 2 OH. Thus, we ran a grid of models varying its branching ratio concerning the competing reactions. We discuss these ﬁndings in 4.1.1 . Likewise, we further analysed the viability of NH 3 + O → NH 2 OH in Section 4.1.2 . (14) HNO + H −→ NO + H 2 (14) Finally, we did not check the inﬂuence of the chemical structural model (Models A and B ) of our quantum chemical calculations in the abundances for the molecular cloud model. We assume Model seems to be of paramount importance and is discussed in the following section. Hydroxylamine chemistry in the ISM ( 1984 ) H + NH 2 OH → NH 3 + OH 0.25 4,903 iii 1 .00 This work (Section 3.2.1 ) H + NH 2 OH → H 2 O + NH 2 0.25 6,206 iii 1 .00 This work (Section 3.2.2 ) H + NH 2 OH → H 2 NO + H 2 0.25 7,439 iii 0 .50 iv This work (Section 3.2.3 ) H + NH 2 OH → HNOH + H 2 0.25 3,218 iii 0 .50 iv This work (Section 3.2.4 ) Notes. (i)This parameter is varied in some models (See Section 4.1.1 ). (ii)NH 3 O is assumed to isomerize to NH 2 OH (See Section 3.2.5 ). (iii)Structural model B values. (See Table 2 ). (iv)To account for the increased tunnelling efﬁciency of H abstraction reactions. B to be more realistic and base the astrophysical conclusions on it. Hence, we continue with the values in Model B throughout this section, indicating that a small contribution from binding sites closer to Model A may inﬂuence variability in our results. We enumerate the main reactions added to Garrod ( 2013 )’s reaction network in Table 6 and a scheme of the main reactions is portrayed in Fig. 11 . particle, with a chemically inactive mantle. Quantum tunnelling of atomic H is taken into account in the e v aluation of the diffusion- reaction mechanism. We explicitly include our o wn deri ved binding energies for NH 2 OH, H 2 NO, and HNOH into the model. Because of the high binding energy of NH 2 OH and related molecules, non- thermal mechanisms returning the molecule to the gas phase are paramount. In our models, we considered photodesorption, from the primary interstellar UV ﬁelds as well as the secondary UV ﬁeld of molecular clouds (Prasad & Tarafdar 1983 ), with a photodesorption yield of 1 × 10 −3 for all molecules. We also considered cosmic- ray-induced desorption due to the stochastic heating of cosmic dust in its Hase ga wa–Herbst formulation (Hase ga wa & Herbst 1993 ), with a peak-temperature per collision event of 70 K for 10 μs. Finally, we also included chemical desorption in our models. For that purpose, we considered two possible scenarios. First, a model with a constant desorption probability per chemical reaction of ∼1 per cent (Garrod, Wakelam & Herbst 2007 ). Hydroxylamine chemistry in the ISM In the second model, we neglected chemical desorption. While the latter model is more physically sound for a molecule as strongly bound as NH 2 OH, the former allows for an increased return of the NH 2 OH molecule formed on grains to the gas phase of molecular clouds. It is important to mention that in the Galactic Centre, where NH 2 OH was recently detected (Rivilla et al. 2020 ), an additional mechanism operates, returning molecules to the gas-phase that are mechanical shocks induced by cloud–cloud collisions; the explicit simulation of this mechanisms is currently out of the scope of our models. 4 CHEMICAL MODELING We included our quantum chemically deri ved acti v ation energies, binding energies, and branching ratios into a three-phase astrochem- ical model (e.g. gas, ice surface, mantle) presented in Furuya et al. ( 2015 ) to simulate a molecular cloud with limited external energy input. Within this model, we considered two temperature settings. First, 10 K, where hydrogenation reactions must dominate, and Figure 10. Binding energy distribution for the main intermediates and NH 2 OH of this study. Left: binding energies of NH 2 OH; Centre: binding energies of HNOH; Right: binding energies of H 2 NO. Figure 10. Binding energy distribution for the main intermediates and NH 2 OH of this study. Left: binding energies of NH 2 OH; Centre: binding energies of HNOH; Right: binding energies of H 2 NO. MNRAS 521, 6061–6074 (2023) 4.1.1 The HNO + H → NO + H 2 reaction 4.1.1 The HNO + H → NO + H 2 reaction Despite the signiﬁcant uncertainties in the last section’s models, they are in line with the values reported in Garrod et al. ( 2022 ), where they report peak abundances in the order of 4 × 10 −6 . In Garrod et al. ( 2022 ), the authors explicitly mention that the abundance of NH 2 OH may be o v erestimated. Hence, we think that NH 2 OH ice abundances of 10 −6 , as the ones we obtained in the previous section, are probably unrealistic and a consequence of very efﬁcient hydrogenation on dust grains. To palliate this, we have deepened the study of reaction ( 14 ). Should this reaction be very efﬁcient then the formation of NH 2 OH would be precluded. This is a general observation of the hydrogenation reaction on ice; more hydrogenations needed to saturate a molecule translate into more possibilities for branches along the hydrogenation sequence. composition and surface co v erage is unfeasible. Therefore, the results in Nguyen et al. ( 2019 ) remain accurate for H 2 O ices. This is the same as saying that in the absence of short-range barriers arising from electronic structure changes in the reactants, e.g. for barrierless reactions, the inﬂuence of the surface in the total reaction rate of a species is very hard to gauge from experiments/theory. A possible way of weighting the importance of a barrierless surface reaction with a marked inﬂuence of surface binding, like reaction ( 14 ), is to run a sensitivity analysis of chemical models to that particular reaction. Since reaction ( 14 ) does not have an entrance barrier, the effect of the surface enters linearly in the rate constant, and it is speciﬁcally reﬂected by the branching ratio of reaction ( α in Table 6 ). Hence, we determined the importance of reaction ( 14 ) in the production of NH 2 OH by running a grid of models varying the branching ratio of the reaction and accordingly adopting scaled values for the competing reactions: In their combined theoretical and experimental study Nguyen et al. ( 2019 ) determined that reaction ( 14 ) does not have an intrinsic acti v ation barrier (e.g. it is barrierless), but its occurrence is forbidden due to orientation effects stemming from the HNO–H 2 O interaction. 4.1 Molecular cloud model seems to be of paramount importance and is discussed in the following section. MNRAS 521, 6061–6074 (2023) 6070 G. Molpeceres et al. 6070 Figure 12. (Top). Time-dependent abundances of main NO-bearing molecules for the molecular cloud model at T Dust = 10 K and using a constant 0.01 chemical desorption probability. (Bottom). Time-dependent abundances of main NO-bearing molecules for the molecular cloud model at T Dust = 10 K and neglecting chemical desorption. The dashed lines represent ice species (surface + mantle), whereas the solid lines represent gas phase species. The abundance here is represented to H nuclei (e.g. H + H 2 ). composition and surface co v erage is unfeasible. Therefore, the results in Nguyen et al. ( 2019 ) remain accurate for H 2 O ices. This is the same as saying that in the absence of short-range barriers arising from electronic structure changes in the reactants, e.g. for barrierless reactions, the inﬂuence of the surface in the total reaction rate of a species is very hard to gauge from experiments/theory. A possible way of weighting the importance of a barrierless surface reaction with a marked inﬂuence of surface binding, like reaction ( 14 ), is to run a sensitivity analysis of chemical models to that particular reaction. Since reaction ( 14 ) does not have an entrance barrier the effect of the surface enters linearly in the rate constant Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Figure 12. (Top). Time-dependent abundances of main NO-bearing molecules for the molecular cloud model at T Dust = 10 K and using a constant 0.01 chemical desorption probability. (Bottom). Time-dependent abundances of main NO-bearing molecules for the molecular cloud model at T Dust = 10 K and neglecting chemical desorption. The dashed lines represent ice species (surface + mantle), whereas the solid lines represent gas phase species. The abundance here is represented to H nuclei (e.g. H + H 2 ). Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Figure 11. Schematic view of the formation and destruction paths of NH 2 OH. Some alternative reactions not leading to hydroxylamine are excluded in the scheme (see the text and Table 6 ). HNO + H −→ HNOH (16) 4.1 Molecular cloud model In solid line, main reaction routes contributing to the reaction network, either at 10 K (Sections 4.1 and 4.1.1 ) or at 20 K, section ( 4.1.2 ). In dashed lines, reactions whose contribution is minor at all temperatures. Figure 12. (Top). Time-dependent abundances of main NO-bearing molecules for the molecular cloud model at T Dust = 10 K and using a constant 0.01 chemical desorption probability. (Bottom). Time-dependent abundances of main NO-bearing molecules for the molecular cloud model at T Dust = 10 K and neglecting chemical desorption. The dashed lines represent ice species (surface + mantle), whereas the solid lines represent gas phase species. The abundance here is represented to H nuclei (e.g. H + H 2 ). 4.1.1 The HNO + H → NO + H 2 reaction We adhere to this picture, observing the effect of the H-bond network in the acti v ation energies of the reactions explicitly considered in this study (See Table 2 ). Under ISM conditions, ho we ver, when ices are made of more components than H 2 O and/or surface co v erage includes more abundant species (e.g. H 2 ), it is possible that not all binding modes of HNO with the surface preclude reaction ( 14 ). We emphasize that theoretically, determining the reaction outcomes of interstellar reactions as a function of the appropriate surface HNO + H −→ HNOH (15) HNO + H −→ H 2 NO (16) (15) MNRAS 521, 6061–6074 (2023) HNO + H −→ H 2 NO The sensitivity analysis results are presented in Fig. 13 . We considered the non-chemical desorption model for this analysis. From the ﬁgure, we found that the production of NH 2 OH markedly MNRAS 521, 6061–6074 (2023) Hydroxylamine chemistry in the ISM 6071 Figure 14. Same as Fig. 12 but with branching ratio of 0.15 for reaction HNO + H → NO + H 2 . Figure 13. Total abundances of NH 2 OH (e.g. gas + ice) as a function of the branching ratio of reaction for the reaction HNO + H → NO + H 2 , reaction ( 14 ). Note that a branching ratio of 0.00 means deacti v ating the reaction. Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Figure 13. Total abundances of NH 2 OH (e.g. gas + ice) as a function of the branching ratio of reaction for the reaction HNO + H → NO + H 2 , reaction ( 14 ). Note that a branching ratio of 0.00 means deacti v ating the reaction. Figure 14. Same as Fig. 12 but with branching ratio of 0.15 for reaction HNO + H → NO + H 2. depends on the efﬁciency of reaction ( 14 ), with abundances that span almost two orders of magnitude. The main reason behind the changes in abundances of hydroxylamine lies in the competition between reaction ( 14 ) and reactions ( 15 ) and ( 16 ). While reaction ( 14 ) is barrierless and has a low branching ratio, reactions ( 15 ) and ( 16 ) present an acti v ation barrier, although for reaction ( 16 ) this is very low. This establishes a pseudo-equilibrium fa v oured by the rapid diffusion of H atoms on the surface in the tunnelling-mediated reaction-diffusion competition between these reaction channels. cademic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 Therefore, we determined that the reaction ( 14 ) is essential and serves as a way of reducing the known problem of the o v erabundance of hydroxylamine in astrochemical models (Garrod 2013 ; He et al. 2015 ; Garrod et al. 2022 ). Ho we ver, we cannot pinpoint the optimal value of the branching ratio. Should NH 2 OH be detected in cold cores, we will have solid evidence to compare with. HNO + H −→ H 2 NO Without that, comparisons with the only source where this molecule was detected, G + 0.693 (Rivilla et al. 2020 ), require careful treatment of the non- thermal mechanisms operating in those environments, which is out of the scope of this work. Figure 14. Same as Fig. 12 but with branching ratio of 0.15 for reaction HNO + H → NO + H 2 . abundance of N 2 O and NO 2 . Ho we v er, our model o v erproduces gas-phase HNO in comparison with G + 0.693. Although a direct comparison with G + 0.693 is out of the scope of this work, it is worth mentioning that, in the absence of chemical desorption (without the signiﬁcant contribution of grain chemistry, where the molecule is formed through NO + H → HNO), gas phase HNO is produced in our model from NH 2 + O → HNO + H and H 2 NO + e −→ HNO + H. New reaction routes may help reduce this molecule’s abundance in the gas phase, bringing together models and observations for this particular molecule in the gas of this source. Concerning the absolute abundance of the parent molecule NO, we observe that the peak abundance of gas NO is n(NO)/n(H 2 ) ∼1 × 10 −7 , in models with and without chemical desorption, and compare relatively well with the abundances of G + 0.693 in Zeng et al. ( 2018 ) of n(NO)/n(H 2 ) ∼3 × 10 −7 . We note that this value is rather consistent with the abundances of the Sgr B2(N) molecular cloud as well (Zeng et al. 2018 ), and previously in other molecular clouds such as L134N, where NO abundances were found to be in between 6 × 10 −8 McGonagle et al. ( 1990 ) and 3 × 10 −7 (Gerin et al. 1992 ). We reiterate that a value of 0.15 for the branching ratio is taken here arbitrarily and is presented for illustrative purposes to highlight the importance of acti v ating or deacti v ating reaction ( 14 ) and provide a solution to the o v erabundance of NH 2 OH on ices (Garrod 2013 ; He et al. 2015 ; Garrod et al. 2022 ). For this reason, we keep this choice in the models discussed in Section 4.1.2 . HNO + H −→ H 2 NO Assuming a compromise value between 0 and 0.33, for example, 0.15, we have computed the molecular abundances of the important NO-bearing molecules with the constant chemical desorption scheme and neglecting chemical desorption. The results can be viewed in Fig. 14 . The change in the branching ratio of reaction ( 14 ) has an important effect on the abundances of both ice and gas NH 2 OH. For the gas, it reduces the peak abundance by around one order of magnitude. More importantly, for the ice’s NH 2 OH abundances, we ﬁnd a decrease of around one or two orders of magnitude in abundance. Such a change promotes the formation of N 2 O through the surface reaction NO + N −→ N2O , (17) (17) NO + N −→ N2O , NO + N −→ N2O , making N 2 O the most abundant NO-bearing molecule on ice and reconciling our models with the lower limit obtained in the IRAS16293-2422 hot-corino (Ligterink et al. 2018 ) in respect of the ratio n(NH 2 OH)/n(N 2 O), with the value of the upper limit derived here, that now is correctly accounted for. Concerning G + 0.693, our models reproduce well the inferred ratios for nitrogen oxides (Rivilla et al. 2020 ), n(NO) > n(N 2 O) ∼n(NO 2 ) > n(HNO) ∼n(NH 2 OH), albeit with some discrepancies. This trend is reasonably reproduced in our models, which also predict a pre v alence of NO as the central nitric oxide at the NO molecules peak (10 6 yr) and an on-par making N 2 O the most abundant NO-bearing molecule on ice and reconciling our models with the lower limit obtained in the IRAS16293-2422 hot-corino (Ligterink et al. 2018 ) in respect of the ratio n(NH 2 OH)/n(N 2 O), with the value of the upper limit derived here, that now is correctly accounted for. Concerning G + 0.693, our models reproduce well the inferred ratios for nitrogen oxides (Rivilla et al. 2020 ), n(NO) > n(N 2 O) ∼n(NO 2 ) > n(HNO) ∼n(NH 2 OH), albeit with some discrepancies. HNO + H −→ H 2 NO This trend is reasonably reproduced in our models, which also predict a pre v alence of NO as the central nitric oxide at the NO molecules peak (10 6 yr) and an on-par We found a general explanation for the reduction of NH 2 OH at low temperatures in the combination of three different surface MNRAS 521, 6061–6074 (2023) MNRAS 521, 6061–6074 (2023) G. Molpeceres et al. 6072 Figure 15. Same as Fig. 12 but at T Dust = 20 K and branching ratio of 0.15 for reaction HNO + H → NO + H 2 . reactions. The most critical reaction is H abstraction from HNO (Reaction 14 ), as explained above, but the H abstraction (Reaction 5 ), and hydrogen addition (Reaction 6 ) loop is also important. At low temperatures, the H abstraction reactions dominate the processing of surface NH 2 OH, with reactions rates up to six orders of magnitude higher than other mechanisms such as photodissociation, either by primary or secondary UV, extracted from rates at 1 Myr. To put it in context, photodissociation and processing through reaction ( 2 ) have similar reaction rates. Finally, it is important to consider the non- thermal effects that can return the molecules to the gas. Especially useful for it is a careful observation of the reaction rates for the model including chemical desorption. The comparison of the reaction rates at 1 Myr, between photodesorption (A v = 5 mag) and chemical desorption for this model, reveals that chemical desorption dominates the release of molecules to the gas phase. Downloaded from https://academic.oup.com/mnras/article/521/4/6061/7084996 by CSIC user on 25 October 2023 The difference between reactions ( 4 ) and ( 5 ) conﬁrms yet another important ﬁnding. The HNOH radical is produced by Reaction ( 5 ), because the acti v ation barrier from HNO (Reaction 15 ) is very high (Nguyen et al. 2020 ). This is indirect evidence that the H- abstraction and H-addition loop enhances the chemical desorption rates of NH 2 OH by nearly two orders of magnitude, a value obtained from the comparison between the rates for the NH 2 OH chemical desorption coming from the HNOH radical (only produced by Reaction 5 ) and the chemical desorption from the H 2 NO radical (produced mainly through HNO hydrogenation, reaction 16 ). 4.1.2 Model for T Dust = 20 K necessary, and dedicated experimental and quantum chemical studies can be pivotal in understanding the mechanism of reaction ( 18 ). In the absence of contamination with 1 O, that was discarded in the e xperiments abo v e based in prior studies by the same group, Jing et al. 2013 , such a step must take place in the 3 O −NH 3 pre-reactant complex. Explicitly simulating that chemical process is out of the scope of this work, but based on this evidence, we think that the (ef fecti ve) acti v ation energy of reaction ( 18 ) must be closer to the upper end of the range of values presented in He et al. ( 2015 ). Therefore, we took 1500 K for this acti v ation barrier. The results of this model can be found in Fig. 15 . Compared to the kinetic models presented in He et al. ( 2015 ), our ﬁnal abundances for NH 2 OH are lower by two to three orders of magnitude, and the dominant NO- bearing molecules on the surface are NO 2 and N 2 O. This is due to the increased efﬁciency at 20 K of both H thermal desorption and the barrierless reactions: necessary, and dedicated experimental and quantum chemical studies can be pivotal in understanding the mechanism of reaction ( 18 ). In the absence of contamination with 1 O, that was discarded in the e xperiments abo v e based in prior studies by the same group, Jing et al. 2013 , such a step must take place in the 3 O −NH 3 pre-reactant complex. Explicitly simulating that chemical process is out of the scope of this work, but based on this evidence, we think that the (ef fecti ve) acti v ation energy of reaction ( 18 ) must be closer to the upper end of the range of values presented in He et al. ( 2015 ). Therefore, we took 1500 K for this acti v ation barrier. The results of this model can be found in Fig. 15 . Compared to the kinetic models presented in He et al. ( 2015 ), our ﬁnal abundances for NH 2 OH are lower by two to three orders of magnitude, and the dominant NO- bearing molecules on the surface are NO 2 and N 2 O. 4.1.2 Model for T Dust = 20 K This is due to the increased efﬁciency at 20 K of both H thermal desorption and the barrierless reactions: We concluded our study of the molecular cloud model by e v aluating the inﬂuence of alternative reactions to NO hydrogenation in the formation of NH 2 OH. As pointed out in He et al. ( 2015 ), a plausible formation route for NH 2 OH formation is the oxidation of ammonia via : NH 3 + O −→ NH 2 OH , (18) (18) NH 3 + O −→ NH 2 OH , a reaction that does not proceed at 10 K due to the low mobility of the oxygen atom on ASW but that starts to contribute at 12 K onwards, reaching asymptotic abundances with respect to the dust temperature at 20 K, irrespective of the not-known activation energy for the reaction (see ﬁg. 9 of He et al. 2015 ). We ran our chemical model, with and without chemical desorption included, for a temperature of 20 K. The selection of the acti v ation energy for the reaction is not trivial, and we tried to constrain such values ab-initio through two possible mechanisms: HNOH + O −→ HNO + OH (21) HNOH + N −→ HNO + NH , (22) HNOH + O −→ HNO + OH (21) (21) NH 3 + 3 O −→ 3 NH 3 O −→ 3 NH 2 OH −→ 1 NH 2 OH (19) NH 3 + 3 O −→ NH 2 + OH −→ NH 2 OH . (20) HNOH + N −→ HNO + NH , (22) (22) (19) that are not competitive at 10 K due to the reduced mobility of the N and O atoms to H.The diffusion activation energies of N and O are 288 and 528 K, e.g fast diffusion. Therefore, the H abstraction reactions from NH 2 OH studied in this work are also important at 20 K. They are the onset for destruction routes not operative at lower temperatures. Since reactions ( 6 ) and ( 7 ) result in H addition rather than abstraction, NH 2 OH is protected thanks that are not competitive at 10 K due to the reduced mobility of the N and O atoms to H.The diffusion activation energies of N and O are 288 and 528 K, e.g fast diffusion. HNO + H −→ H 2 NO Chem- ical desorption is likely the main desorption mechanism in cold environments for NH 2 OH and other molecules that can experience H-abstraction/H-addition loops even if the chemical desorption per reactive event is lower than 1 per cent. Other examples of such loops can be found in H 2 S (Oba et al. 2018 ; Furuya, Oba & Shimonishi 2022a ) or PH 3 (Nguyen et al. 2020 , 2021 ), both molecules with positive conﬁrmation of chemical desorption from ASW. Figure 15. Same as Fig. 12 but at T Dust = 20 K and branching ratio of 0.15 for reaction HNO + H → NO + H 2 . Figure 15. Same as Fig. 12 but at T Dust = 20 K and branching ratio of 0.15 for reaction HNO + H → NO + H 2 . 4.1.2 Model for T Dust = 20 K 4.1.2 Model for T Dust = 20 K Therefore, the H abstraction reactions from NH 2 OH studied in this work are also important at 20 K. They are the onset for destruction routes not operative at lower temperatures. Since reactions ( 6 ) and ( 7 ) result in H addition rather than abstraction, NH 2 OH is protected thanks 5 SUMMARY AND CONCLUSIONS The picture left concerning the pre v alence of NH 2 OH by our quantum chemical and kinetic simulations is as follows. As found in Congiu et al. ( 2012 ); Fedoseev et al. ( 2012 ); and Nguyen et al. ( 2019 ), hydrogenation of NO molecules on dust grains is the primary for- mation path of NH 2 OH under astrophysical conditions. The reaction is very ef fecti ve because it is irreversible at low temperatures once the reaction HNO + H → H 2 NO takes place. Ho we ver, completely neglecting the another branch HNO + H → NO + H 2 results in the known problem of the o v erproduction of NH 2 OH (Garrod 2013 ; He et al. 2015 ; Garrod et al. 2022 ). We found that enabling the reaction, even in small amounts (15 per cent of the available HNO binding sites), modulates the reaction enough to have abundances of NH 2 OH close to the values reported in Rivilla et al. ( 2020 ). Ho we ver, such an agreement can only be reached when signiﬁcant inﬂuence of non-thermal desorption events is considered, which is not expected under molecular cloud conditions. The molecular cloud in the Galactic Centre, G + 0.693, the object where this molecule was detected (Rivilla et al. 2020 ), is subjected to shocks and increased cosmic-ray ﬂuence that violently releases part of the molecular inventory in grains to the gas. When the conditions of the object are milder, we found that hydroxylamine starts to be processed by heavier radicals and atoms, signiﬁcantly reducing its abundance. DATA AVAILABILITY Data supporting this work will be provided upon request to the corresponding author. (20) (20) NH 3 + 3 O −→ NH 2 + OH −→ NH 2 OH . In the abo v e reactions, and based on our calculations (Model B , DSD-PBEP86-D3BJ/def2- TZVP. values), 3 NH 2 OH is a dissociative complex, and the H abstraction in the triplet channel is endothermic. Therefore, to explain experimental ﬁndings of He et al. ( 2015 ), an intersystem crossing from the triplet PES to the singlet one is deemed In the abo v e reactions, and based on our calculations (Model B , DSD-PBEP86-D3BJ/def2- TZVP. values), 3 NH 2 OH is a dissociative complex, and the H abstraction in the triplet channel is endothermic. Therefore, to explain experimental ﬁndings of He et al. ( 2015 ), an intersystem crossing from the triplet PES to the singlet one is deemed MNRAS 521, 6061–6074 (2023) Hydroxylamine chemistry in the ISM 6073 to a loop of addition-abstraction reactions that is broken as soon as heavier radicals can diffuse on the dust surface. It would remain to know if reactions ( 21 ) and ( 22 ) are barrierless; that is an assumption of our model. None the less, alternative reactions with O or N atoms, not explored in this w ork, w ould lead to products different than NH 2 OH, so our conclusion remains valid. Similarly, we expect similar behaviour at higher dust temperatures, providing a sufﬁciently slow warm-up ramp that allows radical chemistry to proceed. In regards to different molecular ratios, we ﬁnd that at 20 K, our predicted n(NH 2 OH)/n(N 2 O) is signiﬁcantly different than at 10 K, as well as other ratios such as n(NH 2 OH)/n(NO 2 ). The variability in these molecular ratios may be helpful in future surv e ys, interpreting source-to-source variations of NH 2 OH. and experimental results (Largo, Redondo & Barrientos 2004 ; Snow et al. 2007 ; Barrientos et al. 2012 ) indicating that it may be a crucial ingredient for the emergence of amino acids and RNA nucleotides in space. ACKNOWLEDGEMENTS GM thanks the Japan Society for the Promotion of Science (Grant P22013) and the Alexander von Humboldt Foundation for their support. Some calculations of this work were performed with the support of the state of Baden-W ¨wrttemberg through bwHPC and the German Research Foundation (DFG) through grant no. INST 40/575-1 FUGG (JUSTUS 2 cluster). VMR has received support from the project RYC2020-029387-I (COOL: Cosmic Origins Of Life) funded by MCIN/AEI /10.13039/501100011033. BM thanks Spain MIC grant PID2020-113084GB-I00. KF acknowledges sup- port from JSPS KAKENHI grants 20H05847 and 21K13967. 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https://openalex.org/W4383745286	http://www.thieme-connect.de/products/ejournals/pdf/10.1055/s-0043-1770964.pdf	English	null	Imaging of Small Bowel Tumors and Mimics	Deleted Journal	2,023	cc-by	6,536	Abstract Small bowel tumors are rare with nonspeciﬁc and protean clinical presentation. Early diagnosis of small bowel tumors is desirable as they can be associated with signiﬁcant morbidity. In malignant small bowel tumors, delayed diagnosis may result in dissemination and metastasis leading to poor clinical outcomes. Imaging evaluation of small bowel can be challenging due to unpredictable luminal distension, peristalsis, and motion. In addition, the lack of distinction between the intraluminal lesions and intraluminal contents can be difﬁcult at times. Computed tomography (CT) and magnetic resonance (MR) enterography are the most common imaging techniques for the evaluation of small bowel tumors. While these techniques may not be able to detect small tumors, they provide comprehensive evaluation of lumen, wall, and extramural structures in tumors more than 2 cm. Acquain- tance of imaging appearance of common benign and malignant small bowel tumors may allow improved detection during evaluation of CT and MR enterography studies. In this review, we discuss the imaging appearances, approach, and differential diagnosis of small bowel tumors on cross-sectional imaging studies. Small bowel tumors are rare with nonspeciﬁc and protean clinical presentation. Early diagnosis of small bowel tumors is desirable as they can be associated with signiﬁcant morbidity. In malignant small bowel tumors, delayed diagnosis may result in dissemination and metastasis leading to poor clinical outcomes. Imaging evaluation of small bowel can be challenging due to unpredictable luminal distension, peristalsis, and motion. In addition, the lack of distinction between the intraluminal lesions and intraluminal contents can be difﬁcult at times. Computed tomography (CT) and magnetic resonance (MR) enterography are the most common imaging techniques for the evaluation of small bowel tumors. While these techniques may not be able to detect small tumors, they provide comprehensive evaluation of lumen, wall, and extramural structures in tumors more than 2 cm. Acquain- tance of imaging appearance of common benign and malignant small bowel tumors may allow improved detection during evaluation of CT and MR enterography studies. In this review, we discuss the imaging appearances, approach, and differential diagnosis of small bowel tumors on cross-sectional imaging studies. Abstract Keywords ►small bowel ►tumors ►CT ►MRI predilection for speciﬁc histologic subtypes tumors, for example, adenocarcinoma is more common in the duode- num and jejunum, and carcinoid tumor is more common in the ileum.1 The risk factors for malignant small bowel tumors are alcohol, tobacco, chronic inﬂammatory diseases includ- ing celiac disease and Crohn’s disease, human immunodeﬁ- ciency virus infection, certain foods (e.g., red meat, smoked, salty, and fatty food), and inherited syndromes including Peutz-Jeghers syndrome (PJS), hereditary nonpolyposis co- lorectal cancer, and familial adenomatous polyposis (FAP).5–9 Imaging of Small Bowel Tumors and Mimics Pankaj Gupta1 Saurav Lamichane1 Harsimran Bhatia1 Manphool Singhal1 Vishal Sharma2 Harjeet Singh3 Rajender Kumar4 Manavjit Singh Sandhu1 1Radiodiagnosis, Post Graduate Institute of Medical Education and Research, Chandigarh, India 2Gastroenterology, Post Graduate Institute of Medical Education and Research, Chandigarh, India 3Surgical Gastroenterology, Post Graduate Institute of Medical Education and Research, Chandigarh, India 4Nuclear Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India Address for correspondence Pankaj Gupta, MD, Department of Radiodiagnosis, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India (e-mail: Pankajgupta959@gmail.com). Address for correspondence Pankaj Gupta, MD, Department of Radiodiagnosis, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India (e-mail: Pankajgupta959@gmail.com). J Gastrointestinal Abdominal Radiol ISGAR 2024;7:55–64. Article published online: 2023-07-10 Article published online: 2023-07-10 Article published online: 2023-07-10 Article published online: 2023-07-10 Article published online: 2023-07-10 Review Article 55 55 article published online July 10, 2023 © 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Thieme Medical and Scientiﬁc Publishers Pvt. Ltd., A-12, 2nd Floor, Sector 2, Noida-201301 UP, India DOI https://doi.org/ 10.1055/s-0043-1770964. ISSN 2581-9933. Introduction Small bowel tumors are rare. They comprise 3 to 6% of gastrointestinal tract neoplasms.1 The diagnosis of small bowel tumors is delayed by the nonspeciﬁc nature of the symptoms (abdominal pain, weight loss, gastrointestinal bleeding) and low clinical suspicion.2 The small bowel neo- plasms can present with complications like intussusception, obstruction, and perforation.3 Thus, early diagnosis is desir- able by accurate interpretation of radiologic ﬁndings. The occurrence of small bowel tumors is more in the proximal small bowel in comparison to the distal small bowel.4 The different segments of the small bowel have Benign small bowel tumors comprise 0.5 to 2% of all gastrointestinal neoplasms.10 Benign small bowel tumors © 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Thieme Medical and Scientiﬁc Publishers Pvt. Ltd., A-12, 2nd Floor, Sector 2, Noida-201301 UP, India DOI https://doi.org/ 10.1055/s-0043-1770964. ISSN 2581-9933. article published online July 10, 2023 article published online July 10, 2023 Imaging of Small Bowel Tumors and Mimics Gupta et al. 56 Table 1 Various diagnostic modalities for small bowel tumors Endoluminal (invasive/ semi-invasive) Radiological (noninvasive) Fluoroscopic Cross-sectional imaging Upper GI endoscopy Colonoscopy Push enteroscopy Balloon enteroscopy Balloon enteroscopy Endoscopic ultrasound Capsule endoscopy SBFT SBE CT enterography MR enterography PET-CT PET-CT Enterography Virtual CT Endoscopy Abb i ti CT t d t h MR ti GI are generally solitary. Multiple tumors are seen in polyposis syndromes.11 Primary malignant small bowel tumors are less common than benign neoplasms.4 The malignant small bowel tumors generally have poor prognosis due to delayed presentation. As small bowel can tolerate mild obstruction, patients usually present late when at least two-thirds of the lumen is obstructed.12 Table 1 Various diagnostic modalities for small bowel tumors At imaging, benign small bowel tumors usually appear round and well circumscribed with smooth margins. The malignant tumors have irregular margins with heteroge- neous enhancement and may be associated with invasion of adjacent structures. Abbreviations: CT, computed tomography; MR, magnetic resonance; GI, gastrointestinal; PET-CT, positron emission tomography-computed to- mography; SBE,smallbowelenteroclysis; SBFT,smallbowelfollow through. Abbreviations: CT, computed tomography; MR, magnetic resonance; GI, gastrointestinal; PET-CT, positron emission tomography-computed to- mography; SBE,smallbowelenteroclysis; SBFT,smallbowelfollow through. Lipoma Despite being a rare tumor, small bowel lipoma is the most common benign lesion of the bowel causing intussusception in adults. Lipoma arises from submucosa and consists of mature adipose tissue surrounded by a thin capsule. Lipoma is usually sessile; however, it can be pedunculated. Almost 50% of lipomas are found in the ileum, and less than 50% of patients with small bowel lipoma are symptomatic.23 Lesions more than 2 cm in diameter can be symptomatic and may cause bowel obstruction or gastrointestinal bleed- ing.24 Small bowel lipoma has no malignant potential. On gastrointestinal contrast studies, lipomas have char- acteristic appearance and demonstrate mobility. They pro- duce a solitary smooth intraluminal ﬁlling defect. They demonstrate a pseudopedicle at their tip.25 CT and MRI features are diagnostic. A well-deﬁned homogeneous mass with fat attenuation (40 to 120 HU) on CT is seen (►Fig. 1).12 The MRI ﬁndings of small bowel lipoma include homogeneous signal intensity corresponding to macroscopic fat without contrast enhancement.13 In comparison to cross-sectional imaging, endoscopy (or enteroscopy) and capsule endoscopy are better for the detection of small intraluminal tumors.15 However, a study reported better sensitivity of CTE over capsule endoscopy for the detection of submucosal lesions.22 Though with the current state of the art endoscopy techniques, the entire small bowel can be evaluated, endoscopic techniques do not provide information regarding the extramural extent of the diseases.3,13 The various imaging techniques utilized in evaluation of small bowel tumors are listed in ►Table 1. The various benign and malignant small bowel tumors are mentioned in ►Table 2. Introduction In general, the imaging detection of small bowel is chal- lenging due to bowel peristalsis, mobility of abdominal struc- tures,respiratory motion,nonuniformdistensionwithluminal agents, and lack of contrast between normal bowel tissue and mass.13 Plain radiograph has no role in patients with small bowel tumors except when they present in emergency with features of small bowel obstruction and perforation. Ultra- sound also has limited role in detection of small bowel tumors. However, in patients at high risk of small bowel tumors like polyposis syndrome, celiac disease, or Crohn’s disease, ultra- sound evaluation of small bowel may be facilitated by luminal distension using protocols similar to computed tomography enterography (CTE). Barium meal follow through is not con- sidered reliable for the detection of small bower tumors. CT is the widely used modality for imaging of small bowel tumors due to its availabilityanditsspeed ofacquisition.Furthermore, CT is lesssusceptible to motion artifacts andprovides excellent spatial resolution.13 However, the ionizing radiation exposure and the need for intravenous iodinated contrast agent are the majorlimitationsofCT.CTEprotocolisthepreferredtechnique for suspected small bowel tumors.14,15 Patients should have at least 4 to 6 hour fasting status before the study.16 Oral contrast is administered for adequate distention of the lumen. Neutral oral contrasts are preferred to positive oral contrast.17 These provide better delineation of mucosal enhancement, mural thickness, and mesenteric vasculature.18 A few studies have evaluated a novel CT technique employing carbon dioxide instillation (virtual CT endoscopy) for the evaluation of small bowel lesions including tumors.19,20 MR enterography is an alternative to CTE. The key beneﬁt of MR enterography is the absence of ionizing radiation. This makes MRI particularly attractive for imaging in children and for repeat examination. However, thelimitations includelong acquisition time, limited availability, and a relative higher cost.21 Table 2 Various benign and malignant small bowel tumors Benign Malignant Lipoma Polyp Leiomyoma Gastrointestinal stromal tumor (GIST) Inﬂammatory ﬁbroid polyp Hemangioma Adenocarcinoma Lymphoma Neuroendocrine tumor Malignant GIST Metastasis Gastrointestinal Neuroectodermal tumors Table 2 Various benign and malignant small bowel tumors Journal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Polyp Polyps comprise one-ﬁfth of the benign small bowel neo- plasms.26 Polyps generally are asymptomatic. However, pol- yps can lead to obstruction or intussusception when they grow large enough. Pathologic subtypes are hamartomatous, hyperplastic, adenomatous, and inﬂammatory.13 Polyps can be solitary or multiple. Numerous polyps are seen in inherited syndromes (►Fig. 2). Polyps appear as small (<2 cm), homo- genously enhancing masses which protrude into the bowel ournal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Imaging of Small Bowel Tumors and Mimics Gupta et al. 57 57 Fig. 1 Lipoma: Axial (A) and coronal sections (B) of contrast-enhanced computed tomography abdomen showing a well-deﬁned fat attenuation lesion in second part of duodenum (white arrows). Fig. 1 Lipoma: Axial (A) and coronal sections (B) of contrast-enhanced computed tomography abdomen showing a well-deﬁned fat attenuation lesion in second part of duodenum (white arrows). Journal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Leiomyoma lumen.11 The adenomatous subtype has increased risk of malignant transformation and is associated with polyposis syndromes.Adenomatouspolypsareseen inpatientswithFAP. Hamartomatous polyps are associated with juvenile polyposis syndrome, PJS, and Cowden syndrome.27 Cronkhite–Canada syndrome is an acquired nonfamilial polyposis syndrome that is characterized by gastrointestinal polyposis, onycholysis, cutaneous pigmentation, and alopecia.28 The risk of malignant transformation is higher for tumors more than 1 cm.23 Size greater than 2 cm and extraserosal extension are highly sug- gestive of malignant degeneration within a polyp.29 Leiomyoma is a rare tumor. It is more common in the jejunum than the ileum.30 Clinical presentation is due to tumor ulceration and bleeding, causing abdominal pain, gastrointestinal bleeding, and chronic anemia. At CT and MRI, it appears as a well-deﬁned homogeneously enhancing mass. Calciﬁcation and ulceration may occur in larger tumors.31 Imaging features may be indistinguishable from gastrointestinal stromal tumor (GIST). Larger tumors (>6 cm) with irregular margins and lymphadenopathy should cause suspicion for malignancy.30 Fig. 2 Polyps: (A) Endoscopic image showing multiple polyps and (B) involvement of ﬁngernails in Cronkhite-Canada syndrome; (C) Axial computed tomography (CT) enterography showing well-deﬁned round homogenously enhancing polyp in the jejunal lumen (arrow); (D) Circumoral muco-cutaneous pigmentation; (E) Barium meal follow through showing round ﬁlling defects (consistent with polyps) in the antrum of stomach (arrows) and (F) Coronal CT enterography section showing multiple enhancing polyps within the small and large bowel (arrows). Fig. 2 Polyps: (A) Endoscopic image showing multiple polyps and (B) involvement of ﬁngernails in Cronkhite-Canada syndrome; (C) Axial computed tomography (CT) enterography showing well-deﬁned round homogenously enhancing polyp in the jejunal lumen (arrow); (D) Circumoral muco-cutaneous pigmentation; (E) Barium meal follow through showing round ﬁlling defects (consistent with polyps) in the antrum of stomach (arrows) and (F) Coronal CT enterography section showing multiple enhancing polyps within the small and large bowel (arrows). Fig. 2 Polyps: (A) Endoscopic image showing multiple polyps and (B) involvement of ﬁngernails in Cronkhite-Canada syndrome; (C) Axial computed tomography (CT) enterography showing well-deﬁned round homogenously enhancing polyp in the jejunal lumen (arrow); (D) Circumoral muco-cutaneous pigmentation; (E) Barium meal follow through showing round ﬁlling defects (consistent with polyps) in the antrum of stomach (arrows) and (F) Coronal CT enterography section showing multiple enhancing polyps within the small and large bowel (arrows). Imaging of Small Bowel Tumors and Mimics Gupta et al. 8 58 Fig. GIST Treatment for resectable GISTs is wide local excision. Systemic treatment in the form of chemotherapy is often administered. Imaging features of GISTs change post-imati- nib therapy and include intralesional hemorrhage, cystic degeneration of tumor, and development of ascites.39 The radiologist must thus be aware of these changes and always acquire a multiphasic CT following a noncontrast scan in follow-up patients with GIST on chemotherapy.39 GISTs are the most frequent mesenchymal tumor arising from gastrointestinal tract.32 More than one-third of the GISTs arise in the small bowel. They are more common in the proximal small bowel.33 GISTs arise from the interstitial cells of Cajal and are characterized by c-KIT (CD117) expres- sion.32 Other mutations include platelet derived growth factor receptor α, succinate dehydrogenase, v-raf murine sarcoma viral oncogene homolog B1, and neuroﬁbromatosis type 1.33 They tend to have a wide spectrum of clinical behavior, ranging from benign tumors (which are inciden- tally discovered) to malignant lesions with considerable overlap in the imaging and microscopic features of both entities.34 All GISTs are potentially malignant.35,36 GISTs are the most frequent mesenchymal tumor arising from gastrointestinal tract.32 More than one-third of the GISTs arise in the small bowel. They are more common in the proximal small bowel.33 GISTs arise from the interstitial cells of Cajal and are characterized by c-KIT (CD117) expres- sion.32 Other mutations include platelet derived growth factor receptor α, succinate dehydrogenase, v-raf murine sarcoma viral oncogene homolog B1, and neuroﬁbromatosis type 1.33 They tend to have a wide spectrum of clinical behavior, ranging from benign tumors (which are inciden- tally discovered) to malignant lesions with considerable overlap in the imaging and microscopic features of both entities.34 All GISTs are potentially malignant.35,36 Leiomyoma 3 Gastrointestinal stromal tumors: (A) Hypervascular intraluminal mass (arrow) in the D-J ﬂexure/proximal jejunum; (B, C): necrotic tumor involving distal jejunum on computed tomography (CT; arrow in B) and ultrasound (arrow in C); (D) Axial contrast-enhanced CT abdomen showing hypoenhancing exophytic mass lesion arising from stomach with multiple foci of calciﬁcation; (E and F) Axial and coronal images showing endophytic jejunal gastrointestinal stromal tumor. Fig. 3 Gastrointestinal stromal tumors: (A) Hypervascular intraluminal mass (arrow) in the D-J ﬂexure/proximal jejunum; (B, C): necrotic tumor involving distal jejunum on computed tomography (CT; arrow in B) and ultrasound (arrow in C); (D) Axial contrast-enhanced CT abdomen showing hypoenhancing exophytic mass lesion arising from stomach with multiple foci of calciﬁcation; (E and F) Axial and coronal images showing endophytic jejunal gastrointestinal stromal tumor. Hemangioma These rare submucosal tumors occur more commonly in the jejunum. They may be sessile or pedunculated.40 On CT, hemangioma appears as enhancing, intraluminal polypoid mass. On MR, hemangiomas show marked T2-weighted hyperintensity with avid nodular enhancement in the arte- rial phase.41 The enhancement is retained in the delayed phase. At imaging, benign GISTs are indistinguishable from other mesenchymal tumors. They are well-circumscribed lesions with a variable enhancement pattern. They commonly ex- tend exophytically from the bowel lumen. Calciﬁcation is a rare ﬁnding (►Fig. 3).37 Smaller lesions (< 2cm) appear as hyper-enhancing lesions. With increasing size, there is de- velopment of necrosis in the tumor core. Larger lesions thus appear as heterogeneously enhancing cavitating lesions. Although imaging features may not be entirely reliable for distinguishing benign and malignant GISTs, larger masses with necrosis, local invasion, and hemorrhage suggest ma- lignant behavior.38 Metastases in such cases commonly occur to the liver, omentum, or peritoneum (►Fig. 4). Signiﬁcant lymphadenopathy is not seen in these cases and favors other malignant neoplasms like lymphoma or metastatic disease. The prognosis of GIST depending upon CT features is men- tioned in ►Table 3. Journal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Adenocarcinoma Adenocarcinoma accounts for 25 to 40% of primary malig- nant tumors of the small bowel.42,43 Proximal jejunum or distal duodenum is the most involved sits.44 Presentation may be nonspeciﬁc or related to malignancy-induced gas- trointestinal bleeding or obstruction. CT appearances include circumferential annular (apple core) mural thickening or eccentric and irregular mass with luminal narrowing (►Fig. 5).45 There may be extension into adjacent fat, vascular invasion, lymphadenopathy, peritoneal and distant metastases (most often to the liver). CT appearances include circumferential annular (apple core) mural thickening or eccentric and irregular mass with luminal narrowing (►Fig. 5).45 There may be extension into adjacent fat, vascular invasion, lymphadenopathy, peritoneal and distant metastases (most often to the liver). ournal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Imaging of Small Bowel Tumors and Mimics Gupta et al. 5 59 Fig. 4 Malignant gastrointestinal stromal tumor: (A) Multiple liver metastases are seen (arrows). The cystic appearance of these lesions is due to imatinib therapy. (B) Marked peritoneal thickening and nodularity (arrows) secondary to peritoneal metastases is seen. stinal stromal tumor: (A) Multiple liver metastases are seen (arrows). The cystic appearance of these lesions is due to d peritoneal thickening and nodularity (arrows) secondary to peritoneal metastases is seen. Fig. 4 Malignant gastrointestinal stromal tumor: (A) Multiple liver metastases are seen (arrows). The cystic appearance of these lesions is due to imatinib therapy. (B) Marked peritoneal thickening and nodularity (arrows) secondary to peritoneal metastases is seen. Neuroendocrine Tumor 40 to 80% of the cases. Mesenteric metastases calcify in 70% of the cases.49 Differential diagnoses include treated lymphoma or retractile mesenteritis. Somatostatin-analog imaging exams have an important role both in diagnosis and staging (►Fig. 6). In-pentetreotide imaging (Octreoscan) has now been replaced by newer analogue agents such as 18F-FDOPA and 68Ga-DOTATATE.51 Gastrointestinal neuroendocrine tumors (GNET) originate from the enterochromafﬁn cells within the gastro-entero- pancreatic system.34 These are the second most common malignant tumors of the small bowel (20–25% of malignant tumors).46 One-third of GNETs originate from the small bow- el.47 Ileum is the most common site of involvement. The characteristic appearance of small bowel GNET is a well- deﬁned solitary avidly enhancing mural mass (►Fig. 6). Smaller lesion may be missed on CT. MRI may allow detection of some of these lesions due to better soft tissue resolution. Multiple GNETs occur in one-fourth of the patients (►Fig. 6).48 Carcinoid tumors may cause asymmetric or nodular mural thickening. The key to correct diagnosis in these cases is the identiﬁcation of spiculated (due to desmoplastic reaction caused by secretion of serotonin) calciﬁed mesenteric lesion with or without liver metastases.49,50 Mesenteric involvement by direct extension or via lymphatics occurs in approximately Journal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Lymphoma Primary gastrointestinal lymphoma is the most common form of extranodal lymphoma.52 Diagnosis can be ascer- tained by the lack of peripheral or mediastinal lymphade- nopathy, normal white blood cell count, and differential leucocyte count without the involvement of liver or spleen.53 Ileum is the most common site of involvement due to abundant lymphoid tissue (►Fig. 7). Lymphoma of the small bowel is categorized into ﬁve forms: pseudoa- neurysmal, polypoid, endoexoenteric, stenosing, and mesenteric (►Fig. 7).54 The most common type of small bowel lymphoma is the polypoidal form with single or multiple polypoidal lesions protruding into the lumen.55 This type of lymphoma may act as a lead point for intus- susception. The pseudoaneurysmal form involves the sub- mucosa and muscularis layers causing mass-like mural thickening. The lack of obstruction is due to complete replacement of the muscle layer with lymphoid tissue.54 The endoexoenteric form or the cavitatory form produces a large soft-tissue mass communicating with the bowel lu- men producing a characteristic air-contrast contrast mate- rial level. Malignant GIST may produce a similar appearance.12 The stenosing form of lymphoma is uncom- mon. It is usually encountered in patients with celiac disease. This form occurs most commonly in the distal duodenum.12 In the mesenteric form, tumor extends into the mesentery from the bowel wall. Table 3 Prognostic features of GIST on imaging CT features Prognosis Site Stomach Duodenum Ileum/jejunum Favorable Intermediate Unfavorable Size <5cm 5–10 cm >10 cm Favorable Intermediate Unfavorable Margin Regular Irregular Favorable Unfavorable Homogeneous enhancement Favorable Hemorrhage Unfavorable Necrosis/cystic degeneration Unfavorable Air Unfavorable Abbreviations: CT, computed tomography; GIST, gastrointestinal stro- mal tumor. Table 3 Prognostic features of GIST on imaging The endoexoenteric form or the cavitatory form produces a large soft-tissue mass communicating with the bowel lu- men producing a characteristic air-contrast contrast mate- rial level. Malignant GIST may produce a similar appearance.12 The stenosing form of lymphoma is uncom- mon. It is usually encountered in patients with celiac disease. This form occurs most commonly in the distal duodenum.12 In the mesenteric form, tumor extends into the mesentery from the bowel wall. Immunophenotypically, lymphomas are of two types: B-cell and T-cell lymphoma. B-cell lymphoma (diffuse large B-cell lymphoma) is the most common type and is usually present is ileum, whereas T-cell lymphoma is usually associ- ated with celiac disease and is present commonly in jejunum Imaging of Small Bowel Tumors and Mimics Gupta et al. 60 Fig. Journal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Metastasis Metastasis to the small bowel is rare. Melanoma, lung cancer, and breast cancer are the tumors that may show small bowel spread.29 Small bowel metastases may be solitary or multiple with a variety of appearances. At one end of the spectrum, they can mimic benign lesions with discrete, smoothly marginated nodules showing homogenous enhancement. At the other end, there are large mass with cavitation, invasion of adjacent structures, and intraperitoneal spread (►Fig. 8).56,57 Metastases must be suspected when a solid Lymphoma Imaging of Small Bowel Tumors and Mimics Gupta et al. 61 Fig. 7 Lymphoma: (A–C): Coronal computed tomography enterography images showing heterogeneously enhancing lesion in relation to terminal ileum (arrow). B: Asymmetric wall thickening involving terminal ileum (arrow); C: Circumferential mural thickening causing luminal stenosis in proximal ileum (arrow); D: Asymmetric mural thickening with exophytic component in terminal ileum (arrow); E: Ileo-colic intussusception secondary to lymphoma (arrow). Fig. 7 Lymphoma: (A–C): Coronal computed tomography enterography images showing heterogeneously enhancing lesion in relation to terminal ileum (arrow). B: Asymmetric wall thickening involving terminal ileum (arrow); C: Circumferential mural thickening causing luminal stenosis in proximal ileum (arrow); D: Asymmetric mural thickening with exophytic component in terminal ileum (arrow); E: Ileo-colic intussusception secondary to lymphoma (arrow). small bowel mass is seen in a patient with malignancy known to metastasize to bowel. Lymphoma 5 Adenocarcinoma: (A) Coronal computed tomography (CT) enterography image showing asymmetric enhancing mural thickening (arow) involving distal jejunum causing intestinal obstruction; (B, C): Coronal and axial CT enterography image showing asymmetric mural thickening involving the duodenum with adjacent invasion (arrow in B) and necrotic retroperitoneal lymph nodes (arrow in C); (D, E): Enteroscopic images showing intraluminal polypoidal (D) and annular growth patterns (E). Fig. 5 Adenocarcinoma: (A) Coronal computed tomography (CT) enterography image showing asymmetric enhancing mural thickening (arow) involving distal jejunum causing intestinal obstruction; (B, C): Coronal and axial CT enterography image showing asymmetric mural thickening involving the duodenum with adjacent invasion (arrow in B) and necrotic retroperitoneal lymph nodes (arrow in C); (D, E): Enteroscopic images showing intraluminal polypoidal (D) and annular growth patterns (E). and proximal ileum.55 T-cell lymphoma isgenerally multifocal and may be associated with complication like perforation. tensive regional and distant adenopathy help in conﬁrming the diagnosis and from other neoplasms. On barium meal follow through, there is irregular wall thickening, fold effacement, aneurysmal bowel dilatation, single, or multiple ﬁlling defects due to polypoid masses. At CT and MRI, the imaging appearance of small bowel lympho- ma parallels the morphological forms described above. Ex- Differentiating lymphoma from primary adenocarcinoma can be challenging. The features favoring lymphoma are distal site of involvement (ileum), marked homogeneous wall thickening usually greater than 2 cm, multifocal involve- ment, and extensive lymphadenopathy.12 Fig. 6 Neuroendocrine tumor: (A) Solitary hypervascular lesion within the duodenal lumen (arrow); (B) Coronal image showing multiple neuroendocrine tumors (NETs) in the stomach (white arrows) and hypervascular lymph nodes (double headed white arrows), (C) Hypervascular NET in the duodenum with calciﬁcations (arrow) with liver metastasis (dashed arrow); (D, E): Somatostatin analogue positron emission tomography images showing avid lesions in the mesentery (arrow); (F) Computed tomography enterography image showing spiculated hypoenhancing mesenteric mass lesion (arrow). Fig. 6 Neuroendocrine tumor: (A) Solitary hypervascular lesion within the duodenal lumen (arrow); (B) Coronal image showing multiple neuroendocrine tumors (NETs) in the stomach (white arrows) and hypervascular lymph nodes (double headed white arrows), (C) Hypervascular NET in the duodenum with calciﬁcations (arrow) with liver metastasis (dashed arrow); (D, E): Somatostatin analogue positron emission tomography images showing avid lesions in the mesentery (arrow); (F) Computed tomography enterography image showing spiculated hypoenhancing mesenteric mass lesion (arrow). Journal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Journal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Small Bowel Diverticulitis Other than duodenal or Meckel’s diverticulitis, small bowel diverticulitis is rare. Jejunum is more commonly involved.58 On CT, there is a thick-walled well-deﬁned mass-like Fig. 8 Small bowel metastases: (A) Multiple enhancing serosal deposits in a case of ovarian cancer; (B) Enteroscopic image showing intraluminal polypoidal hemorrhagic soft tissue mass in jejunum in a patient with choriocarcinoma. Fig. 8 Small bowel metastases: (A) Multiple enhancing serosal deposits in a case of ovarian cancer; (B) Enteroscopic image showing intraluminal polypoidal hemorrhagic soft tissue mass in jejunum in a patient with choriocarcinoma. Journal of Gastrointestinal and Abdominal Radiology ISGAR Vol. 7 No. 1/2024 © 2023. The Author(s). Imaging of Small Bowel Tumors and Mimics Gupta et al. 62 62 Fig. 9 Neoplasms mimics: (A) Jejunal diverticulitis (arrow); (B, C): Dilated terminal ileum with mural thickening (B, arrow) and mural thickening involving cecum with dilated terminal ileum (C, arrow) in a patient with ileocecal tuberculosis; (D) Mural thickening involving jejunal loops in a patient with Henoch–Schonlein purpura. Fig. 9 Neoplasms mimics: (A) Jejunal diverticulitis (arrow); (B, C): Dilated terminal ileum with mural thickening (B, arrow) and mural thickening involving cecum with dilated terminal ileum (C, arrow) in a patient with ileocecal tuberculosis; (D) Mural thickening involving jejunal loops in a patient with Henoch–Schonlein purpura. Fig. 9 Neoplasms mimics: (A) Jejunal diverticulitis (arrow); (B, C): Dilated terminal ileum with mural thickening (B, arrow) and mural thickening involving cecum with dilated terminal ileum (C, arrow) in a patient with ileocecal tuberculosis; (D) Mural thickening involving jejunal loops in a patient with Henoch–Schonlein purpura. Table 4 Differential diagnosis for small bowel tumor Table 4 Differential diagnosis for small bowel tumor Intramural hematoma Anticoagulation, coagulopathy, trauma, vasculitis, mass-like, mural hyperattenuation with luminal narrowing; spontaneous resolution Small bowel diverticulitis Elderly patients, jejunum, ovoid, mass-like structure containing air, ﬂuid, lesion with adjacent fat standing Meckel’s diverticulitis Mass-like structure in continuity with small bowel Eosinophilic gastroenteritis Nodular/Irregular focal, segmental or diffuse thickening vs. lymphoma Giardiasis/Whipple’s disease Fold thickening in duodenum and proximal jejunum Localized lymphangiectasia Low attenuation wall thickening of jejunum Sclerosing mesenteritis versus carcinoid structure containing intestinal contents (debris, fecal mate- rial, and gas). There is associated bowel wall thickening and mesenteric fat stranding. In some patients, additional diver- ticulitis may also be seen at other sites.59,60 phase helps in the differentiation of small bowel hematoma fromothercauses ofbowelwall thickening.65Withagingof the hematoma, the hyperdensity of the bowel may disappear. 1 Neugut AI, Jacobson JS, Suh S, Mukherjee R, Arber N. The epide- miology of cancer of the small bowel. Cancer Epidemiol Biomark- ers Prev 1998;7(03):243–251 Small Bowel Hematoma Small bowel hematoma occurs in the setting of anticoagula- tion, coagulopathies, vasculitis, trauma, and malignancy.63,64 On CT, the thickened bowel shows mural hyperattenuation, andluminalnarrowing.12Themuralhyperattenuationinacute Meckel’s Diverticulum Meckel’s diverticulum is associated with several complica- tions including diverticulitis, perforation, enterolith forma- tion, bowel obstruction, bleeding from ectopic gastric mucosa, and neoplasm.61 On CT, Meckel’s diverticulum may be confused for a bowel origin mass, but evaluation of serial thin sections reveals a visualized as a blind-ending, ﬂuid, or debris-ﬁlled, dilated mass-like structure in continu- ity with the ileum. CT enterography has higher sensitivity in evaluating Meckel’s diverticulum.62 Small Bowel Diverticulitis Complete resolution of hematoma occurs over a few weeks. 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https://openalex.org/W2937855308	https://europepmc.org/articles/pmc6515406?pdf=render	English	null	Finite Element Analysis for Surface Acoustic Wave Device Characteristic Properties and Sensitivity	Sensors	2,019	cc-by	14,017	Received: 15 February 2019; Accepted: 5 April 2019; Published: 12 April 2019 Abstract: The most vital step in the development of novel and existing surface acoustic wave (SAW)-based sensors and transducers is their design and optimization. Demand for SAW devices has been steadily increasing due to their low cost, portability, and versatility in electronics, telecommunications, and biosensor applications. However, a full characterization of surface acoustic wave biosensors in a three-dimensional (3D) ﬁnite element model has not yet been developed. In this study, a novel approach is developed for analyzing shear horizontal Love wave resonator devices. The developed modeling methodology was veriﬁed using fabricated devices. A thorough analysis of the 3D model and the experimental device was performed in this study including scattering parameters (S-parameters), reﬂection coeﬃcient parameters, transmission parameters, and phase velocity. The simulated results will be used as a design guideline for future device design and optimization, which has thus far resulted in close matching between prediction and experimental results. This manuscript is the ﬁrst to demonstrate a 3D ﬁnite element model to correlate the sensitivity of the SAW device with the magnitude of the phase shift, the real and imaginary part of the response, insertion loss, and the frequency shift. The results show that the imaginary part of the response shift has a higher sensitivity compared to other parameters. Keywords: surface acoustic wave (SAW); ﬁnite element method (FEM); sensitivity; IrO2; ZnO sensors sensors sensors Tao Wang 1,2,3 , Ryan Green 2,4, Rasim Guldiken 2,5, Jing Wang 6 , Subhra Mohapatra 1,2,4,* and Shyam S. Mohapatra 1,2,3,* 1 James A Haley VA Hospital, Tampa, FL 33612, USA; taowang@mail.usf.edu Center for Research and Education in Nanobioengineering, University of South Florida, Tampa, FL 33612, USA; rgreen1@health.usf.edu (R.G.); guldiken@usf.edu (R.G.) 2 Center for Research and Education in Nanobioengineering, University of South Florida, Tampa, FL 336 USA; rgreen1@health.usf.edu (R.G.); guldiken@usf.edu (R.G.) 3 Department of Internal Medicine and Pharmacy Graduate Programs, University of South Florida, Tampa, FL 33612, USA 3 Department of Internal Medicine and Pharmacy Graduate Programs, University of South Florida, Tampa, FL 33612, USA 4 Department of Molecular Medicine, University of South Florida, Tampa, FL 33612, USA p y p 5 Microﬂuidics and Acoustics Laboratory, Department of Mechanical Engineering, College of Engineerin University of South Florida, Tampa, FL 33610, USA y p 6 Department of Electrical Engineering, University of South Florida, Tampa, FL 33610, USA; jingw@usf.e * C d h 2@h l h f d (S M) h @h l h f d (S S M ) Department of Electrical Engineering, University of South Florida, Tampa, FL 33610, USA; jingw@usf.edu Correspondence: smohapa2@health.usf.edu (S.M); smohapat@health.usf.edu (S.S.M.); Tel.: +1-813-974-4127 (S.M.); +1-813-974-8568 (S.S.M.) Received: 15 February 2019; Accepted: 5 April 2019; Published: 12 April 2019 www.mdpi.com/journal/sensors Finite Element Analysis for Surface Acoustic Wave Device Characteristic Properties and Sensitivity Tao Wang 1,2,3 , Ryan Green 2,4, Rasim Guldiken 2,5, Jing Wang 6 , Subhra Mohapatra 1,2,4,* and Shyam S. Mohapatra 1,2,3,* 1. Introduction Surface acoustic wave (SAW)-based devices have been mass-produced and widely used in consumer products and communication equipment [1]. The increasing demand for fast and precise simulation tools is driven by their essential role in research and development. The advantages of these tools include reduced time and cost for production as well as improved design optimization and a better understanding of the underlying Multiphysics [2]. Due to their high sensitivity and great portability [3], SAW devices can be utilized in a wide range of applications including electronic actuators [4,5], telecommunication modulators [6,7], as well as biochemical and gas sensors [8–12]. A number of simulation techniques and methods have been developed and applied to acoustic modeling but there is still need for signiﬁcant improvement [2]. The most eﬀective methods are Sensors 2019, 19, 1749; doi:10.3390/s19081749 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 18 Sensors 2019, 19, 1749 based on numerical analysis such as Green’s function and the coupling-of-modes method, which are widely used to analyze and optimize SAW device designs [13–15]. Other methods for analyzing SAW devices are based on impulse response, transmission matrices, and equivalent circuit models, but they are not able to accurately reproduce all the behaviors of SAW devices—especially estimating all the electromechanical eﬀects and multiple order eﬀects on the device’s function [16–19]. Many years ago, a research team proposed the use of cascaded equivalent circuits for the SAW interdigital transducer (IDT) [20]. Over the last few years, research has been conducted on mass loading (the energy storing eﬀect), and this model has been widely used in SAW research and development due to its simplicity and reasonable accuracy [21]. To save computation resources and time, several techniques have been developed to characterize SAW properties for the thin metal layer and metallic grating structures [22]. Eﬀects of the grating ﬁnger thickness have also been studied extensively [23]. Even though the Green’s function and coupling-of-modes methods can be used to estimate the multiple order eﬀects, it becomes more complicated for them to stimulate and estimate the eﬀects of multiple guiding layers, especially with multiple piezoelectric layers. However, because of limited computer resources, early research in this ﬁeld and the modeling tools it employed were relatively simple such as the perturbation theory model [24]. The recent rapid progress of computer hardware and software technologies has made it possible to deal with very complex problems with multiphysics coupling and multilayer metallic grating structures [5,25]. 1. Introduction However, these methods have signiﬁcant limitations and require multiple assumptions that limit their applicability while solving physical constraints, boundary conditions, complex geometry materials, and multiphysics coupling. Previous research [26–28] has led to an eﬀective ﬁnite element method (FEM) analysis technique which utilizes an impulse signal through an applied voltage to propagate at the surface of the device and transfer the energy from input to output interdigital transducers to analyze the frequency response. The results obtained from this technique can help the researcher predict the device’s capability/sensitivity more accurately and to optimize the device coupling with diﬀerent guide layers and samples. Fluids with particles of diﬀerent concentrations and viscosities resulting in frequency shifts in SAW devices are also being investigated using the commercial simulation package (COMSOL Multiphysics) [29] with the FEM. This analysis can easily predict experimental results and optimize the design in an aﬀordable way. However, most of the reports are focused on the wave reﬂection–transmission and mechanical deformation [30,31]. The mass sensitivity and other electromechanical properties have not been demonstrated based on FEM, and its experimental veriﬁcation has also not been adequately reported until now. p In this study, multiple electromechanical properties and mass sensitivity eﬀects determined by the guide layer thickness are discussed. A 3-dimensional (3D) model based on a realistic device is built to obtain 3D wave responses. Due to the limitations of the computational resources available, this model was designed with a trade-oﬀbetween computation speed and precision. By comparing the calculated results from the simulation to measured results from network analysis, we show that the simulation results can be in very close agreement with the measured responses in multiple electrical properties such as S-parameters, reﬂection parameters, transmission parameters, and velocity phase. The calculated phase sensitivity and frequency shift sensitivity are compared in order to optimize signal measurement methods. The most three common electronic conﬁgurations to measure the mass loading are based on the oscillator circuit, vector voltmeter, and network analyzer [32]. Oscillatory circuit systems can be designed to measure the frequency shift or phase shift in a loop system. The vector voltmeter can be used to measure the phase shift. The network analyzer is the instrument used to characterize the device for all information such as phase, frequency, imaginary response, real response, and standing wave ratio. 2.1. Background Recently, an increasing number of research groups have shown interest in ﬁnite element analysis of SAW devices with varying structures and designs. In this study, all simulation models were based on 36◦YX cut lithium tantalate substrates, employed the commercially available ﬁnite element analysis package (COMSOL Multiphysics 5.2), and were analyzed in Matlab©. Two computers with E5-2630 V3 processors and 96 GB RAM were used to calculate the results, and the calculation time for the model was greater than 72 h. The method employed not only provides information on ﬁlter input–output signal levels, phase velocity, wave phase, wave interference, and diﬀraction, it also renders excellent preliminary design information on the response of a SAW sensor [33]. The frequency analysis of a SAW device was conducted through a full three-dimensional (3D) model simulation. The 36◦YX cut lithium tantalate functions to generate the shear horizontal surface acoustic wave. As the guiding layer (such as SiO2 or ZnO) was added to the top surface, the Love wave was generated and propagated at the guiding layer. A fundamental simulation demonstrated the signal response in the form of scattering parameters (S-parameters), reﬂection parameters, transmission parameters and phase. 1. Introduction The optimized measurement method from the simulation can help the researcher determine the optimal measurement conﬁguration for use in the physical device. The sensitivity of the same device at diﬀerent frequencies will be diﬀerently evidenced by its S-parameters. The novel approach proposed herein demonstrates a new path for optimizing SAW sensor models with more accurate parameter selection, which can be used to predict the sensitivity and response of diﬀerent guiding 3 of 18 Sensors 2019, 19, 1749 and mass loading layers for multilayer device optimization. The thorough comparison between phase shift sensitivity, insertion loss changes, and frequency shift sensitivity, shows that phase shift is a more sensitive parameter than the rest. and mass loading layers for multilayer device optimization. The thorough comparison between phase shift sensitivity, insertion loss changes, and frequency shift sensitivity, shows that phase shift is a more sensitive parameter than the rest. 2.2. Model Structures The relationship between piezoelectric stress, strain, electric ﬁeld, and electric displacement ﬁeld was deduced by stress–strain relationship equations in non-piezoelectric materials after the voltage was applied. The electric ﬁeld E will cause a change of piezoelectric materials’ molecular charge distributions, which will result in a surface charge buildup. In the current study, the following equations were used in COMSOL to model surface charge density and mechanical stress relationship in stress-charge form as shown below [34]: D = [e][S] + [ε]E (1) [T] = [c][S] − h eti E (2) D = [e][S] + [ε]E (1) (1) [T] = [c][S] − h eti E (2) (2) In the Equations (1) and (2), [e] represents the piezoelectric constant matrix, E is the applied electric ﬁeld, [c] is the elastic constants, [ε] is the dielectric permittivity, and [S] is the strain matrix. The h eti matrix is the 3 × 6 transposed matrix of the piezoelectric constant matrix [34]. h i The 3D modeling structure in Figure 1c was selected in this study. The simulation model in Figure 1c has 20 pairs of interdigital transducers (IDTs) and 30 pairs of reﬂecting ﬁngers for both receiving and transmitting ports. The realistic device and simulation model have the same wavelength (λ) of 298 µm and same delay line length of 38.25λ. The reﬂecting ﬁngers were located 10.25λ away from the IDTs. Substrate thickness was 500 µm. IDT parameters used in the design are listed in Table 1. The ﬁrst layer of the device was the chrome IDT ﬁngers with a thickness of 100 nm, the second layer was the waveguide layer made of 500 nm-thick ZnO, and the top layer was the IrO2 layer as seen in the fabricated device in Figure 1c. The layer’s properties and fabrication process are shown in our previous report [35]. 4 of 18 Sensors 2019, 19, 1749 Table 1. Device IDT parameters used in both device fabrication and modeling. Table 1. Device IDT parameters used in both device fabrication and modeling. PARAMETERS SETTINGS Wavelength (λ) 298 µm Number of ﬁngers 20 pairs Finger width 74.5 µm Wavelength of reﬂecting ﬁngers 298 µm Number of reﬂecting ﬁngers 30 pairs SAW velocity 4160 m/s Sensors 2019, 19, x FOR PEER REVIEW 4 of (a) (b) (c) (d) (e) Figure 1. (a) Schematic diagram showing the design and structure of the real SAW device. (b) Fabricated device with only the finger layer. 2.2. Model Structures (c) The 3D model of the device used in COMSOL. (d) The completed mesh of the model. (e) Conceptual view of the double guide layer of the SAW device. Table 1. Device IDT parameters used in both device fabrication and modeling. Figure 1. (a) Schematic diagram showing the design and structure of the real SAW device. (b) Fabricated device with only the ﬁnger layer. (c) The 3D model of the device used in COMSOL. (d) The completed mesh of the model. (e) Conceptual view of the double guide layer of the SAW device. 2.3. Boundary Conditions and Meshing (a) (b) (a) (b) (c) (c) (d) (e) Figure 1. (a) Schematic diagram showing the design and structure of the real SAW device. (b) b d d h l h f l ( ) h d l f h d d CO SO (d) h Figure 1. (a) Schematic diagram showing the design and structure of the real SAW device. (b) Fabricated d h l h ﬁ l ( ) h d l f h d d CO SO (d) h l d (d) (c) (d) (e) (d) (c) (e) Figure 1. (a) Schematic diagram showing the design and structure of the real SAW device. (b) Fabricated device with only the finger layer. (c) The 3D model of the device used in COMSOL. (d) The completed mesh of the model. (e) Conceptual view of the double guide layer of the SAW device. Figure 1. (a) Schematic diagram showing the design and structure of the real SAW device. (b) Fabricated device with only the ﬁnger layer. (c) The 3D model of the device used in COMSOL. (d) The completed mesh of the model. (e) Conceptual view of the double guide layer of the SAW device. Table 1. Device IDT param 2.3. Boundary Conditions and Meshing PARAMETERS SETTINGS Wavelength (λ) 298 μm Number of fingers 20 pairs Finger width 74.5 μm Wavelength of reflecting fingers 298 μm A time-domain analysis was conducted to calculate the dynamic characteristics of the device in response to a short impulse signal. The impulse voltage was applied to the input electrodes, where V+ and V−were applied to the even and odd ﬁngers in Equations (3) and (4). For this simulation, a step size of 2 ns from Equation (5) and a total simulation period of 8800 ns were used. The material properties used in the simulation are shown in Table 2. ing fingers 30 pairs 4160 m/s V+ = ( +0.5 V, 0 ≤t ≤2 ns 0 V, t > 2 ns (3) (3) Sensors 2019, 19, 1749 5 of 18 V−= ( −0.5 V, 0 ≤t ≤2 ns 0 V, t > 2 ns (4) Tv < 1 20·fmax (5) (4) Tv < 1 20·fmax (5) (5) The boundary condition of the output electrodes was set to an initial voltage of zero to an outside terminal, which is also connected to the voltmeter and load. The bottom surface and side surface of the substrate were both set to be low reﬂecting surfaces as the absorber. A symmetry boundary was used in the model as illustrated in Figure 1c. Initial body displacement ﬁelds in X, Y, and Z directions (Ux, Uy, and Uz) were set to zero. The measured voltages generated on the output electrodes were used to calculate the frequency response of the device. The output electrode ﬁnger was connected to an electrical circuit via the terminal boundary. A quadrilateral mesh was applied on the ﬁngers, and the triangular mesh was used on the rest of the top surface with swept mesh to the whole device. The device was meshed with diﬀerent node densities to verify the frequency independence from the mesh size. Table 2. Material properties used in COMSOL and Matlab©. Table 1. Device IDT param 2.3. Boundary Conditions and Meshing Material Properties Units Lithium Tantalate ZnO Cr IrO2 Protein Fiber Layer Density (kg/m3) 4700 5680 7150 11660 1350 Young’s Modulus (GPa) 279 322.8 0.07 [36] Poisson’s ratio 0.21 0.33 0.44 Elastic stiﬀness cE 11 ×1010 (N/m2) 23.29 15.7 Elastic stiﬀness cE 12 ×1010 (N/m2) 4.69 8.9 Elastic stiﬀness cE 13 ×1010 (N/m2) 8.02 8.3 Elastic stiﬀness cE 13 ×1010 (N/m2) −1.1 0 Elastic stiﬀness cE 33 ×1010 (N/m2) 27.53 20.8 Elastic stiﬀness cE 13 ×1010 (N/m2) 8.02 4.3 Elastic stiﬀness cE 13 ×1010 (N/m2) 9.30 4.42 Piezoelectric coeﬃcient e15 (C/m2) 2.596 −0.48 Piezoelectric coeﬃcient e22 (C/m2) 1.59 0 Piezoelectric coeﬃcient e31 (C/m2) 0.082 −0.57 Piezoelectric coeﬃcient e33 (C/m2) 1.882 1.32 Table 2. Material properties used in COMSOL and Matlab©. Table 2. Material properties used in COMSOL and Matlab©. The damping eﬀect caused by mass loading is a challenge for accurate model generation because the damping ratio is not only mass dependent, but also a frequency dependent parameter that cannot be readily implemented across an extended range of the frequencies. As the mass of the IrO2 layer increased, the insertion loss (S21) and the diﬀerence between simulation and physical measurements also increased due to a mechanical loss factor being applied to the model in the form of the loss factor (also known as damping ratio) on the IrO2 and lithium tantalate layers in Figure 2. This loss factor was calculated from the experimental results using the equation η = 1/2Q where Q is the quality factor of the device. The performance assessment of most acoustic wave sensors was essentially determined by quality-factor and device sensitivity. The quality factor (Q) is a measure of how capable the acoustic wave device is of retaining its energy during oscillation. Q factor is deﬁned as the ratio of the energy stored in the resonator to the energy dissipated per cycle. The energy losses will be the major parameter aﬀecting the Q factor, which could include anything from a mechanical damping loss to thermal elastic loss, while also depending on the loss in the electrical domain, such as coupling loss, dielectric loss, and conductive loss which is negligibly small in this case. Sensors 2019, 19, 1749 6 of 18 Sensors 2019, 19, x FOR PEER REVIEW 6 of 18 (a) (b) Figure 2. (a) The applied mechanical damping ratio in the substrate layer (lithium tantalate) at different frequencies. Table 1. Device IDT param 2.3. Boundary Conditions and Meshing (b) The applied mechanical damping ratio in the IrO2 layer at different frequencies. Figure 2. (a) The applied mechanical damping ratio in the substrate layer (lithium tantalate) at diﬀerent frequencies. (b) The applied mechanical damping ratio in the IrO2 layer at diﬀerent frequencies. 6 of 18 6 of 18 Sensors 2019, 19, 1749 Sensors 2019, 19, x FOR 6 of 18 6 of 18 (b) (a) (b) (a) Figure 2. (a) The applied mechanical damping ratio in the substrate layer (lithium tantalate) at different frequencies. (b) The applied mechanical damping ratio in the IrO2 layer at different frequencies. Figure 2. (a) The applied mechanical damping ratio in the substrate layer (lithium tantalate) at diﬀerent frequencies. (b) The applied mechanical damping ratio in the IrO2 layer at diﬀerent frequencies. 2 5 Design and Fabrication 2.5. Design and Fabrication 2.5. Design and Fabrication Devices with different guide layers were fabricated and tested to compare their frequency responses to the simulation results. All devices were fabricated using conventional MEMS fabrication processes. The ZnO layer was sputtered by a radio-frequency (RF) sputtering system (AJA) with a 99.9% ZnO target. The oxygen and argon flow ratio were kept at 1:1 and the wafer temperature was set to 180 °C. IrO2 was also sputtered by an RF sputtering system (CRC) with a 99.99% IrO2 target at room temperature in a pure argon environment. The frequency responses of the devices were measured using a vector network analyzer (Anritsu 37369A) of 50 Ω characteristic impedance Devices with diﬀerent guide layers were fabricated and tested to compare their frequency responses to the simulation results. All devices were fabricated using conventional MEMS fabrication processes. The ZnO layer was sputtered by a radio-frequency (RF) sputtering system (AJA) with a 99.9% ZnO target. The oxygen and argon ﬂow ratio were kept at 1:1 and the wafer temperature was set to 180 ◦C. IrO2 was also sputtered by an RF sputtering system (CRC) with a 99.99% IrO2 target at room temperature in a pure argon environment. The frequency responses of the devices were measured using a vector network analyzer (Anritsu 37369A) of 50 Ωcharacteristic impedance. 2.4. Frequency Response Calculation 2.4. Frequency Response Calculation In this study, a discrete fast Fourier transform (FFT) function was performed after the waveform of the output voltage and charge were imported into Matlab©. At the same time, the logarithmic frequency response was converted to the admittance (z) of the device in the frequency domain [27]. After admittance was calculated, a full analysis of the model was performed including the wave phase, linear magnitude, standing wave ratio (SWR), the imaginary part of the response, and real part of the response, which were determined by the equations in Table 3. In this study, a discrete fast Fourier transform (FFT) function was performed after the waveform of the output voltage and charge were imported into Matlab©. At the same time, the logarithmic frequency response was converted to the admittance (z) of the device in the frequency domain [27]. After admittance was calculated, a full analysis of the model was performed including the wave phase, linear magnitude, standing wave ratio (SWR), the imaginary part of the response, and real part of the response, which were determined by the equations in Table 3. Table 3. The formula used for extracting values in the different Cartesian diagrams from the complex measurement. Table 3. The formula used for extracting values in the diﬀerent Cartesian diagrams from the complex measurement. Table 3. The formula used for extracting values in the different Cartesian diagrams from the complex measurement. Table 3. The formula used for extracting values in the diﬀerent Cartesian diagrams from the complex measurement. complex measurement. Trace Format Description Formula Lin Mag Magnitude of z, unconverted \|z\| = sqrt (x2 + y2) Insertion Loss Converted from z to S parameter IL = −20 × log\|S21\| dB Phase Phase of z φ (z) = arctan (y/x) Real Real part of z Re(z) = x Imag Imaginary part of z Im(z) = y SWR (Voltage) Standing Wave Ratio SWR = (1 + \|z\|)/(1 − \|z\|) 2 5 D i d F b i ti p Trace Format Description Formula Lin Mag Magnitude of z, unconverted \|z\| = sqrt (x2 + y2) Insertion Loss Converted from z to S parameter IL = −20 × log\|S21\| dB Phase Phase of z ϕ (z) = arctan (y/x) Real Real part of z Re(z) = x Imag Imaginary part of z Im(z) = y SWR (Voltage) Standing Wave Ratio SWR = (1 + \|z\|)/(1 −\|z\|) 2.5. 2.4. Frequency Response Calculation 2.4. Frequency Response Calculation Design and Fabrication 2 5 Design and Fabrication 2.5. Design and Fabrication 3.1. FEM Analysis of a Multi-Layer SAW Device The propagated shear horizontal waves traveled through the path line as shown in Figure 3. As shown by the modeled elastic displacement in Figure 3, the waves traveled through the active device region and were reﬂected by the edges. A mesh independent study in Figure 4 shows that the 7 of 18 device hat the Sensors 2019, 19, 1749 shown by the mod region and were frequency response was independent of mesh size when more than six mesh nodes were created per wavelength. The device was meshed with a node density of seven nodes per wavelength, and more than 10 × 106 degrees of freedom in total were maintained sweeping throughout all the diﬀerent studies in this manuscript (Average mesh size quality of 0.968). The frequency spectrum was converted from the wave received by the output electrodes and then plotted out to compare with the measurements as shown in Figures 5–7. A guiding layer was applied to the acoustic wave devices to improve the sensitivity, temperature stability, and electromechanical coupling coeﬃcient [37]. As the wave propagates, its phase velocity depends on the properties of the diﬀerent guide layers, the eﬀect of the multi-guide layer material is investigated in Figure 5. A piezoelectric layer with a relatively high permittivity added on top of the piezoelectric layer (Lithium Titanate) increases the electromechanical coupling, thus allowing fabrication of devices with reduced insertion loss [37]. After the ZnO waveguide layer was deposited on top of the surface of the Lithium Tantalite substrate, a layer of IrO2 was deposited on top of the ZnO layer to further increase the sensitivity [35]. The dielectric properties of diﬀerent guide layers will also aﬀect the wave properties in a diﬀerent fashion. The comparison between the simulation results and experimental measurements illustrated in Figures 5–7 shows that the predictions of the 3D model are reasonably comparable to the experimental frequency spectrum of the devices. q y p p p wavelength. The device was meshed with a node density of seven nodes per wavelength, and more than 10 × 106 degrees of freedom in total were maintained sweeping throughout all the different studies in this manuscript (Average mesh size quality of 0.968). The frequency spectrum was converted from the wave received by the output electrodes and then plotted out to compare with the measurements as shown in Figures 5–7. 3.1. FEM Analysis of a Multi-Layer SAW Device A guiding layer was applied to the acoustic wave devices to improve the sensitivity, temperature stability, and electromechanical coupling coefficient [37]. As the wave propagates, its phase velocity depends on the properties of the different guide layers, the effect of the multi-guide layer material is investigated in Figure 5. A piezoelectric layer with a relatively high permittivity added on top of the piezoelectric layer (Lithium Titanate) increases the electromechanical coupling, thus allowing fabrication of devices with reduced insertion loss [37]. After the ZnO waveguide layer was deposited on top of the surface of the Lithium Tantalite substrate, a layer of IrO2 was deposited on top of the ZnO layer to further increase the sensitivity [35]. The dielectric properties of different guide layers will also affect the wave properties in a different fashion. The comparison between the simulation results and experimental measurements illustrated in Figures 5–7 shows that the predictions of the 3D model are reasonably comparable to the experimental frequency spectrum of the devices. (a) (b) Figure 3. (a) Time-lapse elastic displacement distribution in the SAW device after an initial pulse voltage was applied to the input IDTs at T = 0 ns. (b) Time-lapse elastic displacement distribution at T = 300 ns. Figure 3. (a) Time-lapse elastic displacement distribution in the SAW device after an initial pulse voltage was applied to the input IDTs at T = 0 ns. (b) Time-lapse elastic displacement distribution at T = 300 ns. ensors 2019, 19, x FOR PEER REVIEW 8 of 1 (a) (b) (b) (a) Figure 3. (a) Time-lapse elastic displacement distribution in the SAW device after an initial pulse voltage was applied to the input IDTs at T = 0 ns. (b) Time-lapse elastic displacement distribution at T = 300 ns. Figure 3. (a) Time-lapse elastic displacement distribution in the SAW device after an initial pulse voltage was applied to the input IDTs at T = 0 ns. (b) Time-lapse elastic displacement distribution at T = 300 ns. rs 2019, 19, x FOR PEER REVIEW 8 o Figure 4. Effect of node density (mesh node in a wavelength). Figure 4. Eﬀect of node density (mesh node in a wavelength). Figure 4. Effect of node density (mesh node in a wavelength). Figure 4. Eﬀect of node density (mesh node in a wavelength). Figure 4. Effect of node density (mesh node in a wavelength). Figure 4. 3.1. FEM Analysis of a Multi-Layer SAW Device Eﬀect of node density (mesh node in a wavelength). 8 of 18 p sults of Sensors 2019, 19, 1749 14.05 MHz with a l insertion loss of 26.9 dB at 13.98 MHz. Figure 5. Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs only) from 12 MHz to 18 MHz. Figure 5. Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs only) from 12 MHz to 18 MHz. sors 2019, 19, x FOR PEER REVIEW 9 of 18 (a) (b) Figure 6. (a) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer, and an IrO2 layer of 30 nm) from 13 MHz to 16 MHz. The experimental results are obtained from the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 30 nm- thick IrO2 layers. (b) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer and a 50 nm-thick IrO2 layer) from 13 MHz to 16 MHz. Figure 6. (a) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer, and an IrO2 layer of 30 nm) from 13 MHz to 16 MHz. The experimental results are obtained from the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 30 nm-thick IrO2 layers. (b) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer and a 50 nm-thick IrO2 layer) from 13 MHz to 16 MHz. n insertion loss of 26.9 dB at 13.98 MHz. Figure 5. Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs only) from 12 MHz to 18 MHz. Figure 5. Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs only) from 12 MHz to 18 MHz. nsors 2019, 19, x FOR PEER REVIEW 9 of Figure 5. Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs only) from 12 MHz to 18 MHz. Figure 5. Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs only) from 12 MHz to 18 MHz. 3.1. FEM Analysis of a Multi-Layer SAW Device ors 2019, 19, x FOR PEER REVIEW 9 Cr IDTs only) from 12 MHz to 18 MHz. 100 nm Cr IDTs only) from 12 MHz to 18 MHz. (a) (b) Figure 6. (a) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer, and an IrO2 layer of 30 nm) from 13 MHz to 16 MHz. The experimental results are obtained from the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 30 nm- thick IrO2 layers. (b) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer and a 50 nm-thick IrO2 layer) from 13 MHz to 16 MHz. Figure 6. (a) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer, and an IrO2 layer of 30 nm) from 13 MHz to 16 MHz. The experimental results are obtained from the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 30 nm-thick IrO2 layers. (b) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer and a 50 nm-thick IrO2 layer) from 13 MHz to 16 MHz. (b) (a) (b) (a) Figure 6. (a) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer, and an IrO2 layer of 30 nm) from 13 MHz to 16 MHz. The experimental results are obtained from the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 30 nm- thick IrO2 layers. (b) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer and a 50 nm-thick IrO2 layer) from 13 MHz to 16 MHz. Figure 6. (a) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer, and an IrO2 layer of 30 nm) from 13 MHz to 16 MHz. The experimental results are obtained from the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 30 nm-thick IrO2 layers. (b) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer and a 50 nm-thick IrO2 layer) from 13 MHz to 16 MHz. 3.1. FEM Analysis of a Multi-Layer SAW Device The reﬂection behavior diﬀerence contributes to this variation between simulation and experiment on 9 of 18 ith h Sensors 2019, 19, 1749 Figure 6. (a) C the third peak, which may also be interfered by other waves on the physical device that are not present in the simulation study. The measured resonance peak at 14.05 MHz with a 28.7 dB of insertion loss showed a larger diversion from the simulation results of an insertion loss of 26.9 dB at 13.98 MHz. experimental results are obtained from the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 30 nm- thick IrO2 layers. (b) Comparison between simulated and measured frequency responses for the device (with 100 nm Cr IDTs, a 500 nm-thick ZnO layer and a 50 nm-thick IrO2 layer) from 13 MHz to 16 MHz. . Figure 7. Comparison between simulated and measured frequency responses for the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 70 nm/100 nm IrO2 layer from 13 MHz to 20 MHz. Figure 7. Comparison between simulated and measured frequency responses for the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 70 nm/100 nm IrO2 layer from 13 MHz to 20 MHz. Figure 7. Comparison between simulated and measured frequency responses for the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 70 nm/100 nm IrO2 layer from 13 MHz to 20 MHz. Figure 7. Comparison between simulated and measured frequency responses for the device with Cr IDTs, a 500 nm-thick ZnO layer, and a 70 nm/100 nm IrO2 layer from 13 MHz to 20 MHz. As shown in Figure 6a, the simulated frequency characteristic did show a good match with the experimental data in the previous research [35]. In Figures 6 and 7, we show that as the IrO2 layer increased, the insertion loss decreased. The simulation results measuring insertion loss showed a very good match with the measurement for the device with 100 nm Cr IDTs, a 500 nm-thick ZnO, and a 30 nm-thick IrO2 layer. Figure 7 shows that as the layer of the IrO2 increased, the insertion loss increased since the waves began to attenuate or cease propagation. In the experimental measurements, as the layer of the IrO2 increased to 100 nm on top of the 500 nm ZnO layer, the wave propagation vanished. 3.1. FEM Analysis of a Multi-Layer SAW Device At 100 nm thickness all frequency response was eliminated because of IrO2’s electrical properties. The thin layer of the IrO2 had a large resistance, which can be considered as a non-conductive layer. As the IrO2 thickness increased, the conductive IrO2 shorted the device, which can cease the acoustic wave. In this simulation, IrO2 was considered as a nonconductive elastic material to simplify the problem and characterize mass sensitivity. The abrupt transition of IrO2 from high resistance to conductive, which was observed in the experimental results, was not incorporated As shown in Figure 6a, the simulated frequency characteristic did show a good match with the experimental data in the previous research [35]. In Figures 6 and 7, we show that as the IrO2 layer increased, the insertion loss decreased. The simulation results measuring insertion loss showed a very good match with the measurement for the device with 100 nm Cr IDTs, a 500 nm-thick ZnO, and a 30 nm-thick IrO2 layer. Figure 7 shows that as the layer of the IrO2 increased, the insertion loss increased since the waves began to attenuate or cease propagation. In the experimental measurements, as the layer of the IrO2 increased to 100 nm on top of the 500 nm ZnO layer, the wave propagation vanished. At 100 nm thickness all frequency response was eliminated because of IrO2’s electrical properties. The thin layer of the IrO2 had a large resistance, which can be considered as a non-conductive layer. As the IrO2 thickness increased, the conductive IrO2 shorted the device, which can cease the acoustic wave. In this simulation, IrO2 was considered as a nonconductive elastic material to simplify the problem and characterize mass sensitivity. The abrupt transition of IrO2 from high resistance to conductive, which was observed in the experimental results, was not incorporated into the simulation. 3.1. FEM Analysis of a Multi-Layer SAW Device Figure 6. (a) Comparison between simulated and measured frequency responses for the device (with Figure 6. (a) Comparison between simulated and measured frequency responses for the device (with In Figure 5, the simulation results show a very similar resonance frequency (14.03 MHz) compared to the measured results (14.05 MHz). The trend of the simulation results at the ﬁrst two modes with peak frequency at around 14 MHz and 15.3 MHz, were very close to the experimental results, but the third peak frequency did not match the measurement as well as the ﬁrst two modes. The insertion loss of the simulation results was smaller than the experimental measurement. The major reason for the third peak mismatch in Figure 5 is the diﬀerent reﬂecting behavior of the waves reﬂected by the edge of the model compared to the edge of the real device. The real devices had a long distance between the side edges and the IDTs (~40λ). To reduce the required computation time and resources, this distance was changed in the simulated device to 4λ. After reducing the distance between the IDTs to the edge, and applying the absorption boundary, the reﬂection behavior and wave interference were aﬀected. The reﬂection behavior diﬀerence contributes to this variation between simulation and experiment on In Figure 5, the simulation results show a very similar resonance frequency (14.03 MHz) compared to the measured results (14.05 MHz). The trend of the simulation results at the ﬁrst two modes with peak frequency at around 14 MHz and 15.3 MHz, were very close to the experimental results, but the third peak frequency did not match the measurement as well as the ﬁrst two modes. The insertion loss of the simulation results was smaller than the experimental measurement. The major reason for the third peak mismatch in Figure 5 is the diﬀerent reﬂecting behavior of the waves reﬂected by the edge of the model compared to the edge of the real device. The real devices had a long distance between the side edges and the IDTs (~40λ). To reduce the required computation time and resources, this distance was changed in the simulated device to 4λ. After reducing the distance between the IDTs to the edge, and applying the absorption boundary, the reﬂection behavior and wave interference were aﬀected. into the simulation. 3.2. Conversion of Complex into Real Quantities Figure 9 presents the linear magnitude of forwarding transmission vs. frequency (S21). After the data were transferred to the Laplace and Fourier domains, the magnitude and phase responses were obtained—which are commonly referred to as the frequency response. The first two wave modes matched the experimental measurements very well, where the linear magnitude of 0.0248 at the first simulated peak was fairly close 0.0232 from the corresponding measurements. However, the Figure 9 presents the linear magnitude of forwarding transmission vs. frequency (S21). After the data were transferred to the Laplace and Fourier domains, the magnitude and phase responses were obtained—which are commonly referred to as the frequency response. The ﬁrst two wave modes matched the experimental measurements very well, where the linear magnitude of 0.0248 at the ﬁrst simulated peak was fairly close 0.0232 from the corresponding measurements. However, the simulation illustrated additional spurious modes that were not present in the experimental measurements. Sensors 2019, 19, x FOR PEER REVIEW 11 of 18 mulation illustrated additional spurious modes that were not present in the experimental measureme The insertion phase angle versus frequency is another very important characteristic of the dev hich is also critically important for analysis and detection. Most prior work only reported resu sed on phase angle shift to different mass loading [39,40]. After the complex number of opagated wave is calculated from the voltage and current of the output electrodes, the phase an z can be calculated from the equation listed in Table 3. The simulated phase angle is compar ith the experimental measurements to verify the model in Figure 10. The imaginary part of z was converted to the imaginary magnitude of the response a mpared with experimental results and plotted in Figure 11. The simulation results showed v milar characteristics for the first two peaks. The real part of z was converted to the real magnitu admittance response and compared to the experimental results as shown in Figure 12. (a) (b) Figure 9. Comparison between simulated (Blue) and measured (Red) linear magnitude frequency response for the device (with Cr IDTs only) from 13 MHz to 16 MHz. (a) Simulated linear magnitude frequency response of the design. (b) Measured linear magnitude frequency response from the fabricated device. Figure 9. Comparison between simulated (Blue) and measured (Red) linear magnitude frequency response for the device (with Cr IDTs only) from 13 MHz to 16 MHz. into the simulation. 3.2. Conversion of Complex into Real Quantities After the simulation data were generated, a custom written Matlab© program read the ﬁle and converted the data to be used in diﬀerent Cartesian diagrams. The standing-wave ratio (SWR) represents a mathematical expression of the non-uniformity of an electrical ﬁeld throughout a transmission line at radio frequencies. SWR is deﬁned as the ratio of maximum radio-frequency (RF) voltage to minimum RF voltage along the line [38], which is also known as the voltage standing-wave ratio (VSWR). The voltage on a signal transmission line is the same at all points on the line when power losses caused by line resistance and imperfections in the dielectric material separating the line conductors are assumed to be negligible. SWR of the surface acoustic device is mathematically related to the input power and reﬂected power when the device is tested via a network analyzer. In an ideal scenario, SWR is 1:1 when there is no power loss or reﬂected power. Figure 8 shows a reasonable comparison between simulated SWR and experimentally measured results over a range of frequencies. 10 of 18 mentally 10 of 18 mentally Sensors 2019, 19, 1749 power. Figure 8 measured results o easured results over a range of frequencies. (a) (b) Figure 8. Comparison between simulated (Blue) and measured (Red) standing wave ratios of frequency response for the device with Cr IDTs only, without coatings, from 13 MHz to 16 MHz. (a) Simulated standing wave ratios of the design. (b) Measured standing wave ratios from the fabricated d i Figure 8. Comparison between simulated (Blue) and measured (Red) standing wave ratios of frequency response for the device with Cr IDTs only, without coatings, from 13 MHz to 16 MHz. (a) Simulated standing wave ratios of the design. (b) Measured standing wave ratios from the fabricated device. (a) (b) (b) (a) Figure 8. Comparison between simulated (Blue) and measured (Red) standing wave ratios of frequency response for the device with Cr IDTs only, without coatings, from 13 MHz to 16 MHz. (a) Simulated standing wave ratios of the design. (b) Measured standing wave ratios from the fabricated Figure 8. Comparison between simulated (Blue) and measured (Red) standing wave ratios of frequency response for the device with Cr IDTs only, without coatings, from 13 MHz to 16 MHz. (a) Simulated standing wave ratios of the design. (b) Measured standing wave ratios from the fabricated device. device. into the simulation. 3.2. Conversion of Complex into Real Quantities (a) Simulated linear magnitude frequency response of the design. (b) Measured linear magnitude frequency response from the fabricated device. ulation illustrated additional spurious modes tha The insertion phase angle versus frequency is ch is also critically important for analysis and ed on phase angle shift to different mass lo pagated wave is calculated from the voltage an can be calculated from the equation listed in h the experimental measurements to verify the The imaginary part of z was converted to mpared with experimental results and plotted ilar characteristics for the first two peaks. The dmittance response and compared to the expe (a) t were not present in the experimental measurem another very important characteristic of the de detection. Most prior work only reported res ading [39,40]. After the complex number of d current of the output electrodes, the phase a Table 3. The simulated phase angle is compa model in Figure 10. the imaginary magnitude of the response in Figure 11. The simulation results showed v real part of z was converted to the real magnit rimental results as shown in Figure 12. (b) (b) (a) Figure 9. Comparison between simulated (Blue) and measured (Red) linear magnitude frequency response for the device (with Cr IDTs only) from 13 MHz to 16 MHz. (a) Simulated linear magnitude frequency response of the design. (b) Measured linear magnitude frequency response from the fabricated device. Figure 9. Comparison between simulated (Blue) and measured (Red) linear magnitude frequency response for the device (with Cr IDTs only) from 13 MHz to 16 MHz. (a) Simulated linear magnitude frequency response of the design. (b) Measured linear magnitude frequency response from the fabricated device. The insertion phase angle versus frequency is another very important characteristic of the device, which is also critically important for analysis and detection. Most prior work only reported results based on phase angle shift to diﬀerent mass loading [39,40]. After the complex number of the propagated wave is calculated from the voltage and current of the output electrodes, the phase angle of z can be calculated from the equation listed in Table 3. The simulated phase angle is compared with the experimental measurements to verify the model in Figure 10. The imaginary part of z was converted to the imaginary magnitude of the response and compared with experimental results and plotted in Figure 11. into the simulation. 3.2. Conversion of Complex into Real Quantities The simulation results showed very similar 11 of 18 Sensors 2019, 19, 1749 characteristics for the ﬁrst two peaks. The real part of z was converted to the real magnitude of admittance response and compared to the experimental results as shown in Figure 12. g p ( ) ( ) g q y response for the device (with Cr IDTs only) from 13 MHz to 16 MHz. (a) Simulated linear magnitude frequency response of the design. (b) Measured linear magnitude frequency response from the fabricated device. response for the device (with Cr IDTs only) from 13 MHz to 16 MHz. (a) Simulated linear magnitude frequency response of the design. (b) Measured linear magnitude frequency response from the fabricated device. Figure 10. Comparison between simulated (Blue) and measured (Red) phase angle responses from the device (with Cr IDTs only). Figure 10. Comparison between simulated (Blue) and measured (Red) phase angle responses from the device (with Cr IDTs only). Figure 10. Comparison between simulated (Blue) and measured (Red) phase angle responses from the device (with Cr IDTs only). Figure 10. Comparison between simulated (Blue) and measured (Red) phase angle responses from the device (with Cr IDTs only). Figure 10. Comparison between simulated (Blue) and measured (Red) phase angle responses from the device (with Cr IDTs only). Figure 10. Comparison between simulated (Blue) and measured (Red) phase angle responses from the device (with Cr IDTs only). (a) (a) (a) (b) Figure 11. Comparison between simulated (Blue) and measured (Red) imaginary magnitudes for the frequency response of the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated imaginary magnitude of the response from the design. (b) Measured imaginary magnitude of the response from the fabricated device. (a) (b) Figure 11. Comparison between simulated (Blue) and measured (Red) imaginary magnitudes for the frequency response of the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated imaginary magnitude of the response from the design. (b) Measured imaginary magnitude of the response from the fabricated device. Figure 11. Comparison between simulated (Blue) and measured (Red) imaginary magnitudes for the frequency response of the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated imaginary magnitude of the response from the design. (b) Measured imaginary magnitude of the response from the fabricated device. 3.3.1. Frequency Shift Detection The fundamental biosensing technique using surface acoustic waves measures changes in propagation velocity, resonant frequency, phase angle, or to a lesser degree amplitude of reﬂection or transmission signals. Variations in these parameters of the acoustic wave can be attributed to intrinsic factors such as material properties: density, elasticity, phase transformation, viscosity, conductivity, permittivity, as well as changes in carrier concentration and mobility [35]. In our previous research [8,30], we show that the sensor’s structure is a delay line two-port resonator device, conﬁgured as a gained controlled RF oscillator system. In this oscillation setup system, the frequency is determined by the transfer function of the transducers and ampliﬁers through a closed-loop feedback conﬁguration. After the two oscillation condition requirements are satisﬁed, which include loop gain over 0 dB and loop phase equal to 0 degrees, any change (∆v) in the phase velocity give rise to a frequency shift ∆f in the output oscillation frequency f, given by [41]: ∆f f0 = ∆v v0 = 1 v( ∂v ∂m∆m + ∂v ∂σ∆σ + ∂v ∂c ∆c + ∂v ∂ε∆ε + ∂v ∂T ∆T + ∂v ∂P∆P + ∂v ∂ρ∆ρ + · · ·) (6) (6) The above Equation (6) assumes that any other external perturbations listed below are negligibly small; where ∆m is the change in mass load, ∆σ the change in conductivity, ∆c the change in mechanical constant, ∆ε the change in dielectric constant, ∆T the change in temperature, ∆P the change in pressure, and ∆ρ the change in density. The above Equation (6) assumes that any other external perturbations listed below are negligibly small; where ∆m is the change in mass load, ∆σ the change in conductivity, ∆c the change in mechanical constant, ∆ε the change in dielectric constant, ∆T the change in temperature, ∆P the change in pressure, and ∆ρ the change in density. into the simulation. 3.2. Conversion of Complex into Real Quantities Sensors 2019, 19, x FOR PEER REVIEW 12 of 18 (b) (b) (a) (a) (b) (b) Figure 11. Comparison between simulated (Blue) and measured (Red) imaginary magnitudes for the frequency response of the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated imaginary magnitude of the response from the design. (b) Measured imaginary magnitude of the response from the fabricated device. Figure 11. Comparison between simulated (Blue) and measured (Red) imaginary magnitudes for the frequency response of the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated imaginary magnitude of the response from the design. (b) Measured imaginary magnitude of the response from the fabricated device. Figure 11. Comparison between simulated (Blue) and measured (Red) imaginary magnitudes for the frequency response of the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated imaginary magnitude of the response from the design. (b) Measured imaginary magnitude of the response from the fabricated device. Sensors 2019, 19, x FOR PEER REVIEW 12 of 18 (a) (a) (b) Figure 12. Comparison between simulated (Blue) and measured (Red) real magnitude responses for the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated real magnitude of the response from the design. (b) Measured real magnitude of the response from the fabricated device. 3 3 Effect of Layer Sensitivity Figure 12. Comparison between simulated (Blue) and measured (Red) real magnitude responses for the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated real magnitude of the response from the design. (b) Measured real magnitude of the response from the fabricated device. (b) (b) (a) Figure 12. Comparison between simulated (Blue) and measured (Red) real magnitude responses for the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated real magnitude of the response from the design. (b) Measured real magnitude of the response from the fabricated device. Figure 12. Comparison between simulated (Blue) and measured (Red) real magnitude responses for the device (with Cr IDTs only) from 12 MHz to 16 MHz. (a) Simulated real magnitude of the response from the design. (b) Measured real magnitude of the response from the fabricated device. 12 of 18 Sensors 2019, 19, 1749 3.3. Eﬀect of Layer Sensitivity For SAW-based biosensors, the sensitivity is a more important parameter for evaluating the overall performance of an acoustic sensor. To determine the mass sensitivity of the design, the frequency shift, phase shift, and insertion loss changes per mass loading are the primary parameters to evaluate. It is a sophisticated system for predicting the design and obtain a similar real sensitivity on test cases with a range of diﬀerent mass loading. In our case, the actual wave propagation problem on the piezoelectric substrate involved multiple anisotropic layers, a double piezoelectricity layer, and three-dimensional wave diﬀraction. It is also not possible for other methods to analyze all the properties of the wave mode such as phase, electrical perturbation, mechanical mass loading, and wave transmission. The ﬁnite element method provides a more suitable and aﬀordable method for characterizing the design compared to the much more costly and labor-intensive experimental approaches. 3.3.1. Frequency Shift Detection The sensitivity of an acoustic wave sensor, Sr to any external perturbation y is deﬁned as: Sr = lim ∆y→0 ∆f f∆y = df fdy (7) (7) For the sensitivity speciﬁed based on other parameters such as phase, SWR, insertion loss and linear magnitude can be deﬁned as: For the sensitivity speciﬁed based on other parameters such as phase, SWR, insertion loss and linear magnitude can be deﬁned as: df fdy = dp pdy = dS Sdy = dI Idy = dL Ldy (8) (8) where p is the phase of the device, S is the value of the SWR, I is the insertion loss, and L is the linear magnitude of the response. 3.3.3. Sensitivity Comparison Acoustic propagation mass sensing within multi-thin-layer systems is essential for optimizing gravimetric sensors. The mass sensitivity will vary by diﬀerent thickness of the ZnO and IrO2 layer. A design created in COMSOL supports a parameter sweep to determine the relationship between thickness and sensitivity. All mass loadings were set up as a protein layer of approximately 35.64 ng protein at a ﬁxed area (8 mm × 55 mm) with a thickness of 0.06 µm on top of the device as shown in Figure 1a, with varied layers of ZnO and IrO2 in terms of their thicknesses. g y From Equation (8), the relative mass sensitivity can be measured in terms of diﬀerent parameters such as phase, SWR, insertion loss, and linear magnitude. Figure 13 presents a comparison with respect to sensitivity based on the measurements of diﬀerent parameters inﬂuencing sensitivity, including: the imaginary and real magnitude of the response, insertion loss S21, SWR, the total magnitude of phase, the linear magnitude of S21, and the frequency shift. After the protein layer was added to the top of the device surface, the received signal was compared to the one from the device without the added protein layer using Matlab©. The responses of the frequency shift, insertion loss change, and magnitude of phase have all been well studied in previous research. However, there is no prior work focusing on the sensitivity of other parameters. The veriﬁed model system developed herein can be used to deﬁne the device’s mass sensitivity with speciﬁc mass loading which is useful to predict the device’s capabilities and improve the achievable sensitivity. In Figure 13a,b, the imaginary phase shift shows the largest sensitivity to the mass loading. Even with only 100 nm ZnO layer on top of the device, it still has −0.0023 ppm of normalized sensitivity to mass loading compared to its frequency sensitivity which was only 1.57 × 10−6. The frequency in the oscillator system needs to adjust the loop phase to 0 degrees and then determine the frequency shift by comparing the two frequencies. Therefore, the data were imported to Matlab© to match the loop phase, and were then compared to obtain the frequency shift. 3.3.2. Phase Shift Detection The operation of the phase shift technique has been described in previous research, and the magnitude of the phase shift is more sensitive than the frequency shift [28,42,43]. The SAW device is conﬁgured as a delay line and fed by a radio frequency excitation signal. The phase of the signals at 13 of 18 Sensors 2019, 19, 1749 the input and output of the line are compared to obtain their diﬀerence ϕ, which is the phase delay of the acoustic line [41]: 1 lf ϕ = 2π 1 λ = 2πlf v (9) (9) ∆ϕ = −2πlf v ∆v v = −ϕ0 ∆v v (10) (10) Here, l is the length of the line corresponding to the center-to-center distance of the IDTs in Equations (9) and (10), λ is the acoustic wavelength at the operating frequency f, and v is the acoustic phase velocity. Any change ∆v in the velocity is detected as a change ∆ϕ in the phase delay ϕ0 of the wave. The expression shows how the output signal, which is proportional to ∆ϕ, can be magniﬁed by increasing phase delay compared to frequency shift. On the other hand, it is necessary to consider the 2π period ranges in the response of the phase detector, which limit the upper levels of the dynamic range of the device. 3.3.3. Sensitivity Comparison Negative sensitivity means that as the mass loading increases or guiding layer increases, the resonant phase, resonant insertion loss, and operating frequency of the device decrease. to the frequency sensitivity, which can be confirmed by Equation (11). ∇𝑓= 1 𝜏 ∆ø 360° = 1 𝐿஽ 𝑣஽+ 𝐿ூ஽் 𝑣ூ஽் ∆ø 360° (11) where τ is the delay time across the device, vD is the acoustic velocity in the delay line path, vIDT is the acoustic velocity in the IDT region, LD is the delay line path length, LIDT is the propagation path length in the IDT region, and ∆ø is the phase shift across the device. The velocity decrease caused by the guiding layer will increase the delay time. The simulation results confirmed that with the same amount of frequency shift at the device, phase shift should increase. (a) (b) Figure 13. Comparison of normalized sensitivity defined based on different device parameters of the simulated SAW device with only ZnO layer. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 13. Comparison of normalized sensitivity deﬁned based on diﬀerent device parameters of the simulated SAW device with only ZnO layer. (a) Full view of the plot, and (b) zoomed ﬁgure without Imaginary data. Sensors 2019, 19, x FOR PEER REVIEW 15 of 18 (a) (b) (a) (b) Figure 13. Comparison of normalized sensitivity defined based on different device parameters of the simulated SAW device with only ZnO layer. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 13. Comparison of normalized sensitivity deﬁned based on diﬀerent device parameters of the simulated SAW device with only ZnO layer. (a) Full view of the plot, and (b) zoomed ﬁgure without Imaginary data. ors 2019, 19, x FOR PEER REVIEW 15 o In Figure 14, the sensitivity of the response to the protein layer is compared, showing that all the nsitivities increased slightly compared to the results shown in Figure 13, where the 50 nm IrO2 layer as absent. This sensitivity increase has also been proven in our recent publication [35] by rturbation analysis, showing that a device with multiple guiding layers on top can achieve proved sensitivity. Figures 13 and 14 show the phase sensitivity, the real part of admittance nsitivity, and the linear magnitude of S21. 3.3.3. Sensitivity Comparison The relationship between the sensitivity of all these parameters indicated that the normalized sensitivity gradually decreased when it was deﬁned based upon the imaginary magnitude of the response, magnitude of the phase, real magnitude of the response, linear magnitude S21, insertion loss S21 (dB), frequency with phase matched, or SWR. The magnitude of phase sensitivity was ~100 folds larger than the frequency shift at the ZnO thickness of 1000 µm. As the thickness of the guiding layer increased, the phase sensitivity increased compared to the frequency sensitivity, which can be conﬁrmed by Equation (11). ∇f = 1 τ ∆o 360◦= 1 LD vD + LIDT vIDT ∆o 360◦ (11) (11) where τ is the delay time across the device, vD is the acoustic velocity in the delay line path, vIDT is the acoustic velocity in the IDT region, LD is the delay line path length, LIDT is the propagation path Sensors 2019, 19, 1749 compared to obta parameters indicat 14 of 18 these d based 14 of 18 l these d based length in the IDT region, and ∆ø is the phase shift across the device. The velocity decrease caused by the guiding layer will increase the delay time. The simulation results conﬁrmed that with the same amount of frequency shift at the device, phase shift should increase. p g y g p g p g response, linear magnitude S21, insertion loss S21 (dB), frequency with phase matched, or SWR. The magnitude of phase sensitivity was ~100 folds larger than the frequency shift at the ZnO thickness of 1000 μm. As the thickness of the guiding layer increased, the phase sensitivity increased compared In Figure 14, the sensitivity of the response to the protein layer is compared, showing that all the sensitivities increased slightly compared to the results shown in Figure 13, where the 50 nm IrO2 layer was absent. This sensitivity increase has also been proven in our recent publication [35] by perturbation analysis, showing that a device with multiple guiding layers on top can achieve improved sensitivity. Figures 13 and 14 show the phase sensitivity, the real part of admittance sensitivity, and the linear magnitude of S21. The device exhibits a passive sensitivity, meaning that as the mass loading is applied to the device, the values of these parameters increase. 3.3.3. Sensitivity Comparison The device exhibits a passive sensitivity, meaning that the mass loading is applied to the device, the values of these parameters increase. Negative nsitivity means that as the mass loading increases or guiding layer increases, the resonant phase, sonant insertion loss, and operating frequency of the device decrease. Figure 15 shows the frequency sensitivity comparison between the device with only a ZnO layer d the device with a multilayer of ZnO and a 50 nm-thick IrO2. The frequency sensitivity of the ultilayer device was larger than that with only ZnO due to the additional IrO2 layer added on top, hich increases the confinement of acoustic energy within the guiding ZnO layer. Figure 16 shows at the sensitivity increased as the thickness of the IrO2 increased from 10 nm to 80 nm. (a) (b) Figure 14. The simulated results based on different sensitivity parameters for SAW devices with 50 nm IrO2 layer and a ZnO layer of varied thicknesses. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 14. The simulated results based on diﬀerent sensitivity parameters for SAW devices with 50 nm IrO2 layer and a ZnO layer of varied thicknesses. (a) Full view of the plot, and (b) zoomed ﬁgure without Imaginary data. In Figure 14, the sensitivity of the response to t ensitivities increased slightly compared to the resu was absent. This sensitivity increase has also b erturbation analysis, showing that a device wi mproved sensitivity. Figures 13 and 14 show th ensitivity, and the linear magnitude of S21. The d s the mass loading is applied to the device, the ensitivity means that as the mass loading increas esonant insertion loss, and operating frequency of Figure 15 shows the frequency sensitivity com nd the device with a multilayer of ZnO and a 5 multilayer device was larger than that with only Z which increases the confinement of acoustic energ hat the sensitivity increased as the thickness of the (a) he protein layer is compared, showing that all the ts shown in Figure 13, where the 50 nm IrO2 layer een proven in our recent publication [35] by h multiple guiding layers on top can achieve e phase sensitivity, the real part of admittance vice exhibits a passive sensitivity, meaning that values of these parameters increase. Negative s or guiding layer increases, the resonant phase, the device decrease. 3.3.3. Sensitivity Comparison arison between the device with only a ZnO layer nm-thick IrO2. The frequency sensitivity of the O due to the additional IrO2 layer added on top, within the guiding ZnO layer. Figure 16 shows IrO2 increased from 10 nm to 80 nm (b) been th m e ph evic e va es or the was ertu mpr ensi s th ensi ZnO la 10 nm (b) nfinem eased (a) y Figure 14. The simulated results based on different sensitivity parameters for SAW devices with 50 nm IrO2 layer and a ZnO layer of varied thicknesses. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 14. The simulated results based on diﬀerent sensitivity parameters for SAW devices with 50 nm IrO2 layer and a ZnO layer of varied thicknesses. (a) Full view of the plot, and (b) zoomed ﬁgure without Imaginary data. Figure 15 shows the frequency sensitivity comparison between the device with only a ZnO layer and the device with a multilayer of ZnO and a 50 nm-thick IrO2. The frequency sensitivity of the 15 of 18 Sensors 2019, 19, 1749 multilayer device was larger than that with only ZnO due to the additional IrO2 layer added on top, which increases the conﬁnement of acoustic energy within the guiding ZnO layer. Figure 16 shows that the sensitivity increased as the thickness of the IrO2 increased from 10 nm to 80 nm. (a) ( ) Figure 14. The simulated results based on different sensitivity parameters for SAW devices with 50 nm IrO2 layer and a ZnO layer of varied thicknesses. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 14. The simulated results based on different sensitivity parameters for SAW devices with 50 nm IrO2 layer and a ZnO layer of varied thicknesses. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. (a) (b) Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 15. 3.3.3. Sensitivity Comparison The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. g q y y p y y g layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. (a) (b) Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed figure without Imaginary data layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. (a) (b) Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed ﬁgure without Imaginary data. (a) (b) Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed figure without I i d t (a) (b) Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed ﬁgure without Imaginary data. (b) (b) (a) (a) (a) (a) (b) (b) ( ) ( ) Figure 16. 3.3.3. Sensitivity Comparison Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed figure without I i d t Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed ﬁgure without Imaginary data. 3.3.3. Sensitivity Comparison The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. (a) (b) Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed figure without Imaginary data. Figure 16. Simulation results of the SAW device with a 300 nm ZnO layer and an extra coating IrO2 layer of varied thickness from 10nm to 80nm. (a) Full view of the plot, and (b) zoomed ﬁgure without Imaginary data. Figure 15 The frequency sensitivity comparison between a SAW-based device with only varying ZnO Fi 15 Th f iti it i b t SAW b d d i ith l i Z O Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO l d d i ith 50 I O l t f th i Z O l Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. Figure 15. The frequency sensitivity comparison between a SAW-based device with only varying ZnO layer and a device with 50 nm IrO2 layer on top of the varying ZnO layer. Figure 15. g y 4. Summary and Conclusions C ﬂi f I Th h d l ﬂi f i nowledgments: The authors wish to thank Christine Bouchard for her assistance in editing the manuscript Acknowledgments: The authors wish to thank Christine Bouchard for her assistance in editing the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. g g p Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. s of Interest: The authors declare no conﬂict of interest. References 1. Hashimoto, K. Surface Acoustic Wave Devices in Telecommunications; Springer: Berlin/Heidelberg, Germany, 2000; ISBN 978-3-642-08659-5. 1. Hashimoto, K. Surface Acoustic Wave Devices in Telecommunications; Springer: Berlin/Heidelberg, Germany, 2000; ISBN 978-3-642-08659-5. 2. Hashimoto, K. Simulation of Surface Acoustic Wave Devices. Jpn. J. Appl. Phys. 2006, 45, 4423–4428. [CrossRef] 2. Hashimoto, K. Simulation of Surface Acoustic Wave Devices. Jpn. J. Appl. Phys. 2006, 45, 4423–4428. [CrossRef] 2. Hashimoto, K. Simulation of Surface Acoustic Wave Devices. Jpn. J. Appl. Phys. 2006, 45, 4423–4428. [CrossRef] 3 Go D B ; Atashbar M Z ; Ramshani Z ; Chang H -C Surface acoustic wave devices for chemical sensing . Go, D.B.; Atashbar, M.Z.; Ramshani, Z.; Chang, H.-C. Surface acoustic wave devices for chemical sen and microﬂuidics: A review and perspective. Anal. Methods 2017, 9, 4112–4134. [CrossRef] Guldiken, R.; Jo, M.C.; Gallant, N.D.; Demirci, U.; Zhe, J. 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A Urinary Bcl-2 Surface Acoustic Wave Biosensor for Early Ovarian Cancer Detection. Sensors 2012, 12, 7423–7437. [CrossRef] [PubMed] 9. Onen, O.; Ahmad, A.A.; Guldiken, R.; Gallant, N.D. Surface Modiﬁcation on Acoustic Wave Biosensors for Enhanced Speciﬁcity. Sensors 2012, 12, 12317–12328. [CrossRef] [PubMed] 10. Zhang, Y.; Yang, F.; Sun, Z.; Li, Y.-T.; Zhang, G.-J. A surface acoustic wave biosensor synergizing DNA-mediated in situ silver nanoparticle growth for a highly speciﬁc and signal-ampliﬁed nucleic acid assay. Analyst 2017, 142, 3468–3476. [CrossRef] y y 11. Jakubik, W.; Powro´znik, P.; Wrotniak, J.; Krzywiecki, M. Theoretical analysis of acoustoelectrical sensitivity in SAW gas sensors with single and bi-layer structures. Sens. Actuators B Chem. 2016, 236, 1069–1074. [CrossRef] 12. Marcu, A.; Viespe, C. g y 4. Summary and Conclusions In this study, we developed and evaluated a novel approach for characterizing and analyzing the SAW-based resonator device. This model was then veriﬁed by comparison to experimental data taken from fabricated devices. Our results show that the ﬁnite element model used herein reduces the computation requirements and time consumption while maintaining suﬃcient accuracy for the targeted research applications of acoustic sensor optimization to detect mass-loading eﬀects. The simulation results of the sensor’s frequency spectrum were plotted, showing a clear trend and ﬁt of the experimentally measured results. A thorough sensitivity analysis of the 3D model including S-parameters, reﬂection parameter, transmission parameter, and velocity phase were compared in this study. The results of this study can be used to determine and optimize the measurement conﬁguration of SAW sensors. By comparing the sensitivity between the frequency, phase, and imaginary response of diﬀerent guiding layers and structure designs, one can set up the appropriate measurement system Sensors 2019, 19, 1749 16 of 18 16 of 18 such as an oscillatory circuit system or voltmeter and network analyzer system. However, a further study to reduce the spurious modes and increase the accuracy of the model will be investigated in the future. This study and its results will be used as feedback for the experimental device design in order to optimize the device in the future as this study shows that the simulation can accurately replicate experimental data. Author Contributions: T.W.: Conducting research, analysis of data and writing of the manuscript. R.G. (Ryan Green): Writing of the manuscript. R.G. (Rasim Guldiken): Research design and writing of the manuscript. J.W.: Writing of the manuscript. S.M.: Research design and writing of the manuscript. S.S.M.: Research design and writing of the manuscript. Funding: This work is supported by Veterans Aﬀairs Merit Review grant (BX003413) to Subhra Mohapatra, and Research Career Scientist Awards to Subhra Mohapatra (IK6BX004212) and Shyam Mohapatra (IK6 BX003778). Though this report is based upon work supported, in part, by the Department of Veterans Aﬀairs, Veterans Health Administration, Oﬃce of Research and Development, the contents of this report do not represent the views of the Department of Veterans Aﬀairs or the United States Government. Acknowledgments: The authors wish to thank Christine Bouchard for her assistance in editing the manuscript. References Surface Acoustic Wave Sensors for Hydrogen and Deuterium Detection. Sensors 2017, 17, 1417. [CrossRef] [PubMed] 13. Hashimoto, K.; Endoh, G.; Yamaguchi, M. Coupling-of-modes modelling for fast and precise simulation of leaky surface acoustic wave devices. 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https://openalex.org/W3087610062	https://link.springer.com/content/pdf/10.1007/s00417-020-04939-7.pdf	English	null	Tendon elongation with bovine pericardium in strabismus surgery—indications beyond Graves’ orbitopathy	Graefe's archive for clinical and experimental ophthalmology	2,020	cc-by	8,754	ERROR: type should be string, got "https://doi.org/10.1007/s00417-020-04939-7\nGraefe's Archive for Clinical and Experimental Ophthalmology (2021) 259:145–155 https://doi.org/10.1007/s00417-020-04939-7\nGraefe's Archive for Clinical and Experimental Ophthalmology (2021) 259:145–155 NEUROPHTHALMOLOGY * Andrea Hedergott\nandrea.hedergott@uk-koeln.de Abstract Background For some patients with complex ocular motility disorders, conventional strabismus surgery is insufficient. Surgery\nwith tendon elongation allows correction of larger angles and maintains a sufficient arc of contact for rectus muscles. This study\nreports results for tendon elongation with bovine pericardium (Tutopatch®) in indications other than Graves’ orbitopathy in\nwhich it is already widely used. Methods We reviewed the records of all patients who underwent surgery with Tutopatch® in our institution. Angles of squint\nand head postures were analyzed preoperatively, on the first postoperative day, and in the long term (median 9 weeks after the\noperation). Patients with Graves’ orbitopathy were excluded. Results From 2011 to 2018, the procedures on 58 eyes of 54 patients (35 females, median age 35 years (3–75)) met the inclusion\ncriteria. Horizontal rectus muscle surgery (53 eyes) was conducted on patients with residual strabismus (13), Duane’s retraction\nsyndrome with eso- (type I: 16)/exodeviation (type II: 2, type III: 1), 6th (7)/3rd nerve palsy (7), Möbius syndrome (2), congenital\nfibrosis of the extraocular muscles type 3A (CFEOM3A, TUBB3 mutation) (4), and orbital apex syndrome (1). Vertical rectus\nmuscle surgery (5 eyes) was conducted on patients with myasthenia (1), vertical tropia after orbital floor fracture (1), CFEOM1\n(2), and Parry-Romberg syndrome (1). 42 eyes had prior eye muscle surgery (1–5 procedures, median 1). Out of 45 patients with\npostoperative long-term data, 43 showed an angle reduction. Fifty-one percent had an angle of 10Δ (prism diopter) or less, one\nhad a significant over-effect, and 10 had revision surgery. For the heterogeneous group of residual eso- and exotropias, the\nmedian absolute horizontal angle was reduced from 35Δ (16 to 45Δ) to 9Δ (0 to 40Δ), for Duane’s retraction syndrome from\n27.5Δ (9 to 40Δ) to 7Δ (0 to 40Δ), and for sixth and third nerve palsies from 43Δ (20 to 75Δ) to 18Δ (4 to 40Δ). For 3 patients\nwith vertical rectus muscle surgery, the median absolute vertical angle was reduced from 30Δ (20 to 45Δ) to 4Δ (1 to 22Δ). The\nmotility range was shifted in the direction contrary to the elongated muscle in all subgroups. A considerable reduction of the\nexcursion into the field of action of the elongated muscle had to be registered. Conclusions Strabismus surgery with bovine pericardium introduces new surgical options for complicated revisions and for rare and\ncomplex oculomotor dysfunctions. 1\nDepartment of Ophthalmology, Faculty of Medicine and University\nHospital Cologne, University of Cologne, Kerpener Strasse 62,\n50937 Cologne, Germany Abstract Yet, it has to be recognized that this type of surgery aiming at maximum effects, despite preservation\nor restitution of the arc of contact, leads to reduction of the excursion into the field of action of the elongated muscle. Furthermore, dose\nfinding can be difficult depending on the underlying pathology and more than one intervention might be necessary for optimal results. Keywords Bovine pericardium . Tutopatch® . Strabismus surgery . Tendon elongation This study was presented at the annual meeting of the German Society of\nOphthalmology (DOG) 2019, 26.09. – 29.09., Berlin, Germany. Andrea Hedergott1\n& Ursula Pink-Theofylaktopoulos1 & Antje Neugebauer1 & Julia Fricke1 Received: 29 April 2020 /Revised: 18 August 2020 /Accepted: 10 September 2020\n# The Author(s) 2020\n/ Published online: 19 September 2020 Introduction Electronic supplementary material The online version of this article\n(https://doi.org/10.1007/s00417-020-04939-7) contains supplementary\nmaterial, which is available to authorized users. Primarily used in cardiothoracic, vascular, and neurosurgery,\nbovine pericardium has been introduced into ophthalmosurgery\naround the turn of the millennium [1, 2]. Its use was described\nfor coating hydroxyapatite implants in enucleation surgery, cov-\nering Ahmed implants in glaucoma surgery, or as a corneal\npatch in corneal ulcers [3–7]. In eye muscle surgery recessions are limited: the recessed\nmuscles should not be placed far behind the equator to Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 146 Key messages\nTendon elongation with bovine pericardium (Tutopatch®) is widely used in patients with Graves´ Orbitopathy. This study proofs that tendon elongation with Tutopatch® is a safe surgical method beyond this indication. Tutopatch® introduces new surgical options for complex and/or rare oculomotor dysfunctions, especially for \ncomplicated revisions, large angles of squint and residual head postures, where conventional eye muscle surgery has \nlimits. Key messages\nTendon elongation with bovine pericardium (Tutopatch®) is widely used in patients with Graves´ Orbitopathy. This study proofs that tendon elongation with Tutopatch® is a safe surgical method beyond this indication. Tutopatch® introduces new surgical options for complex and/or rare oculomotor dysfunctions, especially for \ncomplicated revisions, large angles of squint and residual head postures, where conventional eye muscle surgery has \nlimits. Tendon elongation with bovine pericardium (Tutopatch®) is widely used in patients with Graves´ Orbitopathy. This study proofs that tendon elongation with Tutopatch® is a safe surgical method beyond this indication. Tutopatch® introduces new surgical options for complex and/or rare oculomotor dysfunctions, especially for \ncomplicated revisions, large angles of squint and residual head postures, where conventional eye muscle surgery has \nlimits. Tutopatch® introduces new surgical options for complex and/or rare oculomotor dysfunctions, especially for \ncomplicated revisions, large angles of squint and residual head postures, where conventional eye muscle surgery has \nlimits. Orthoptic and ophthalmologic examination The motility of each eye was recorded with regard to\nover- or underactions in the main fields of action of all\nsix eye muscles (The patient was asked to follow a pen-\nlight with his eyes without moving the head. Monocular\nexcursions in the six main fields of action of the eye\nmuscles were estimated in degrees by an experienced\northoptist) The motility of each eye was recorded with regard to\nover- or underactions in the main fields of action of all\nsix eye muscles (The patient was asked to follow a pen-\nlight with his eyes without moving the head. Monocular\nexcursions in the six main fields of action of the eye\nmuscles were estimated in degrees by an experienced\northoptist) Within this study, we report results and experiences of\ntendon elongation with bovine pericardium (Tutopatch®) in\na large series with 58 eyes of 54 patients for various indica-\ntions beyond Graves’ orbitopathy. Measurement at the tangent screen of Harms was per-\nformed in the six fields of action of the eye whenever possible\n(especially patients with paresis and restrictive disorders). Due\nto young age and/or suppression of one eye, measurements\nwere not possible in all patients. Therefore, the values are not\ngiven within this study. Orthoptic and ophthalmologic examination preserve motility. The effect of rectus muscle recession is\ninitially linear, but increases with enlarging recession distance\nand approach to the equator. Yet if the muscle is placed behind\nthe equator, increasing duction limitations can be expected\n[8]. Surgery with tendon elongation allows correction of larg-\ner angles and maintains a sufficient arc of contact. Various\nmaterials were used for tendon elongation such as homolo-\ngous scleral grafts, polytetrafluoroethylene (Goretex), sili-\ncone, or fascia lata [9–12]. For this study the following pre- and postoperative measure-\nments have been considered from our medical records: –\nWhen a head posture was adopted: the head posture mea-\nsured with a hand-held goniometer with the patient fixing\nthe smallest age-appropriate target distinguishable for the\npatient at distance under binocular conditions with\nBCVA (best-corrected visual acuity) In 2011, Esser and Eckstein were the first to publish their\nexperiences (dating back to 1996) with bovine pericardium\n(Tutopatch®) in eye muscle surgery for tendon elongation in\npatients with Graves’ orbitopathy [13, 14]. They reported it to\nbe a safe surgical method which also showed satisfying long-\nterm results in correction of large angles of squint in this\nrestrictive disorder [14, 15]. Further, bovine pericardium was\nreported to be replaced by tendon-like tissue over time [13, 14,\n16]. Squint angles cannot be satisfactorily reduced by conven-\ntional eye muscle surgery not only in patients with Graves’\norbitopathy but also in other patients with secondary, com-\nplex, restrictive, and rare oculomotor dysfunctions. Van Rijn\net al. reported tendon elongation with Tutopatch® for 31 pa-\ntients with exotropia (two of them oculomotor nerve paresis/\npalsy), 7 patients with esotropia (two of them abducens nerve\npalsy), and one patient with hypotropia [16]. They showed\nsatisfactory results with fairly good alignment and high patient\nsatisfaction, but duction limitations in the direction of the\nelongated muscle. –\nTests for binocular function (Bagolini striated glasses test,\nLang I or II stereo test, Titmus stereo test) with the head\nposture adopted with BCVA Measurement of the full extent of deviation in primary\nposition by alternate prism and cover-uncover test at far\nfixation with BCVA (in setting of poor vision in one eye,\nthe angle was measured by Krimsky test). In restrictive\nand paralytic strabismus, measurement of the primary an-\ngle instead of the secondary angle is essential. This was\nachieved by strictly placing the prism in front of the eye\nwith worse motility. Patients’ characteristics From 2011 to 2018, 58 eyes of 54 patients (35 females, 19\nmales, median age 35 years (3 to 75 years)) underwent stra-\nbismus surgery with tendon elongation with bovine\npericardium. In all cases, the surgical procedure was performed by one\nof the authors (J.F.). Surgery was performed under general\nanesthesia. For tendon elongation, the affected muscle was\nheld by a muscle clamp and disinserted. Then, the posterior\nend of the implant (Tutopatch®) was sutured to the detached\nend of the muscle with non-absorbable sutures (Mersilene®\n5.0). In cases where the muscle was very tight and therefore\nthe use of a muscle clamp was not possible, the posterior end\nof the implant was attached by two sutures directly to the\nmuscle closely behind the insertion and then the muscle was\ndisinserted. In all cases, the anterior end of the implant was\nsutured to the sclera at a median distance of 3 mm behind the\nphysiological insertion (sutures should not be visible through\nthe conjunctiva). In early cases non-absorbable sutures\n(Mersilene® 5.0) were used for this step as well. Recently,\nwe switched to absorbable sutures (Resorba® 6.0). These\nwere only used in one patient within this study (patient 32). When a larger recession had already taken place on the muscle\nin question, the insertion of the elongated tendon thus was\n“advanced” while the original muscle tendon was recessed/\nelongated, as we aimed at weakening the muscle but enhanc-\ning the arc of contact. Indications involving the horizontal rectus muscles (53\neyes) were residual strabismus, Duane’s retraction syndrome\n(DRS) with eso- (type I)/exodeviation (type II, type III), par-\ntial abducens nerve palsy, partial oculomotor nerve palsy,\nMöbius syndrome, congenital fibrosis of the extraocular mus-\ncles type 3A (CFEOM3A, TUBB3 mutation), and orbital apex\nsyndrome (supplementary Table 1). Indications involving the\nvertical rectus muscles (5 eyes) comprised myasthenia gravis,\npersisting vertical tropia after orbital floor fracture, CFEOM1,\nand Parry-Romberg syndrome (supplementary Table 1). In 42\ncases, the procedure was performed on a muscle which al-\nready had been operated on, whereas in 16 cases it was a\nprimary procedure. The median number of previous proce-\ndures on the concerned eyes with previous surgery was 1\n(range 1–5). Results primary position, reduction of a head posture, elimination of\ndiplopia in primary position, and improvement of binocular\nfunctions. Therefore, the full extent of deviation in primary\nposition pre- and postoperatively was used to calculate the\ndose-effect. Statistical analysis Statistical analysis was performed using Excel (Microsoft\nExcel for Mac, Version 15.29.1, Microsoft, USA) and SPSS\n(IBM SPSS Statistics, Version 23.0.0.2, IBM, USA). For met-\nric data, median, minimum, and maximum values were calcu-\nlated. Differences between groups were evaluated with\nStudent’s paired t test for data with normal distribution, oth-\nerwise with the Wilcoxon test. Postoperative data Long-term data were available for 45 patients (83%), after a\nmedian of 9 weeks post operation (5 to 261 weeks). In 43\npatients the squint angle was reduced (96%); in 23 out of the\n43 patients the angle was reduced to 10Δ or less (51%). One\npatient had a significant over-effect. The patients’ binocular\nfunctions were either the same or better than before surgery\n(for detailed patients’ characteristics of all patients with long-\nterm data, see supplementary Table 1). Surgery In this single-center retrospective study, we reviewed the med-\nical records of all patients who underwent strabismus surgery\nwith tendon elongation with bovine pericardium in our insti-\ntution. Patients with Graves’ orbitopathy were excluded from\nthe study. Choice of procedure was made on an individual basis and\ndependent on the patients’ horizontal and vertical deviation,\nmotility, and head turn. The total amount of surgery was based\non the dosages used for each motility disorder for regular\nrevision surgery. Aim of surgery was ocular alignment in Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 147 Patients’ characteristics The tendon of the medial rectus muscle was elon-\ngated in 32 eyes (24 of them with previous surgery), of the\nlateral rectus muscle in 21 eyes (16 of them with previous\nsurgery), of the inferior rectus muscle in four eyes (1 of them\nwith previous surgery), and of the superior rectus muscle in\none eye (with previous surgery). Based on the surgical reports, the following data was de-\ntermined: length of the implant, amount of surgery in compar-\nison to preoperative localization of the muscle (“effective re-\ncession distance”), and insertion site of the implant. The dose-\neffect relation of the tendon elongation was calculated for\ngroups with at least 5 patients with the same preoperative\ndiagnosis and with long-term results. The groups had to be\nhomogeneous with regard to whether or not additional opera-\ntions were performed on other eye muscles. For calculation of\nthe dose-effect relation, the difference between largest preop-\nerative and long-term squint angle in primary position was\ncalculated (prism diopter Δ). This value was divided by the\ntotal amount of surgery (mm). For calculating the total amount\nof surgery in combined surgery, the effective recession dis-\ntance was added to the effective tucking/advancing distance of\nthe antagonist. Surgery Twenty-six patients had tendon elongation with bovine peri-\ncardium as single muscle surgery. Twenty-three patients had\nfurther procedures on the same eye, 3 had further procedures\non the partner eye, and 2 had further procedures on the same\nand on the partner eye. Patients with residual eso- and exotropia All of the 5 patients with long-term measurement showed an\nangle reduction with three showing an angle of ≤10Δ (60%). Median, minimum, and maximum squint angles are displayed\nin Table 1. Pre- and postoperative squint angles of each patient\nare shown in Fig. 1. For residual esotropia, the medial rectus muscle tendon was\nelongated with a median implant length of 12 mm (8 to 12\nmm). The median effective recession distance was 5 mm (3 to\n6 mm). In 3 eyes, the lateral rectus muscle was simultaneously\ntucked (patients 35, 36, 38). Thus, the median total amount of\nhorizontal eye muscle surgery for all eyes was 7.5 mm (4 to 11\nmm). In one case, the superior oblique muscle was additionally\nrecessed (patient 37). All patients with residual esotropia\nshowed a reduction in squint angle on the first day after surgery,\nbut in the long term, one of 5 patients showed angle enlarge-\nment nearly shifting back to the preoperative position. Three of\nthem had an angle of ≤10Δ (60%). Median, minimum, and\nmaximum squint angles are displayed in Table 1. Pre- and\npostoperative squint angles of each patient are shown in Fig. 1. The horizontal motility range has been narrowed. The me-\ndian preoperative abduction of the concerned eye was 35° (12\nto 40) with a median adduction of 50° (40 to 50). In the long The horizontal motility range was narrowed by the proce-\ndure. The median preoperative adduction of the concerned eye\nwas 50° (40 to 50) while the median abduction was 45° (30 to Table 1\nPatients with horizontal rectus muscle tendon elongation and\nlong-term follow-up: full extent of deviation in primary position (in prism\ndiopters) preoperatively, at the first postoperative day, in the long term\n(convergent angles are given in positive, divergent angles in negative values), and angle improvement for residual esotropia (5 patients), resid-\nual exotropia (5 patients), oculomotor nerve palsy (7 patients), abducens\nnerve palsy (5 patients), esotropic Duane’s retraction syndrome (13 pa-\ntients), exotropic Duane’s retraction syndrome (3 patients) Patients with long-term\nmeasurement\nHorizontal angle of squint (Δ)\nMedian (minimum to maximum)\nLong-term measurement\n(weeks)\nMedian (minimum\nto maximum)\nPreop. Postop. first day\nLong term\nDifference preop. -postop. Patients with residual eso- and exotropia Long-term data are available for 5 of the 6 patients with\nesotropia and for 5 of the 7 patients with exotropia. The squint\nangle showed statistically significant postoperative reduction Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 148 in the long term (Wilcoxon test p = 0.008). One patient with\ntendon elongation for secondary esotropia after traumatic in-\njury as a child had an enucleation of the painful and blind eye\n4 years after eye muscle surgery. Due to rubeotic glaucoma\nand PVR retinal detachment, the eye was enucleated after\nunsuccessful retinal surgery. The patient, one of two with\npostoperative over-effect after tendon elongation, was there-\nfore not available for long-term results. term, we measured a median abduction of 30° (20 to 50) and\nno median gain in abduction: −5° (−15 to 15). The median\nadduction was reduced to 30° (20 to 50) with a median loss in\nadduction of 10° (0 to 30). Up to date, the one patient (patient 38) without angle im-\nprovement in the long term and another patient (patient 36)\nwith residual esotropia and vertical tropia had a revision\nsurgery. All of the patients had previously had surgery. The esotropic\npatients had residual esotropia after esotropia surgery. The\nexotropic patients had residual exotropia after exotropia surgery\n(two of them initially had esotropia surgery). The median num-\nber of procedures on the concerned eye for residual esotropia\nwas 2 (1 to 5), for residual exotropia 1 (1 to 5). For residual exotropia, the lateral rectus muscle tendon was\nelongated with a median implant length of 10 mm (8 to 13\nmm). The median effective recession distance was 5 mm (1 to\n6 mm). In the case with an effective recession of only 1 mm,\nintraoperatively the lateral rectus muscle was found to insert in\na distance of 17 mm behind the limbus. Adhesions to the\nglobe over a longer section were solved before inserting the\ntendon elongation. The medial rectus muscle was simulta-\nneously tucked in four eyes respectively advanced in two eyes\nwith previous medial rectus muscle resection and insertion\nfound behind the original insertion (patients 1–3, 5–7). Thus, the median total amount of horizontal eye muscle sur-\ngery for all eyes was 9.5 mm (5 to 12 mm). All but one patient\nshowed a reduction in squint angle on the first day after sur-\ngery. The remaining patient showed a reduction after 8 weeks. Patients with oculomotor/abducens nerve palsy Up to date, one patient with bilateral oculomotor nerve\npalsy had a revision surgery. Long-term data are available for all 7 patients with oculomotor\nnerve palsy and for 5 of 7 patients with abducens nerve palsy. The squint angle showed statistically significant reduction in\nthe long term (Wilcoxon test p = 0.002). Two of the patients\nwith oculomotor nerve palsy had bilateral palsy. One of them\nas well as four of the unilateral palsies had prior surgery. One\nof the patients with abducens nerve palsy had bilateral palsy. This patient as well as 5 of the unilateral palsies had prior\nsurgery. For oculomotor nerve palsy, the lateral rectus muscle\nhad tendon elongation with a median implant length of 10 mm\n(8 to 14 mm); for abducens nerve palsy the medial rectus\nmuscle tendon was elongated with a median implant length\nof 9 mm (7 to 10 mm). In patients with abducens nerve palsy, the median absolute\nrecession distance was 6 mm (5 to 12 mm). In 4 eyes, the\nlateral rectus muscle was simultaneously tucked/resected (pa-\ntients 27–29, 33). Thus, the median total amount of horizontal\neye muscle surgery for all eyes was 11 mm (5 to 20 mm). In\none eye, the superior oblique muscle was tucked due to addi-\ntional trochlear nerve palsy (patient 32). Median, minimum,\nand maximum squint angles are displayed in Table 1, and pre-\nand postoperative squint angles of each patient in Fig. 2. All\npatients showed a reduction in squint angle postoperatively\n(first day and long term). In the long term, three of five had\nan angle ≤10Δ (60%). The horizontal motility has been shifted towards abduction\nand narrowed. The median abduction of the concerned eye\nwas 0° preoperatively (−15 to 40) while the median adduction\nwas 45° (40 to 50). In the long term, all except of one patient\nshowed an improved abduction with a median abduction of 7°\n(0 to 30) and a median improvement of abduction of 7° (−10\nto 15). The median adduction was reduced to 30° (10 to 50)\nwith a median loss in adduction of 15° (0 to 35). For patients with oculomotor nerve palsy, the median ef-\nfective recession distance was 6 mm (4 to 12 mm). In 6 eyes,\nthe medial rectus muscle was simultaneously tucked or\nresected (patients 41–46). Patients with residual eso- and exotropia first day\nDifference preop.-\nlong term\nResidual esotropia\n5/6 patients\n35\n(16 to 40)\n12\n(0 to 35)\n6\n(−8 to 40)\n16\n(0 to 23)\n19\n(0 to 43)\n188 (6 to 260)\nResidual exotropia\n5/7 patients\n−35\n(−18 to −45)\n−16\n(−4 to −18)\n−10\n(1 to −25)\n19\n(0 to 41)\n20\n(8 to 46)\n8 (8 to 261)\nOculomotor nerve palsy\n7/7 patients\n−45\n(−25 to −75)\n−10\n(0 to −50)\n−30\n(−8 to −40)\n31\n(19 to 40)\n15\n(5 to 42)\n16 (6 to 40)\nAbducens nerve palsy\n5/7 patients\n41\n(20 to 53)\n8\n(2 to 40)\n8\n(4 to 35)\n14\n(12 to 48)\n18\n(12 to 46)\n8 (7 to 16)\nEsotropic Duane’s retraction\nsyndrome 13/16 patients\n25\n(9 to 40)\n8\n(−2 to 20)\n2\n(−8 to 20)\n14\n(5 to 37)\n22\n(7 to 48)\n8 (5 to 260)\nExotropic Duane’s retraction\nsyndrome\n3/3 patients\n−30\n(−12 to −30)\n−12\n(−4 to −14)\n−19\n(−5 to −40)\n16\n(8 to 18)\n6\n(−10 to 11)\n11 (9 to 16) Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 149 -50\n-40\n-30\n-20\n-10\n0\n10\n20\n30\n40\n50\nPreop\npostop ﬁrst day\npostop longterm\n1\n2\n3\n4\n5\n6\n7\n34\n35\n36\n37\n38\n39\nFig. 1 Pre- and postoperative full\nextent of deviation in primary\nposition for all patients with\nresidual esotropia and exotropia\n(prism diopters; esotropia is given\nin positive, exotropia in negative\nvalues). Patients with\nsimultaneous surgery on the\ncontralateral horizontal rectus\nmuscle: 1–3, 5–7, respectively 35,\n36, 38\n149\nGraefes Arch Clin Exp Ophthalmol (2021) 259:145–155 50). In the long term, one patient showed a gain in adduction\nwhile four showed no relevant change in adduction. The me-\ndian postoperative adduction was 50° (47.5 to 50) and the\nmedian gain in adduction 0 (0 to 7.5). The median abduction\nwas reduced to 30° (20 to 45), median loss in abduction of 15°\n(−15 to 30). minimum, and maximum squint angles are displayed in\nTable 1. Pre- and postoperative squint angles of each patient\nare shown in Fig. 2. The horizontal motility was shifted towards adduction. The\nmedian preoperative adduction of the concerned eye was 0° (−\n15 to 25) while the median abduction was 50° (40 to 50). Patients with residual eso- and exotropia In\nthe long-term course, all but one patient showed a gain in\nadduction with a median adduction of 10° (0 to 35) and a\nmedian gain in adduction of 5° (−10 to 35). The median\nabduction was reduced to 40° (5 to 50), median loss in abduc-\ntion 5° (0 to 40). Up to date, one patient (patient 6) with secondary exotropia\ndue to traumatic injury in childhood had two revision surger-\nies for remaining exotropia. Patients with Duane’s retraction syndrome dose-effect relation was 2.6Δ/mm (1.1 to 5.2Δ/mm; all 13\npatients with long-term data). Considering only patients with\nprevious medial rectus muscle surgery, the median effect was\n2.7Δ/mm (10 patients). Long-term data are available for 13 of 16 patients with\nesotropic DRS type I (the data of some of these patients\nhave already been published in a separate paper [17]),\nand for 3 of 3 patients with exotropic DRS (type II: 2;\ntype III: 1). Two patients had marked bilateral motility\nrestrictions. Eleven of 16 eyes with esotropic DRS and\ntwo out of three eyes with exotropic DRS had previous-\nly undergone surgery. The horizontal motility was shifted towards abduction and\nconsiderably narrowed. The median abduction of the con-\ncerned eye was 0° preoperatively (−3 to 30), the median\nadduction 50° (30 to 50). In the long term, 8 out of 13 patients\nshowed a gain in abduction. The median postoperative abduc-\ntion was 7° (0 to 30) and the median gain in abduction was 5°\n(0 to 20). The median adduction was reduced to 20° (7 to 45)\nwith a median loss in adduction of 25° (5 to 35°). For esotropic DRS, the medial rectus muscle tendon\nwas elongated with a median implant length of 10 mm\n(8 to 12 mm). The median absolute recession distance\nwas 6 mm (4 to 13 mm). None of the patients had\nsimultaneous surgery on another eye muscle. The abso-\nlute head turn (far distance) was reduced from median\n20° preoperatively (5 to 30°; turn to the left: 13; turn to\nthe right: 2; no head turn: 1) to 0° in the long term (−\n5 to 15°, negative value indicating over-effect). For exotropic DRS the lateral rectus muscle tendon was\nelongated with an implant length of 10 mm in all eyes. The\nhead turn was reduced from median 20° preoperatively (12 to\n25) to 12° in the long term (0 to 20). The median absolute\nrecession distance was 6 mm (6 to 10 mm). Median, minimum,\nand maximum squint angles are displayed in Table 1. Pre- and\npostoperative squint angles of each patient are shown in Fig. 3. All patients showed a reduction in squint angle on the first day\npostoperatively, but one patient showed no improvement in the\nlong term (patient 22). In the long term, one out of three patients\nwith exotropic DRS had an angle ≤10Δ (33%). Patients with oculomotor/abducens nerve palsy Thus, the median total amount of\nhorizontal eye muscle surgery for all eyes was 14 mm (5 to 22\nmm). In one eye, the superior rectus muscle was simulta-\nneously tucked (patient 40). All patients showed a reduction\nin squint angle postoperatively (first day and long term), but in\nthe long term, only one had an angle ≤10Δ (17%). Median, Up to date, one patient had a revision surgery. Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 150 -80\n-70\n-60\n-50\n-40\n-30\n-20\n-10\n0\n10\n20\n30\n40\n50\n60\nPreop\npostop ﬁrst day\npostop longterm\n27\n28\n29\n30\n31\n32\n33\n40\n41\n42\n43\n44\n45\n46\nFig. 2 Pre- and postoperative full\nextent of deviation in primary\nposition for all patients with NIII\nand NVI palsy (prism diopters;\nesotropia is given in positive,\nexotropia in negative values),\nincluding patient 29 with bilateral\nNVI palsy and patients 43 and 44\nwith bilateral NIII palsy. Patients\nwith simultaneous surgery on the\ncontralateral horizontal rectus\nmuscle: 27–29, 33, respectively\n41–46 Other patients with horizontal rectus muscle tendon\nelongation Surgery on horizontal rectus muscles (4 patients, 7 eyes)\nwas conducted in patients with Mobius syndrome (pa-\ntient 48: bilateral medial rectus muscle tendon elonga-\ntion), congenital fibrosis of the extraocular muscles (pa-\ntients 50 and 51: bilateral lateral rectus muscle elonga-\ntion, patient 51 with previous surgery on the concerned\nmuscles), and orbital apex syndrome (patient 54: unilat-\neral medial rectus tendon elongation, previous surgery\non the concerned muscle). The median implant length\nwas 10 mm (6 to 10). The median absolute recession\ndistance was 10 mm (5 to 13 mm). Surgery on vertical rectus muscles (4 patients, 5 eyes) was\nconducted in patients with orbital floor fracture (patient 52:\nsuperior rectus muscle tendon elongation), myasthenia gravis,\nand Parry-Romberg syndrome (patients 47 and 53: inferior\nrectus muscle tendon elongation), furthermore in a patient\nwith a chin-up head posture due to congenital fibrosis of the\nextraocular muscles (patient 49: surgery on both inferior rec-\ntus muscles). Two patients had prior surgery of the affected\nmuscle (patients 52 and 53). The rectus muscle tendon was\nelongated with a median implant length of 8 mm (6 to 10). The\nmedian absolute recession distance was 10 mm (3.5 to 12\nmm). All patients showed a reduction in squint angle respec-\ntively head posture on the first postoperative day and in the\nlong term. Two out of three patients with unilateral vertical\nrectus muscle tendon elongation showed a maximum angle of\n10Δ in the long term. Median, minimum, and maximum\nsquint angles of the three patients are displayed in Table 2\n(due to the small and heterogeneous sample size, only descrip-\ntive statistics based on absolute values are provided). The\nvertical motility range was shifted to the direction contrary\nto the elongated muscle in all cases. In patients with inferior\nrectus muscle tendon elongation, the preoperative elevation of\nthe concerned eye was 15° below midline in patient 47 and\n10° above midline in patient 53. In the long term, the elevation\nwas almost to midline in patient 47 and almost without limi-\ntation in patient 53. In the patient with superior rectus muscle\ntendon elongation, the depression was possible only to 10°\nbefore midline in adduction and to midline in abduction before\nthe operation. Patients with Duane’s retraction syndrome Median, minimum, and maximum squint angles for all pa-\ntients with esotropic DRS are displayed in Table 1. Pre- and\npostoperative squint angles of each patient are shown in Fig. 3. Considering patients with esotropic DRS without previous\nsurgery only (patients 16, 18, 19, 21, 25), the median preop-\nerative squint angle was 40Δ (35 to 45Δ), the median abso-\nlute preoperative head turn was 25° (7 to 30°), and the abduc-\ntion was before or just up to midline (median abduction −5°,\nfrom −10 to 0°). The horizontal motility was shifted towards adduction. The\nmedian adduction of the concerned eye was 10° preoperative-\nly (−15 to 45) while the median abduction was 25° (20 to 45). In the long term, two patients showed improved adduction. The median postoperative adduction was 15° (−10 to 25) and\nthe median improvement in adduction was 5° (−30 to 15). The median abduction was reduced to 20° (20 to 40) while the\nmedian loss in abduction was 5° (0 to 5). All patients with esotropic DRS showed a reduction in\nsquint angle on the first day postoperatively and in the long-\nterm course (long term: Wilcoxon test p = 0.001, statistically\nsignificant reduction). In the long term, 9 of the 13 patients\nwith esotropic DRS (69%) had an angle ≤10Δ. The median Of all patients with DRS, two patients had a revision\nsurgery. Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 151 -40\n-30\n-20\n-10\n0\n10\n20\n30\n40\n50\nPreop\npostop ﬁrst day\npostop longterm\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n25\n26\n22\n23\n24\nFig. 3 Pre- and postoperative full\nextent of deviation in primary\nposition for all patients with\nDuane’s retraction syndrome\n(prism diopters; esotropia is given\nin positive, exotropia in negative\nvalues)\n151\nGraefes Arch Clin Exp Ophthalmol (2021) 259:145 155 -40\n-30\n-20\n-10\n0\n10\n20\n30\n40\n50\nPreop\npostop ﬁrst day\npostop longterm\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n25\n26\n22\n23\n24\nFig. 3 Pre- and postoperative full\nextent of deviation in primary\nposition for all patients with\nDuane’s retraction syndrome\n(prism diopters; esotropia is given\nin positive, exotropia in negative\nvalues) Patients with vertical rectus muscle tendon\nelongation Other patients with horizontal rectus muscle tendon\nelongation Surgical effect midline. Patient 53 (Parry-Romberg syndrome) had additional\neye lid surgery a few months later. Patients with complex, rare, restrictive, large, and residual\nstrabismus types, as well as paretic strabismus types with pre-\nvious surgery were selected for tendon elongation with bovine\npericardium. Results showed a reduction of squint angle in all\nexcept of two patients. In the postoperative long term, 51% of\npatients had a squint angle of 10Δ or less, before any kind of\nrevision surgery. Only one patient had a significant over-ef-\nfect. The effect of surgery showed a high variability, probably\ncaused by the very heterogeneous patient characteristics. Possible explanations for this are previous surgery of various\namounts, adherence of the affected muscle to the sclera, re-\nstriction of motility, paresis of various extents, or\ndysinnervation. Furthermore, some patients had a combined\nsurgery on both horizontal rectus muscles. Another patient (patient 49) with congenital fibrosis of the\nextraocular muscles had bilateral inferior rectus muscle reces-\nsion due to chin-up head posture. The head posture of 18°\npreoperatively (also due to bilateral ptosis) was reduced to\n15° on the first day after surgery. In the long term, after addi-\ntional eye lid surgery, the patient showed no longer chin-up or\nchin-down head posture. Preoperatively, the eye elevation was\nunder midline. Postoperatively, elevation was possible a short\ndistance above midline for both eyes. Adverse events and revision surgery One patient showed increasing conjunctival injection and\nchemosis 4 days after the operation. Patient was treated with\nsystemic antibiotics and steroids. Because of persistent swelling,\nwe decided to explore the surgical site 12 days after the tendon\nelongation. During revision surgery, there was no infection nor\nimplant rejection visible. Therefore, the implant was not\nexplanted but the surgical site was rinsed with antibiotics. Eventually the conjunctival chemosis and injection resolved un-\nder steroid therapy. Nine weeks post operation, the patient was\nfree of complaints and the postoperative site was unremarkable. As mentioned above, Esser et al. primarily published their\nexperience with tendon elongation with bovine pericardium\nfor eye muscle surgery in 2011 [13] (when dose-effects in\nstudies referred to in this discussion were given in °/mm, we\nconverted the numbers also into Δ/mm for better comparison\nas to be seen in brackets). For 10 patients with large vertical\nsquint angles due to Graves’ orbitopathy (GO), they reported\nan angle reduction of 2.0°/mm (3.5Δ/mm) using bovine peri-\ncardium for tendon elongation of the inferior rectus muscle. Good results were achieved for patients with/without previous\ninferior rectus muscle recession and with/without previous\norbital decompression. Up to now in 9 cases, a revision surgery was performed on\na muscle which had been elongated by bovine pericardium. The pericardium had been transformed into tendon-like mate-\nrial which could be picked up with a strabismus hook. Except\nfor the suture, the material usually showed no adhesion to the\nsclera. In 8 cases, the pseudo-tendon was recessed, and in one\ncase resected. The revisions could be performed without\ncomplications. Recently, the same study group reported results for medial\nrectus tendon elongation after orbital decompression surgery\nin GO patients. All patients showed lower dose effects com-\npared with medial rectus muscle recessions without prior de-\ncompression. Considering patients with unilateral and bilater-\nal tendon elongation of the medial rectus muscle, a median\ndose-effect of 1.8 ± 0.6Δ/mm was achieved [14]. Unilateral\nrecessions had a mean effect of 1.2 ± 0.4°/mm (2.1Δ/mm),\nbilateral of 1.0 ± 0.3°/mm (1.8Δ/mm). Unilateral medial rec-\ntus muscle tendon elongation with simultaneous simple reces-\nsion of the medial rectus muscle on the partner eye had a mean\neffect of 0.92 ± 0.3°/mm (1.6Δ/mm) and bilateral tendon\nelongation of 0.87 ± 0.3°/mm (1.5Δ/mm) [15]. Interestingly, Other patients with horizontal rectus muscle tendon\nelongation Postoperatively, the depression was 10° over The child with Mobius syndrome had a reduction of the\nhorizontal angle from 25Δ to 0Δ the first day after surgery,\nbut an over-effect of −25Δ in the long term which required a\nrevision surgery. In one child with congenital fibrosis of the extraocular\nmuscles (patient 51), the head turn was reduced from 30° to\n0° and the squint angle was reduced from −100Δ to −55Δ in\nthe long term; respectively in another child (patient 50), the\nhead turn was reduced from 35° to < 5° and the squint angle\nfrom −30° to −20° in the long term (Hirschberg test, near\nfixation). Patient 50 had further surgery to correct patient’s\nvertical head turn. One adult patient had an orbital apex syndrome due to\nmetastasis of a leiomyosarcoma at the side of her only seeing\neye. Patient’s head turn to the right of 40° preoperatively was\nreduced to 20° on the first day after surgery. The squint angle\nshowed a reduction from 75Δ to 40Δ. Patient was not avail-\nable for long-term results. Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 152 Table 2\nPatients with vertical rectus muscle surgery to improve a vertical angle of squint and long-term follow-up: full extent of deviation in primary\nposition (in prism diopters, absolute values) preoperatively, at the first postoperative day, in the long term, and improvement (3 patients)\nPatients with long-term\nmeasurement\nVertical angle of squint (Δ)\nMedian (minimum to maximum)\nLong-term measurement\n(weeks)\nMedian (minimum\nto maximum)\nPreop. Postop. first day\nLong term\nDifference\npreop.-postop. first day\nDifference\npreop.- long term\nVertical rectus muscle surgery to\nimprove a vertical angle of squint\n3/3 patients\n30 (20 to 45)\n8 (3 to 18)\n4 (1 to 22)\n22 (17 to 27)\n19 (8 to 41)\n12 (9 to 40) Discussion In this study tendon elongation with bovine pericardium was\nconsidered for a heterogeneous group of patients which was\nunlikely to show satisfying results with conventional eye mus-\ncle surgery only. Thereby the spectrum of indications for this\nspecial procedure has been broadened [13–18]. 153 Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 esotropic DRS (type I) preoperatively showed a median ad-\nduction of 50° with a very limited median abduction of 0°. Postoperatively, the abduction improved to median 7°, but the\nmedian postoperative adduction declined to 20°. A median\nloss of adduction of 25° was detected. None of the other\ngroups in our study showed a median loss of more than 15°\nin motility. the effect was stronger for inferior rectus muscle than for\nmedial rectus muscle tendon elongation. Wipf et al. reported a much higher dose-effect relation of\n3.6Δ/mm surgery. Within their study only five GO patients\nwith bilateral medial rectus muscle elongation but up to 10\nyears follow-up were described. Four out of the patients had\nprevious orbital decompression surgery [18]. In DRS, secondary structural muscle alterations occur. The\nhypo- or aplasia of the 6th nerve and its nucleus cause para-\ndoxical innervation of the lateral rectus muscle by the 3rd\nnerve. The cocontraction of medial and lateral rectus muscle\nleads to secondary fibrotic alterations in the medial rectus\nmuscle. Interestingly, a similar loss in adduction was found\nin patients with GO, another disease with fibrotic/alterated\nmuscle structure [14]. For patients with GO and decompres-\nsion surgery, Oeverhaus et al. reported a substantial narrowing\nof the horizontal motility after tendon elongation of the medial\nrectus muscle: abduction was improved by 12° in the unilat-\neral and 20° in the bilateral group, while adduction deteriorat-\ned by 20° in the unilateral and 40° in the bilateral group [14]. Especially for correction of exotropia, Van Rijn et al. also\nreported duction limitations in the direction of the involved\nmuscle. The duction limitations “had little effect on the cos-\nmetic appearance and patient satisfaction and decreased dur-\ning follow-up” [16]. This data might implicate that postoper-\native duction limitations are most pronounced in restrictive\nmotility disorders. Van Rijn et al. Biocompatibility Beside the otherwise mostly positive reports about bovine\npericardium in ophthalmic surgery, there is also one publica-\ntion with a critical view by De Vries et al. [21]. Following\nserious complications for 4 cases were described after using\nthis material as a wrap for acrylic implants in enucleation\nsurgery: orbital cellulitis, extrusion, and rapid resorption. It\nis unknown whether the acrylic material or bovine pericardi-\num caused these complications. However, beyond this publication the literature does not\nreport gross complications, especially not after rectus muscle\ntendon elongation with bovine pericardium [13, 14, 16, 17]. One of our patients showed a prolonged wound healing,\nbut no infection nor implant rejection was detected when re-\nvising the muscle. However, beyond this publication the literature does not\nreport gross complications, especially not after rectus muscle\ntendon elongation with bovine pericardium [13, 14, 16, 17]. One of our patients showed a prolonged wound healing,\nbut no infection nor implant rejection was detected when re-\nvising the muscle. Discussion performed tendon elongation with bovine\npericardium in a heterogeneous group of patients with second-\nary or consecutive eso- and exotropia (7 esotropic, respective-\nly 31 exotropic patients, also including two patients with N III\nand two with N VI palsy, and one patient with hypotropia). They described the dose-effect of muscle elongation as gen-\nerally stronger than in conventional recessions [16]. Angle\nreductions from −21.8 ± 5.7° to −3.3 ± 5.9° were reported\nfor correction of exotropia, from + 19.1 ± 5.4° to + 0.2 ± 0.5°\nfor correction of esotropia. For esotropic DRS, our study showed a median effect of\n2.6Δ/mm (respectively 2.7Δ/mm considering only patients with\nprevious medial rectus muscle surgery). This effect was stronger\nthan the effect described for medial rectus recession without\ntendon elongation as a primary procedure (2Δ/mm surgery)\n[19]. Altogether, the effect of tendon elongation with bovine\npericardium showed a high variability, probably depending\non the underlying pathology and muscle structure. The effect\ntends to be stronger than in conventional eye muscle surgery. Our study is limited by the short follow-up time of median\n9 weeks post operation. Oeverhaus et al. described excellent\nlong-term stability (up to 15 years) for patients with Graves’\norbitopathy and medial rectus tendon elongation [14]. In\nneuroparalytic strabismus though, the paretic muscle can fur-\nther stretch out. In fibrosis, muscles may remain tight and in\nother subgroups lack of fusion may destabilize the eye posi-\ntion [20]. Therefore, these patient groups may be more prone\nto recurrence of a squint angle. This is also reflected in the\nnumber of previous surgery for each subgroup of our study. Patients with residual strabismus (most of them without any\nbinocular functions) and patients with N. III/N. VI paresis had Altogether, the effect of tendon elongation with bovine\npericardium showed a high variability, probably depending\non the underlying pathology and muscle structure. The effect\ntends to be stronger than in conventional eye muscle surgery. tends to be stronger than in conventional eye muscle surgery. Our study is limited by the short follow-up time of median\n9 weeks post operation. Oeverhaus et al. described excellent\nlong-term stability (up to 15 years) for patients with Graves’\norbitopathy and medial rectus tendon elongation [14]. In\nneuroparalytic strabismus though, the paretic muscle can fur-\nther stretch out. In fibrosis, muscles may remain tight and in\nother subgroups lack of fusion may destabilize the eye posi-\ntion [20]. Discussion Therefore, these patient groups may be more prone\nto recurrence of a squint angle. This is also reflected in the\nnumber of previous surgery for each subgroup of our study. Patients with residual strabismus (most of them without any\nbinocular functions) and patients with N. III/N. VI paresis had\nthe highest number of previous surgery and thus the highest\nrisk of recurrence of the squint angle. References 1. Li X, Guo Y, Ziegler KR, Model LS, Eghbalieh SD, Brenes RA,\nKim ST, Shu C, Dardik A (2011) Current usage and future direc-\ntions for the bovine pericardial patch. Ann Vasc Surg 25(4):561–\n568. https://doi.org/10.1016/j.avsg.2010.11.007 2. Parizek J, Mericka P, Husek Z, Suba P, Spacek J, Nemecek S,\nNemeckova J, Sercl M, Elias P (1997) Detailed evaluation of\n2959 allogeneic and xenogeneic dense connective tissue grafts (fas-\ncia lata, pericardium, and dura mater) used in the course of 20 years\nfor duraplasty in neurosurgery. Acta Neurochir (Wien) 139(9):827–\n838 Although the arc of contact is preserved or restituted, this\ntype of surgery—aiming at maximum effects—reduces the\nexcursion into the field of action of the elongated muscle. Dose finding can be difficult depending on the underlying\npathology. Patients also have to know that more than one\nintervention might be necessary for optimal results. 3. Gupta M, Lyon F, Singh AD, Rundle PA, Rennie IG (2007) Bovine\npericardium (Tutopatch) wrap for hydroxyapatite implants. Eye\n(Lond) 21(4):476–479. https://doi.org/10.1038/sj.eye.6702227 Author contributions All authors contributed to the study conception and\ndesign. Material preparation and data collection and analysis were per-\nformed by Andrea Hedergott, Ursula Pink-Theofylaktopoulos, Antje\nNeugebauer, and Julia Fricke. The first draft of the manuscript was written\nby Andrea Hedergott and all authors commented on previous versions of\nthe manuscript. All authors have read and approved the final manuscript. 4. Gupta M, Puri P, Rennie IG (2002) Use of bovine pericardium as a\nwrapping material for hydroxyapatite orbital implants. Br J\nOphthalmol 86(3):288–289. https://doi.org/10.1136/bjo.86.3.288 5. Quaranta L, Riva I, Floriani IC (2013) Outcomes of using a\nsutureless bovine pericardial patch graft for Ahmed glaucoma valve\nimplantation. Eur J Ophthalmol 23(5):738–742. https://doi.org/10. 5301/ejo.5000260 Funding Open Access funding enabled and organized by Projekt DEAL. Funding Open Access funding enabled and organized by Projekt DEAL. 6. Gayre GS, Debacker C, Lipham W, Tawfik HA, Holck D, Dutton\nJJ (2001) Bovine pericardium as a wrapping for orbital implants. Ophthalmic Plast Reconstr Surg 17(5):381–387 Data availability The authors have full control of all primary data and\nthey agree to allow Graefes Archive for Clinical and Experimental\nOphthalmology to review their data upon request 7. Alio JL, Rodriguez AE, Martinez LM (2013) Bovine pericardium\nmembrane (tutopatch) combined with solid platelet-rich plasma for\nthe management of perforated corneal ulcers. Cornea 32(5):619–\n624. https://doi.org/10.1097/ICO.0b013e31825a6d9a Conclusions This study shows that tendon elongation with bovine pericar-\ndium is a safe surgical method not only in Graves’\norbitopathy. Strabismus surgery with bovine pericardium in-\ntroduces new surgical options for complex and/or rare oculo-\nmotor dysfunctions, especially for complicated revisions and\nlarge angles of squint and residual head postures, where con-\nventional eye muscle surgery has limits. Side effects Large recessions with placement of a muscle posterior to the\nequator lead to limited ocular ductions [8]. Maintaining the arc\nof contact by tendon elongation instead of large or repeated\nrecessions could have less effect on motility. In our patients,\nthe motility range was shifted in the direction contrary to the\nelongated muscle in all groups of indications. Yet, a consid-\nerable reduction of the excursion into the field of action of the\nelongated muscle had to be registered. This effect was most\nprominent for patients with esotropic DRS. Our patients with Oeverhaus et al. described the non-absorbable sutures of\nthe implant to penetrate the conjunctiva over time. Therefore,\nthey recommended using absorbable sutures [15]. Awadein\net al. reported a high incidence of over-effect for large hang-\nback medial rectus muscle recessions with absorbable sutures. This was due to inadequate anchoring of the medial rectus\nmuscle [22]. The problem did not occur with non-absorbable\nsutures. Nevertheless, we also switched to absorbable sutures\nfor anchoring of the implant recently, but only one patient Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 154 study, the Declaration of Helsinki and applicable national regulations and\nlaws were observed. with absorbable sutures was considered in this study. Seven\nweeks after surgery, the patient did not have an over-effect. Future studies will need to assess the safety of absorbable\nsutures in tendon elongation with bovine pericardium. Consent to participate\nNot applicable Consent to participate\nNot applicable Consent for publication\nNot applicable Consent for publication\nNot applicable Code availability\nVia corresponding author Code availability\nVia corresponding author Code availability\nVia corresponding author Open Access This article is licensed under a Creative Commons\nAttribution 4.0 International License, which permits use, sharing,\nadaptation, distribution and reproduction in any medium or format, as\nlong as you give appropriate credit to the original author(s) and the\nsource, provide a link to the Creative Commons licence, and indicate if\nchanges were made. The images or other third party material in this article\nare included in the article's Creative Commons licence, unless indicated\notherwise in a credit line to the material. If material is not included in the\narticle's Creative Commons licence and your intended use is not\npermitted by statutory regulation or exceeds the permitted use, you will\nneed to obtain permission directly from the copyright holder. To view a\ncopy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Like other study groups, we observed the bovine pericardium\nto be eventually replaced by tendon-like tissue. It usually\nshowed no adherence to the sclera behind the insertion\n[14–16]. Nevertheless, Oeverhaus reported one case with re-\nvision surgery where the implant was not distinguishable from\nsurrounding fibrotic scar tissue. In our study, revision surgery\nwas feasible in all cases. Compliance with ethical standards 8. Coats D, Olitsky SE (2017) Recession of the rectus muscle. In:\nLambert S, Lyons CJ (eds) Taylor & Hoyt’s Pediatric\nOphthalmology and Strabismus, vol 5. Elsevier, Edinburgh,\nLondon, New York, Oxford, Philadelphia, St. Louis, Sydney,\nTotonto, p 885 Conflict of interest\nThe authors declare that they have no conflict of\ninterest. Ethics approval\nAccording to regional medical regulations on retrospec-\ntive single-center clinical studies (§15 of the Professional code of con-\nduct, General Medical Council for the Northern Rhine in accordance with\nthe General Data Protection Regulation GDPR of the European Union),\nthe Ethics Committee of the University of Cologne ruled that approval\nwas not required for this retrospective analysis. Throughout the whole 9. Talebnejad MR, Eghtedari M, Owji N, Alavi A (2008) Super\noblique tendon elongation with fascia lata. J AAPOS 12(5):507–\n509. https://doi.org/10.1016/j.jaapos.2008.02.014 10. Wright KW (2000) Results of the superior oblique tendon elonga-\ntion procedure for severe Brown's syndrome. Trans Am\nOphthalmol Soc 98:41–48. discussion 48-50 Graefes Arch Clin Exp Ophthalmol (2021) 259:145–155 155 11. Langmann A, Lindner S, Wackernagel W, Koch M, Horantner R\n(2006) Polytetrafluoroethylene (Goretex) for muscle elongation in\nthe surgical treatment of strabismus with restricted motility. Acta\nOphthalmol Scand 84(2):250–253. https://doi.org/10.1111/j.1600-\n0420.2005.00578.x 17. Hedergott AM, Fricke J, Neugebauer A (2014) Medial rectus ten-\ndon elongation with bovine pericardium for type 1 Duane’s retrac-\ntion syndrome. Klin Monbl Augenheilkd 231(10):980–987. https://\ndoi.org/10.1055/s-0034-1383070 18. Wipf M, Berg BI, Palmowski-Wolfe A (2018) Medial rectus tendon\nelongation with bovine pericard (Tutopatch(R)) in thyroid-\nassociated orbitopathy: a long-term follow-up including\noculodynamic MRI. J Ophthalmol 2018:1294761. https://doi.org/\n10.1155/2018/1294761 12. Dyer JA (1976) The oculorotary muscles in Graves’ disease. Trans\nAm Ophthalmol Soc 74:425–456 13. Esser J, Schittkowski M, Eckstein A (2011) Graves’ orbitopaty:\ninferior rectus tendon elongation for large vertical squint angles that\ncannot be corrected by simple muscle recession. Klin Monbl\nAugenheilkd 228(10):880–886. https://doi.org/10.1055/s-0031-\n1281776 19. Fricke J, Neugebauer A, Russmann W (2006) Surgical options in\nretraction syndrome. Klin Monbl Augenheilkd 223(1):42–47. https://doi.org/10.1055/s-2005-858857 20. Guyton DL (2006) The 10th Bielschowsky Lecture. Changes in\nstrabismus over time: the roles of vergence tonus and muscle length\nadaptation. Binocul Vis Strabismus Q 21(2):81–92 14. Oeverhaus M, Fischer M, Hirche H, Schluter A, Esser J, Eckstein\nAK (2018) Tendon elongation with bovine pericardium in patients\nwith severe esotropia after decompression in Graves’ orbitopathy-\nefficacy and long-term stability. Strabismus 26(2):62–70. https://\ndoi.org/10.1080/09273972.2018.1450430 21. Publisher’s note Springer Nature remains neutral with regard to jurisdic-\ntional claims in published maps and institutional affiliations. Compliance with ethical standards De Vries FR, Notting IC, Marinkovic M, Schalij-Delfos NE,\nLuyten GP (2012) Complications due to bovine pericardium used\nto cover acrylic implants after enucleation and tubes of aqueous\ndevices. Eye (Lond) 26(2):336. https://doi.org/10.1038/eye.2011. 294 15. Oeverhaus M, Fischer M, Schluter A, Esser J, Eckstein A (2018)\nCorrective surgery of severe esotropia after decompression in pa-\ntients with Graves’ orbitopathy - comparison of tendon elongation\nand recession of the medial rectus muscle. Klin Monbl Augenheilkd\n235(10):1105–1114. https://doi.org/10.1055/a-0719-5354 22. Awadein A, Marsh JD, Guyton DL (2016) Nonabsorbable versus\nabsorbable sutures in large, hang-back medial rectus muscle reces-\nsions. J AAPOS 20(3):206–209. https://doi.org/10.1016/j.jaapos. 2016.03.009 16. van Rijn LJ, van De Ven SJ, Krijnen JS, Jansen SM, Bakels AJ,\nLangenhorst AM (2016) Tendon elongation with bovine pericardi-\num (Tutopatch(R)) when conventional strabismus surgery is not\npossible. Eur J Ophthalmol 26(3):193–202. https://doi.org/10. 5301/ejo.5000689 Publisher’s note Springer Nature remains neutral with regard to jurisdic-\ntional claims in published maps and institutional affiliations."
https://openalex.org/W3009588415	https://www.research-collection.ethz.ch/bitstream/20.500.11850/413423/3/jcm-09-00720-v3.pdf	English	null	Corticospinal Control of Human Locomotion as a New Determinant of Age-Related Sarcopenia: An Exploratory Study	Journal of clinical medicine	2,020	cc-by	15,304	ETH Library Author(s): Gennaro, Federico; Maino, Paolo; Kaelin-Lang, Alain; De Bock, Katrien; de Bruin, Eling 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. Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Federico Gennaro 1,* , Paolo Maino 2 , Alain Kaelin-Lang 3,4,5, Katrien De Bock 1 and Eling D. de Bruin 1,6 Federico Gennaro 1,* , Paolo Maino 2 , Alain Kaelin-Lang 3,4,5, Katrien De Bock 1 and Eling D. de Bruin 1,6 1 Department of Health Sciences and Technology, Institute of Human Movement Sciences and Sport, ETH Zurich, 8093 Zurich, Switzerland; katrien-debock@ethz.ch (K.D.B.); eling.debruin@hest.ethz.ch (E.D.d.B.) 2 Pain Management Center, Neurocenter of Southern Switzerland, Regional Hospital of Lugano, 6962 Lugano S it l d l i @ h 1 Department of Health Sciences and Technology, Institute of Human Movement Sciences and Sport, ETH Zurich, 8093 Zurich, Switzerland; katrien-debock@ethz.ch (K.D.B.); eling.debruin@hest.ethz.ch (E.D.d.B.) 2 Pain Management Center, Neurocenter of Southern Switzerland, Regional Hospital of Lugano, 6962 Lugano, Switzerland; paolo.maino@eoc.ch 3 Neurocenter of Southern Switzerland, Regional Hospital of Lugano, 6900 Lugano, Switzerland; alain.kaelin@eoc.ch 4 Faculty of Biomedical Sciences, Università della Svizzera Italiana, 6900 Lugano, Switzerland 5 Medical faculty, University of Bern, 3008 Bern, Switzerland 6 Department of Neurobiology, Division of Physiotherapy, Care Sciences and Society, Karolinska Institutet, 171 77 Stockholm, Sweden 6 Department of Neurobiology, Division of Physiotherapy, Care Sciences and Society, Karolinska Institute 171 77 Stockholm, Sweden * Correspondence: federico.gennaro@hest.ethz.ch * Correspondence: federico.gennaro@hest.ethz.ch Keywords: sarcopenia; dynapenia; corticomuscular coherence; corticospinal control; connectivity; EEG; EMG; gait; walking; locomotion Corticospinal Control of Human Locomotion as a New Determinant of Age-Related Sarcopenia: An Exploratory Study Federico Gennaro 1,* , Paolo Maino 2 , Alain Kaelin-Lang 3,4,5, Katrien De Bock 1 and Eling D. de Bruin 1,6 1. Introduction Sarcopenia is an age-related progressive decline in skeletal muscle mass and function [1], which was only very recently oﬃcially recognized as a muscle disorder by the World Health Organization (WHO) with a speciﬁc code (M62.84) from the 10th revision of the International Classiﬁcation of Diseases (ICD-10) [2]. Since the ﬁrst term of sarcopenia was coined in 1988 [3], several sarcopenia deﬁnitions have been developed, among which the most used were developed by the European Working Group on Sarcopenia in Older People (EWGSOP) [4,5], the International Working Group on Sarcopenia (IWGS) [6], the Society of Sarcopenia, Cachexia and Wasting Disorders (SCWD) [7] and the Foundation for the National Institutes of Health Biomarkers Consortium Sarcopenia Project (FNIH) [8]. However, although some authors undertook comparisons between diﬀerent sarcopenia algorithms [9–11] a global consensus on the best deﬁnition is still lacking. Moreover, the Centers for Disease Control and Prevention (CDC, USA) has recently recognized that sarcopenia is in need of improved measures for diagnosis and screening [2]. Muscle weakness is well-known to place older adults at an increased risk of mobility limitations and mortality [12] and sarcopenia can be present in up to ~20% of community-dwelling older adults above 65 years old, and up to ~50% in those aged above 80 [2,6,13]. For several decades, it was initially believed that sarcopenia was mainly due to poor muscle mass. However, recent studies demonstrated that muscle atrophy is a relatively small contributor to the loss of muscle strength, since the latter is lost at a substantially faster rate than muscle atrophy and gaining muscle mass does not necessarily prevent the aging-related loss of muscle strength [14]. For this reason, the new term dynapenia (from Greek: dyna- = power/strength and -penia = loss) has been suggested to replace the traditionally used term sarcopenia (from Greek: sarco- = meat/muscle and -penia = loss) [1,14–16]. Interestingly, ongoing theoretical reasoning [17,18] and mounting evidence points to changes in central nervous system (CNS) function and/or the intrinsic force-generating properties of skeletal muscle as contributors to muscle weakness and motor dysfunction [18]. That is, a not well-functioning neuromuscular system in sarcopenia might be due to an impaired corticospinal interaction [18], which, in turn, may contribute to the muscle microenvironment leading to the loss of muscle mass, strength and functionality. Received: 21 January 2020; Accepted: 2 March 2020; Published: 6 March 2020 Abstract: Sarcopenia is a muscle disease listed within the ICD-10 classiﬁcation. Several operational deﬁnitions have been created for sarcopenia screening; however, an international consensus is lacking. The Centers for Disease Control and Prevention have recently recognized that sarcopenia detection requires improved diagnosis and screening measures. Mounting evidence hints towards changes in the corticospinal communication system where corticomuscular coherence (CMC) reﬂects an eﬀective mechanism of corticospinal interaction. CMC can be assessed during locomotion by means of simultaneously measuring Electroencephalography (EEG) and Electromyography (EMG). The aim of this study was to perform sarcopenia screening in community-dwelling older adults and explore the possibility of using CMC assessed during gait to discriminate between sarcopenic and non-sarcopenic older adults. Receiver Operating Characteristic (ROC) curves showed high sensitivity, precision and accuracy of CMC assessed from EEG Cz sensor and EMG sensors located over Musculus Vastus Medialis [Cz-VM; AUC (95.0%CI): 0.98 (0.92–1.04), sensitivity: 1.00, 1-speciﬁcity: 0.89, p < 0.001] and with Musculus Biceps Femoris [Cz-BF; AUC (95.0%CI): 0.86 (0.68–1.03), sensitivity: 1.00, 1-speciﬁcity: 0.70, p < 0.001]. These muscles showed signiﬁcant diﬀerences with large magnitude of eﬀect between sarcopenic and non-sarcopenic older adults [Hedge’s g (95.0%CI): 2.2 (1.3–3.1), p = 0.005 and Hedge’s g (95.0%CI): 1.5 (0.7–2.2), p = 0.010; respectively]. The novelty of this exploratory investigation is the hint toward a novel possible determinant of age-related sarcopenia, derived from corticospinal control of locomotion and shown by the observed large diﬀerences in CMC when sarcopenic and non-sarcopenic older adults are compared. This, in turn, might represent in future a potential treatment target to counteract sarcopenia as well as a parameter to monitor the progression of the disease and/or the potential recovery following other treatment interventions. Keywords: sarcopenia; dynapenia; corticomuscular coherence; corticospinal control; connectivity; EEG; EMG; gait; walking; locomotion J. Clin. Med. 2020, 9, 720; doi:10.3390/jcm9030720 www.mdpi.com/journal/jcm J. Clin. Med. 2020, 9, 720 2 of 22 2.1. Participants A total of ~2500 community-dwelling older adults were informed about the study through a letter with a pre-paid response letter send by ATTE, an association of older adults of the Canton Ticino (ATTE, Bellinzona, Switzerland) as well as through word of mouth and with the help of other local associations and institutions that were approached in person. Of these, ~500 community-dwelling older adults informed the experimenter to be interested in participating. Community-dwelling volunteers interested to participate were included if they were ≥65 years old, were able to walk without any walking aid and when having a body mass index (BMI) ≤30 kg/m2. Volunteers interested to participate were excluded if they had a self-reported history or clinical signs or symptoms of severe, uncontrolled or unstable diabetic, cardio-circulatory, respiratory, liver, renal, thyroidal, neurological, neuromuscular, peripheral arterial disease(s) or autoimmune disease. Moreover, they were excluded if they had recent lower limb fracture or lower limb surgeries in the previous 10 years or pain in either left or right lower limb or any other condition that could be considered a contraindication for muscle or walking/gait testing. Additionally, interested participants were excluded if they had a current or past (within 10 years) history of malignancy (excluding non-melanoma skin cancer). A total of 203 participants were included for the present study, however, of which ﬁve participants had to be excluded from further analysis for health reasons. Therefore, a total of ~198 participants (120 females; age: 73 ± 6 years; range: 65–97 years; Table 1) were included and completed all the experimental procedures. The study protocol was approved by the cantonal ethics committee of Ticino (2018-00040 CE 3316) and an informed consent in accordance with the Declaration of Helsinki was obtained signed by the participants before starting the experimental procedures. Table 1. Descriptive statistics of the study population represented as mean (± standard deviation). Table 1. Descriptive statistics of the study population represented as mean (± standard deviation). 1. Introduction Corticomuscular coherence (CMC) is a measure of synchronization between motor cortical areas and spinal motor neurons and it reﬂects an eﬀective mechanism of corticospinal interaction and central drive to skeletal muscle [19–23]. The loss of CMC has been deﬁned as “the hallmark of aging” [24], and a weaker synchronization between supraspinal and subspinal structures has been observed in older compared to young adults [25,26]. Walking is a precision task involving both motor and cognitive eﬀorts which become more demanding in older adults and, thus, impaired walking is believed to be one of the causes leading to an increased fall risk [27]. Gait is a locomotor task driven by neural inputs directed to skeletal muscles [28,29]. Interestingly, sarcopenic older adults, who often exhibit cognitive impairments [16,30–33], also show gait speed decline [34]. However, the neuronal and nervous dynamics occurring in older adults during walking are only partly investigated and understood. Investigating the corticospinal role in the control of gait can be achieved within the so-called mobile brain/body imaging (MoBI) research framework [35–37], which encompasses concurrent recordings of brain activity, for example by means of Electroencephalography (EEG), and other neurophysiological activity; e.g., muscle activity by means of Electromyography (EMG) or biomechanical parameters. The latter can be done by employing camera-based motion capture or through kinematic assessment using inertial measurement units. MoBI can help in ﬁnding insights about the corticospinal control available during locomotion tasks and how this relates to biomechanical, neurophysiological and/or cognitive aspects [38–40]. Corticospinal control of gait has been assessed in clinical settings in people suﬀering from spinal cord injury [41], and in neuromuscular diseases and movement disorders [42–46]. Moreover, corticospinal control of gait has been investigated also in healthy young and older participants [28,29,47–49]. When diﬀerent age groups are compared it seems that CMC is diﬀerently modulated in older compared to younger adults when diﬀerent gait task modalities are employed [48,50]. CMC assessed during walking may represent one approach that J. Clin. Med. 2020, 9, 720 3 of 22 could ﬁt the CDC recommendations [2] on improving the current state of the art of screening for and diagnosing of sarcopenia. Furthermore, a possible neurogenic contribution in this ICD-10 recognized muscle disorder may shed a light on a potentially disrupted pyramidal system as contributing factor related to the loss of muscle mass, strength and functionality in sarcopenia. 1. Introduction Measures of central drive to skeletal muscle, such as CMC, have been shown to potentially serve as putative biomarker able to distinguish several neurological and neuromuscular diseases, such as stroke [51], motor neuron disease [46] and dystonia [52]. However, to the best of our knowledge it seems that using neurophysiological measures of neuromuscular functioning to distinguish between sarcopenic and non-sarcopenic individuals is so far not available. Therefore, the aim of this exploratory observational study was to investigate the corticospinal control of gait and explore its possible relation with sarcopenia by comparing older adult individuals with against those without sarcopenia. We, furthermore, explore whether corticomuscular coherence between EEG and EMG recordings over several lower limbs muscles during gait can distinguish the presence of sarcopenia. 2.3.2. Muscle Strength Muscle strength was assessed using a handheld dynamometer (Saehan Co., Yangdeok-Dong, Masan, South Korea). Participants were comfortably seated holding the handgrip with the left/right hand with wrist in neutral position, thumb facing upward and resting the arm and forearm over the ipsilateral leg. The experimenter was always the same person seated in front of the participant and supporting the weight of the dynamometer during each muscle strength trial. The participant was asked to perform an isometric maximum voluntary contraction with each side (left/right) for ~5 s. Each participant always started with the right side and continued with the left-side as soon as the ﬁrst side trial was terminated. Participants rested ~120 s between right-left side pairs of trials and a total of three (pairs) of trials were performed. The highest value from all trials was used as muscle strength value. Verbal encouragement was provided during each muscle strength trial in a standardized fashion. 2.1. Participants Total (n = 198) Women (n = 120) Men (n = 78) Age (years) 73 (6) 73 (6) 74 (6) Height (m) 1.65 (0.09) 1.61 (0.06) 1.73 (0.06) Weight (kg) 67 (11) 62 (8) 75 (9) BMI (kg/m2) 24.5 (2.8) 24.1 (2.9) 25.2 (2.6) Total Body fat (kg) 19 (6) 20 (6) 18 (5) ALM (kg) 19 (4) 16 (2) 24 (3) Muscle strength (kg) 32 (10) 26 (5) 42 (7) Gait speed (m·s−1) 1.08 (0.21) 1.05 (0.21) 1.14 (0.20) ALM = Appendicular Lean Muscle Mass; BMI = Body Mass Index. J. Clin. Med. 2020, 9, 720 4 of 22 2.3.1. Body Composition Body composition, and speciﬁcally, skeletal muscle mass and total body fat were assessed by bioelectrical impedance analysis (BIA; Inbody 120, InBody Co., Ltd. Seoul, Korea). Skeletal muscle mass was represented by appendicular lean muscle mass (ALM), further adjusted by height squared or by BMI depending on the operational deﬁnition of sarcopenia (see Section 2.3.4. below). Participants were asked to stand barefoot on the BIA platform while holding with both hands the handlebar connected to the device after cleaning both hand palms and foot soles provided for this purpose by the BIA manufacturer. When possible, participants were asked to remove metallic objects and participants having a pacemaker were excluded from body composition and further measurements. Although BIA is not considered the “gold standard” muscle mass measuring method, it has been accepted as an alternative method to the reference (and more valid) dual-energy X-ray absorptiometry (DXA), representing a good trade-oﬀbetween method validity and clinical practice convenience [4,5,53,54]. 2.2. Experimental Protocol Experimental measurements were performed in two separate sessions. After signing the informed consent, height and weight were measured, and BMI was calculated for each participant dividing the weight in kilograms by the height in meters squared. Afterwards, for the purpose of screening for the presence of sarcopenia, three measurements were performed always in the following order: muscle strength, skeletal muscle mass and physical performance. In the second session, only the older adults identiﬁed as having sarcopenia (see Section 2.3) were asked to participate, together with a sample-matched control group chosen at random from the entire sample of participants (see Section 2.3. below). In this second session, CMC has been measured between EEG and EMG recorded from several lower limb muscles during overground walking (see Section 2.5.3. below). 2.3. Sarcopenia Screening 2.3. Sarcopenia Screening 2.3.4. Operational Deﬁnition of Sarcopenia Given that a “gold standard” operational deﬁnition of sarcopenia is still lacking, we decided to opt for using a multi-algorithms strategy to deﬁne and detect “sarcopenic” older adults from the participants of this study, in line with previous studies [55,56]. More speciﬁcally, we used the 1st and 2nd version of the EWGSOP, respectively EWGSOP1 [4] and EWGSOP2 [5], the IWGS [6], the SCWD [7] and the FNIH [8]. All these deﬁnitions take speciﬁc thresholds into account derivable from some parts or from all the measurements of skeletal muscle mass, muscle strength and physical performance taken from the individuals (see Table 2 for further details of thresholds used). Additionally, the EWGSOP1 was further employed by using three diﬀerent thresholds of ALM (see Table 2 for further details of thresholds used). We speciﬁcally used the ALM deﬁnition from Baumgartner (EWGSOP1BAUM) [57], the ﬁrst deﬁnition from Delmonico (EWGSOP1DELM1) [58] and the second deﬁnition from Delmonico (EWGSOP1DELM2) [59]. Table 2. EWGSOP = European Working Group on Sarcopenia in Older People; IWGS = International Working Group on Sarcopenia; SCWD = Society of Sarcopenia, Cachexia and Wasting Disorders; FNIH = Foundation for the National Institutes of Health Biomarkers Consortium Sarcopenia Project; ALM = Appendicular lean muscle mass calculated by summing lean muscle mass of upper and lower limbs; ALM/BMI = ALM adjusted by BMI; ALM/height2 = ALM adjusted by height squared; * ALMPLM = gender-speciﬁc predicted linear model of ALM following ALM linear regression by height and total body fat mass. ♀: ALMPLM = −13.21 + 14.76 × height + 0.23 × total fat mass \| ♂: ALMPLM = −22.59 + 24.21 × height + 0.21 × total fat mass. 2.3.3. Physical Performance Physical performance was assessed by measuring gait speed over a course of ~5 m. Participants were asked to walk at their natural self-paced gait speed by wearing comfortable shoes usually worn in their daily walking activities. High-heel shoes were not allowed for the gait speed test. The operator gave verbal instruction relative to both start and end of the gait trial, which was considered completed when participants performed three times the entire gait course of ~5 m. Gait speed was measured and recorded with an inertial measurement unit (G-WALK, BTS Bioengineering, Milan, Italy) placed on the lower back at the height of the second lumbar spinal process below the imaginary line connecting the left and right most prominent bone of the posterior superior iliac spine. J. Clin. Med. 2020, 9, 720 5 of 22 2.3.4. Operational Deﬁnition of Sarcopenia Operational Deﬁnition Skeletal Muscle Mass 1O Muscle Strength 2O Physical Performance 3O Deﬁnition Criteria Prevalence (%) Low ALM Cut-OﬀPoints Low Handgrip (kg) Low Gait Speed (m/s) FNIH ALM/BMI: ♀≤0.512 \| ♂≤0.789 ♀< 16 \| ♂< 26 — 1O + 2O — EWGSOP1BAUM ALM/height2: ♀≤5.45 kg/m2 \| ♂≤7.26 kg/m2 ♀< 20 \| ♂< 30 <0.8 1O + 2O + 3O 4 (~2) EWGSOP1DELM1 ALM/height2: ♀≤5.67 kg/m2 \| ♂≤7.25 kg/m2 ♀< 20 \| ♂< 30 <0.8 1O + 2O + 3O 6 (~3) EWGSOP1DELM2 ALM −ALMPLM < 20th percentile of the gender-speciﬁc * distribution of residuals ♀< 20 \| ♂< 30 <0.8 1O + 2O + 3O 8 (~4) EWGSOP2 ALM/height2: ♀≤6.00 kg/m2 \| ♂≤7.00 kg/m2 ♀< 16 \| ♂< 27 ≤0.8 1O + 2O + 3O 2 (~1) IWGS ALM/height2: ♀≤5.67 kg/m2 \| ♂≤7.23 kg/m2 — <1.0 1O + 3O 8 (~4) SCWD ALM/height2: ♀≤5.18 kg/m2 \| ♂≤6.81 kg/m2 — <1.0 1O + 3O 3 (~2) FNIH + EWGSOP1BAUMGARTNER + EWGSOP1DELMONICO1 + EWGSOP1DELMONICO2 + EWGSOP2 + IWGS + SCWD 17 (~9) 2.4. Experimental Protocol 2.4. Experimental Protocol Electrophysiologic measurements were carried out only in older adults diagnosed to be sarcopenic by the operational deﬁnitions of sarcopenia and a sample-matched randomly selected control group of older adults from the participants of this study. From the seventeen sarcopenic older adults found after screening, eleven participated at the second session of the electrophysiologic measurements. From the remaining six sarcopenic older adults two participants were not able to participate for health reasons and four participants for personal reasons. Therefore, the electrophysiologic assessments were performed in eleven sarcopenic older adults and eleven healthy older adults (SARCO: n = 11 and CTRL: n = 11, respectively; see Table 3 for further details). Cortical and muscular activity was recorded during overground walking in a ﬁgure-8 gait course. The ﬁgure-8 gait course was structured by two custom-built parallelepiped-shaped structures with an in-between distance of ~5 m. Participants were asked to walk continuously without stopping by turning around each of these two structures. On top of each structure, a big easy-to-spot arrow was placed to indicate the direction and side of turning (depicted in Figure 1). 6 of 22 6 f 24 J. Clin. Med. 2020, 9, 720 J Cli M d FOR Table 3. Descriptive statistics of the study population represented as mean (± standard deviation). und each of these two structures. On top of each structure, a big easy-to-spot arrow was plac Table 3. Descriptive statistics of the study population represented as mean (± standard deviation). 2.4. Experimental Protocol Sarcopenic (n = 11) Non-Sarcopenic (n = 11) Total (n = 11) Women (n = 9) Men (n = 2) Total (n = 11) Women (n = 6) Men (n = 5) Age (years) 75 (7) 73 (6) 85 (1) 72 (4) 74 (4) 71 (4) Height (m) 1.57 (0.09) 1.54 (0.07) 1.69 (0.04) 1.69 (0.07) 1.64 (0.07) 1.74 (0.03) Weight (kg) 57 (9) 55 (7) 68 (12) 69 (11) 64 (9) 75 (12) BMI (kg/m2) 23.2 (3.1) 23 (3.2) 23.9 (3.3) 24.2 (2.8) 23.7 (2.4) 24.8 (3.3) Total Body fat (kg) 18 (5) 18 (5) 20 (9) 18 (6) 19 (5) 17 (7) ALM (kg) 15 (3) 14 (2) 19 (2) 21 (4) 18 (3) 24 (2) Muscle strength (kg) 23 (5) 22 (4) 31 (4) 36 (11) 28 (4) 46 (6) Gait speed (m·s−1) 0.82 (0.10) 0.79 (0.09) 0.94 (0.04) 1.07 (0.08) 1.04 (0.07) 1.10 (0.08) ALM = Appendicular Lean Muscle Mass; BMI = Body Mass Index. around each of these two structures. On top of each structure, a big easy-to-spot arrow was placed to indicate the direction and side of turning (depicted in Figure 1). Table 3. Descriptive statistics of the study population represented as mean (± standard deviation). Sarcopenic (n = 11) Non-Sarcopenic (n = 11) Total (n = 11) Women (n = 6) Men (n = 5) Total (n = 11) Women (n = 6) Men (n = 5) Age (years) 75 (7) 73 (6) 85 (1) 72 (4) 74 (4) 71 (4) Height (m) 1.57 (0.09) 1.54 (0.07) 1.69 (0.04) 1.69 (0.07) 1.64 (0.07) 1.74 (0.03) Weight (kg) 57 (9) 55 (7) 68 (12) 69 (11) 64 (9) 75 (12) BMI (kg/m2) 23.2 (3.1) 23 (3.2) 23.9 (3.3) 24.2 (2.8) 23.7 (2.4) 24.8 (3.3) Total Body fat (kg) 18 (5) 18 (5) 20 (9) 18 (6) 19 (5) 17 (7) ALM (kg) 15 (3) 14 (2) 19 (2) 21 (4) 18 (3) 24 (2) Muscle strength (kg) 23 (5) 22 (4) 31 (4) 36 (11) 28 (4) 46 (6) Gait speed (m·s-1) 0.82 (0.10) 0.79 (0.09) 0.94 (0.04) 1.07 (0.08) 1.04 (0.07) 1.10 (0.08) ALM = Appendicular Lean Muscle Mass; BMI = Body Mass Index. Gait speed (m·s-1) 0.82 (0.10) 0.79 (0.09) 0.94 (0.04) 1.07 (0.08) 1.04 (0.07) 1.10 (0.08) ALM = Appendicular Lean Muscle Mass; BMI = Body Mass Index. gure 1. 2.4. Experimental Protocol A participant is depicted while approaching to turn around one of the two parallelepiped-shaped uctures with an easy-to-spot big black arrow on top showing the turning direction. The figure-8 gait path composed by two structures as depicted and walking is performed by turning around each structure ntinuously. The participant walked in the figure-8 gait course, while wearing an EEG cap and EMG nsors over eight muscles of both left and right leg. Moreover, two footswitches were placed under the le of the foot before wearing socks and shoes. The backpack served to store the amplifier of the EEG cap d additional elements (i.e., cables), however EEG signals were monitored real time remotely. Figure 1. A participant is depicted while approaching to turn around one of the two parallelepiped-shaped structures with an easy-to-spot big black arrow on top showing the turning direction. The figure-8 gait path is composed by two structures as depicted and walking is performed by turning around each structure continuously. The participant walked in the figure-8 gait course, while wearing an EEG cap and EMG sensors over eight muscles of both left and right leg. Moreover, two footswitches were placed under the sole of the foot before wearing socks and shoes. The backpack served to store the amplifier of the EEG cap and additional elements (i.e., cables), however EEG signals were monitored real time remotely. ure 1. A participant is depicted while approaching to turn around one of the two parallelepiped-shaped ctures with an easy-to-spot big black arrow on top showing the turning direction. The figure-8 gait path omposed by two structures as depicted and walking is performed by turning around each structure inuously. The participant walked in the figure-8 gait course, while wearing an EEG cap and EMG ors over eight muscles of both left and right leg. Moreover, two footswitches were placed under the of the foot before wearing socks and shoes. The backpack served to store the amplifier of the EEG cap additional elements (i.e., cables), however EEG signals were monitored real time remotely. Figure 1. A participant is depicted while approaching to turn around one of the two parallelepiped-shaped structures with an easy-to-spot big black arrow on top showing the turning direction. The figure-8 gait path is composed by two structures as depicted and walking is performed by turning around each structure continuously. 2.4. Experimental Protocol The participant walked in the figure-8 gait course, while wearing an EEG cap and EMG sensors over eight muscles of both left and right leg. Moreover, two footswitches were placed under the sole of the foot before wearing socks and shoes. The backpack served to store the amplifier of the EEG cap and additional elements (i.e., cables), however EEG signals were monitored real time remotely. Participants started the gait trial from one of these two structures, which was always kept the same, and whenever they were comfortable and ready to start. Start was always after a verbal “start” call and subjects were expected to walk continuous until a subsequent verbal “stop” call was verbally expressed by the experimenter Participants were asked to walk at a self-selected preferred walking Participants started the gait trial from one of these two structures, which was always kept the same, and whenever they were comfortable and ready to start. Start was always after a verbal “start” call and subjects were expected to walk continuous until a subsequent verbal “stop” call was verbally 7 of 22 J. Clin. Med. 2020, 9, 720 expressed by the experimenter. Participants were asked to walk at a self-selected preferred walking speed. The gait trial was considered completed when the participant performed a total of 15 ﬁgure-8 loops. A total of three trials were performed with an in-between rest of ~5 min. Counting of ﬁgure-8 gait loops was performed by the experimenter and not by the participant in order to avoid any possible dual-task cognitive additional load. A tape was applied on the ground at ~1 m distance from the structure to manually trigger beginning and ending of straight walking parts of the gait path by manually pressing computer keyboard speciﬁc keys. Participants were instructed to walk naturally as soon as possible to maintain ecological validity of the experimental protocol, but, at the same time, they were asked to maintain their gaze straight towards the arrow placed on top of each structure in front of them as much as possible during the straight part of the walking trial. When walking the curved part of the ﬁgure-8 path no instruction relative to the gaze was provided. 2.5.1. Data Acquisition Surface EEG activity was recorded at a sampling frequency of 1000 Hz by a high-density 64-channel EEG system (eego sport, ANT Neuro, Enschede, The Netherlands). Three EEG cap sizes were employed in order to accommodate diﬀerent head circumferences (waveguard, ANT Neuro, Enschede, The Netherlands) and EEG electrodes were placed according the 10-10 international system [60]. EEG reference and ground were placed over the left and right mastoid, respectively. An electrodes impedance ≤5 kΩwas required before EEG recordings. Surface EMG activity was recorded at a sampling frequency of 1000 Hz (FREEEMG 1000, BTS Bioengineering, Milan, Italy) by means of pairs of bipolar Ag-AgCl electrodes (H124SG Covidien, Minneapolis, MN, USA) placed with an inter-electrodes distance of ~2 cm accordingly to SENIAM guidelines [61] over eight muscles of interest in both left and right leg: Vastus Lateralis (VL), Vastus Medialis (VM), Rectus Femoris (RF), Biceps Femoris (BF), Tibialis Anterior (TA), Soleus (SOL), Gastrocnemius Medialis (GM) and Gastrocnemius Lateralis (GL). The skin was properly cleaned and, when necessary, shaved before placing the EMG sensors. The same inertial measurement unit (G-WALK, BTS Bioengineering, Milan, Italy) used for gait speed analysis during the screening of sarcopenia in the ﬁrst testing session was utilized also during this measurement but not further analyzed for the present study. Heel Strike’s onsets were detected by placing two footswitches approximately on the midpoint of the calcaneus in each foot. The two sensors were stacked on top of each other in order to provide a redundant backup of the heel strike onsets recordings, in case one of the two footswitch had technical problems. EEG and EMG recordings were synchronized by sending an analog square wave pulse to both EEG and EMG system from a custom-made device equipped with Transistor-Transistor-Logic (TTL) ports in order to align both time series in the subsequent data analysis. 2.5. Electrophysiology 2.5. Electrophysiology 2.4. Experimental Protocol Before executing the gait trial, participants were asked to perform a familiarization walking trial of ~5 min as warm up, followed by a ~2 min standing EEG recordings which preceded the beginning of the ﬁrst gait trial and served for further analysis and data preprocessing steps. 2.5.2. Data Pre-Processing All signal processing was performed using custom-made scripts and Fieldtrip, an open-source toolbox for electrophysiological data analysis [62] for Matlab (Mathworks Inc., Natick, MA, USA). An overview of the adopted pipeline for data pre-processing, including spectral analysis (see Section 2.5.3. below) can be retrieved in Figure 2. After alignment of the EEG and EMG data according to the TTL pulse, EMG data was high pass ﬁltered (two pass Butterworth ﬁlter, 4th order, 20 Hz cutoﬀ) and powerline noise, as well as its harmonics were ﬁltered out using a notch ﬁlter based on Discrete Fourier Transformation (DFT). Filtered EMG data was then full wave rectiﬁed using the Hilbert transform as a widely used preprocessing step before undertaking further coherence analysis [63]. Only straight parts of the ﬁgure-8 gait path were hence considered for analysis of the 8 of 22 J. Clin. Med. 2020, 9, 720 aligned EEG/EMG data. After removing mastoid electrodes from further analysis (M1 and M2), EEG data was then bandpass ﬁltered (two pass blackman-windowed sync ﬁlter, 5500 order, 1.5–48 Hz cutoﬀ) and concurrently demeaned as well as detrended. Powerline noise and harmonics were ﬁltered out as described above. Noisy channels were detected and removed if they were ﬂat for > 5 s or the correlation between neighboring channels was < 0.6. On average, ~1 channel was removed. Artifactual activity (e.g., movement artifacts) was attenuated with the following strategy. Firstly, a non-stationary method was employed to clean occasionally large amplitude noise and increase the stationarity of EEG data in preparation of the next Independent Component Analysis (ICA) cleaning step. For this purpose, a sliding window adaptive approach based on Principal Component Analysis (PCA) decomposition was used by means of the Riemannian modiﬁed version of the Artifact Subspace Reconstruction (rASR) method [64]. The entire data was used as calibration data and a lax threshold was chosen as parameter (30 standard deviations), as previously recommended, to be large enough to reduce artifactual activity from EEG data while preserving cerebral activity [29,65]. The combined used of ICA and Artifact Subspace Reconstruction has been suggested to represent an eﬀective strategy to remove artifactual signals from EEG data [66] and it has been largely used in studies involving cleaning of EEG data acquired during human locomotion tasks such as gait [29,37,67–70]. Portions of data not completely repaired by rASR were removed if more than 30% of channels were noisy in that data segment. 2.5.2. Data Pre-Processing Previously rejected noisy channels were then interpolated using spline interpolation and afterwards EEG data was re-referenced to an average reference and then EEG signals were decomposed into temporally maximally independent components (ICs) by applying on the remaining rank of the data Adaptive Mixture ICA (AMICA) with enabled online artifacts rejection using a threshold of ﬁve standard deviations in ﬁve iterations intervals starting after the ﬁrst ﬁve iterations and the whole procedure repeated ﬁve times. AMICA algorithm was chosen given that it has been shown to outperform other ICA algorithms [71]. After AMICA, a machine learning based approach was used to identify cerebral ICs by employing the ICLabel classiﬁer [72]. On average, ~6 cerebral ICs were identiﬁed by ICLabel which was in line with the suggested ~5–15 range of brain ICs that can be detected reliably [73]. The respective ICA weights and spheres matrices of the retained cerebral ICs were conveyed to an EEG dataset identical but processed using a more conventional ﬁltering approach (high pass ﬁlter: two pass hamming-windowed sync ﬁlter, 6600 order, cutoﬀ0.5 Hz; powerline noise ﬁltered as in the EMG analysis described above). In the present study, we have chosen to focus on the EEG Cz electrode for further spectral analysis of CMC. This vertex located sensor is widely employed to assess CMC during gait, as well as during isometric contraction tasks using lower limbs’ muscle, such as ankle dorsiﬂexors [28,47,48,50,74–77] 9 of 22 J. Clin. Med. 2020, 9, 720 J. Clin. Med. 2020, 9, x FOR PEER REVIEW 9 of 24 Figure 2. Overview of the pipeline adopted to perform data pre-processing and spectral analysis; Data pre-processing steps are depicted by the boxes, whereas spectral analysis steps are depicted by greyish boxes. TTL: Transistor-Transistor Logic; EEG: Electroencephalography; EMG: E lectromyography; DFT: Discrete Fourier Transform; ASR: Artifact Subspace Reconstruction; ICA: Independent component analysis; MTMFFT: multitaper frequency transformation. 2.5.3. Spectral Analysis Figure 2. Overview of the pipeline adopted to perform data pre-processing and spectral analysis; Data pre-processing steps are depicted by the boxes, whereas spectral analysis steps are depicted by greyish boxes. TTL: Transistor-Transistor Logic; EEG: Electroencephalography; EMG: E lectromyography; DFT: Discrete Fourier Transform; ASR: Artifact Subspace Reconstruction; ICA: Independent component analysis; MTMFFT: multitaper frequency transformation. Figure 2. Overview of the pipeline adopted to perform data pre-processing and spectral analysis; Data pre-processing steps are depicted by the boxes, whereas spectral analysis steps are depicted by greyish boxes. 2.5.2. Data Pre-Processing TTL: Transistor-Transistor Logic; EEG: Electroencephalography; EMG: E lectromyography; DFT: Discrete Fourier Transform; ASR: Artifact Subspace Reconstruction; ICA: Independent component analysis; MTMFFT: multitaper frequency transformation. Figure 2. Overview of the pipeline adopted to perform data pre-processing and spectral analysis; Data pre-processing steps are depicted by the boxes, whereas spectral analysis steps are depicted by greyish boxes. TTL: Transistor-Transistor Logic; EEG: Electroencephalography; EMG: E lectromyography; DFT: Discrete Fourier Transform; ASR: Artifact Subspace Reconstruction; ICA: Independent component analysis; MTMFFT: multitaper frequency transformation. 2.5.3. Spectral Analys The lea ed e 2.5.3. Spectral Analysis The cleaned preprocessed EEG and EMG data were then segmented according to the swing phase from 650-ms to 50-ms before heel strike onsets in analogy to previous studies[28,47,48,76,77], avoiding inclusion of any potentially remaining artifact due to the collision of the foot with the ground. On average, ~212 gait segments, considering the sum of any left and right heel strikes, were used for coherence estimation. Spectral analysis was performed as previously described[76]. Briefly, data segments were zero-padded up to 2 s and tapered with a variable set of discrete prolate spheroidal (Slepian) sequences by applying a multi taper frequency transform yielding to a broad 1– 60 Hz frequency band power- and cross-spectra with a frequency resolution set to 1 Hz. The frequencies of interest (FOI) for this analysis focused on the entire beta frequency band (i.e., 13–35 The cleaned preprocessed EEG and EMG data were then segmented according to the swing phase from 650-ms to 50-ms before heel strike onsets in analogy to previous studies [28,47,48,76,77], avoiding inclusion of any potentially remaining artifact due to the collision of the foot with the ground. On average, ~212 gait segments, considering the sum of any left and right heel strikes, were used for coherence estimation. Spectral analysis was performed as previously described [76]. Brieﬂy, data segments were zero-padded up to 2 s and tapered with a variable set of discrete prolate spheroidal (Slepian) sequences by applying a multi taper frequency transform yielding to a broad 1–60 Hz frequency band power- and cross-spectra with a frequency resolution set to 1 Hz. The frequencies of interest (FOI) for this analysis focused on the entire beta frequency band (i.e., 13–35 Hz) gathered with the lower bound of the gamma frequency band (i.e., 36–48 Hz), since it appears that, at individual level, the maximum amount of coherence (e.g., peak) related to the stance phase of gait that can be present in 10 of 22 J. Clin. Med. 2020, 9, 720 a beta-to-lower gamma FOI (i.e., ~13–50 Hz) [28,48,78]. Therefore, we adopted ten tapers resulting in a spectral smoothing of ±9 Hz. With this strategy, we assured to encompass our beta-to-lower gamma FOI using a total bandwidth of ~18 Hz and, therefore, including the entire beta frequency band, where the entire bandwidth is usually found to be ~10 Hz, but also, partially, the gamma frequency band, where the entire bandwidth is reported to be ~25 Hz [23]. 2.6. Statistics In order to test the ability of CMC in distinguishing between sarcopenic and not sarcopenic older adults, Receiver Operating Characteristics (ROC) and relative accuracy (i.e., Area Under the Curve; AUC) statistics was estimated using easyROC [82]. This statistic procedures allowed to determine precision (i.e., 1-speciﬁcity), sensitivity and AUC with the following equations: 1 −Speciﬁcity = Tp Tp + Fp (1) Sensitivity = Tp Tp + Fn (2) AUC = Tp + Tn Tp + Tn + Fn + Fp (3) (1) (2) AUC = Tp + Tn Tp + Tn + Fn + Fp (3) (3) where Tp denotes true positives (i.e., sarcopenic older adults in this study), Fp denotes false negative and Tn denotes true negatives (i.e., healthy control older adults in this study). Standard Error (SE) and Conﬁdence Intervals of the AUC was estimated using the nonparametric approach of DeLong (1998) and signiﬁcance level was set to α = 0.05. Additionally, the Yoden method has been employed in order to estimate optimal cut-oﬀpoints and criterion from ROC curves. Furthermore, CMC estimates were compared between groups as well as relative eﬀect sizes using DABEST, a data analysis strategy, which uses estimation statistics [83]. Estimation statistics is considered a superior statistic compared to dichotomous signiﬁcance testing, focusing on eﬀect sizes and relative precision [83]. P-values are reported respective the observed eﬀect size and conﬁdence intervals (CI). Estimation of the 95% CI mean diﬀerence was calculated by performing 5000 bootstrapping resamples. For the between groups comparisons, a non-parametric Mann-Whitney test was used within the Data Analysis with Bootstrapped ESTimation (DABEST) framework. Magnitude of the eﬀect was calculated as Hedge’s g, which is similar to Cohen’s d, but corrected for small-sample bias. As in Cohen’s d, an eﬀect size ≥0.2 was considered small, an eﬀect size ≥0.5 medium and an eﬀect size ≥0.8 was considered large. where Tp denotes true positives (i.e., sarcopenic older adults in this study), Fp denotes false negative and Tn denotes true negatives (i.e., healthy control older adults in this study). Standard Error (SE) and Conﬁdence Intervals of the AUC was estimated using the nonparametric approach of DeLong (1998) and signiﬁcance level was set to α = 0.05. Additionally, the Yoden method has been employed in order to estimate optimal cut-oﬀpoints and criterion from ROC curves. 2.6. Statistics Furthermore, CMC estimates were compared between groups as well as relative eﬀect sizes using DABEST, a data analysis strategy, which uses estimation statistics [83]. Estimation statistics is considered a superior statistic compared to dichotomous signiﬁcance testing, focusing on eﬀect sizes and relative precision [83]. P-values are reported respective the observed eﬀect size and conﬁdence intervals (CI). Estimation of the 95% CI mean diﬀerence was calculated by performing 5000 bootstrapping resamples. For the between groups comparisons, a non-parametric Mann-Whitney test was used within the Data Analysis with Bootstrapped ESTimation (DABEST) framework. Magnitude of the eﬀect was calculated as Hedge’s g, which is similar to Cohen’s d, but corrected for small-sample bias. As in Cohen’s d, an eﬀect size ≥0.2 was considered small, an eﬀect size ≥0.5 medium and an eﬀect size ≥0.8 was considered large. 2.5.3. Spectral Analys The lea ed e 2.5.3. Spectral Analysis Furthermore, it has been shown that the central drive to muscles during gait is largely and broadly present in this FOI [28,41,47,48,77,79–81]. Coherence estimates were considered signiﬁcant if they exceeded a conﬁdence limit (CL) with a probability of 95% (α = 0.05), related to the number of segments used for the coherence calculation (i.e., heel strikes, which consisted in a variable quantity for each participant, multiplied by the number of tapers used in the multi-tapered spectral analysis). CMC was estimated for the left and right side separately, to take into account the unequal number of segments between sides and per participant. However, for further statistics analysis, the maximum CMC estimate between left and right side was used, and, in case only one side was used for spectral analysis (i.e., because of technical problems in a speciﬁc footswitch and side), then only one side was considered for further analysis. 3. Results ROC curve analysis of the logarithmically transformed sum of coherence above signiﬁcant conﬁdence limits, between EEG Cz sensors and eight lower limbs muscle, yielded to signiﬁcant accuracy above 85% of chance in diﬀerentiating between sarcopenic and non-sarcopenic older adults, with high sensitivity and precision, when EEG Cz sensor is coupled with Vastus Medialis [Cz-VM; AUC (95.0%CI): 0.98 (0.92–1.04), sensitivity: 1.00, 1-speciﬁcity: 0.89, p < 0.001] or with Biceps Femoris [Cz-BF; 11 of 22 J. Clin. Med. 2020, 9, 720 AUC (95.0%CI): 0.86 (0.68–1.03), sensitivity: 1.00, 1-speciﬁcity: 0.70, p < 0.001]. For these two CMC estimates, Yoden methods suggested optimal cut-oﬀpoint of the log-transformed sum of coherence for diﬀerentiating sarcopenic from non-sarcopenic older adults [−5.2 and −6.2, respectively] with an estimated cut-oﬀoptimal criterion of 0.89 and 0.70, respectively. Moreover, Cz-VM and Cz-BF showed to be signiﬁcantly diﬀerent between sarcopenic and not sarcopenic older adults with large eﬀect sizes in the DABEST analysis [Hedge’s g (95.0%CI): 2.2 (1.3–3.1), p = 0.005 and Hedge’s g (95.0%CI): 1.5 (0.7–2.2), p = 0.010; respectively]. In all the other EEG Cz sensor–EMG electrode pair, ROC curves analysis did not show accuracy chance above 50% and signiﬁcant or DABEST large eﬀects sizes with signiﬁcant results. ROC curve and DABEST analysis are depicted for the group of muscles located in the upper part of the lower limbs and for the group of muscles located in the lower part of the leg in Figures 3 and 4, respectively. ROC curve and DABEST analysis are extensively presented for all EEG Cz sensor-muscle pairs in Table 4. Figure 3. Results relative to the log-transformed coherence area (sum of coherence above signiﬁcant conﬁdence limits) between the EEG Cz sensor and the muscles located in the upper part of the lower limb: Vastus Lateralis (Cz-VL), Vastus Medialis (Cz-VM), Biceps Femoris (Cz-BF) and Rectus Femoris (Cz-RF). (A) shows the ROC curve with the respective accuracy (i.e., AUC estimate) depicted within the plot, precision (i.e., 1-speciﬁcity) shown on the x-axis while sensitivity presented in the y-axis. The dotted diagonal line depicts the 50% chance of diﬀerentiating between sarcopenic and non-sarcopenic older adults. (B) Cumming estimation plots showing mean diﬀerences of the log-transformed coherence area plotted in the upper axes separately for both sarcopenic (SARC, darkish color) and healthy control older adults (CTRL, light color). 3. Results Each mean diﬀerence is represented by dots and plotted on the (C) lower axes as a bootstrap sampling distribution, while the ends of the vertical error bars denote the 95% conﬁdence intervals. Figure 3. Results relative to the log-transformed coherence area (sum of coherence above signiﬁcant conﬁdence limits) between the EEG Cz sensor and the muscles located in the upper part of the lower limb: Vastus Lateralis (Cz-VL), Vastus Medialis (Cz-VM), Biceps Femoris (Cz-BF) and Rectus Femoris (Cz-RF). (A) shows the ROC curve with the respective accuracy (i.e., AUC estimate) depicted within the plot, precision (i.e., 1-speciﬁcity) shown on the x-axis while sensitivity presented in the y-axis. The dotted diagonal line depicts the 50% chance of diﬀerentiating between sarcopenic and non-sarcopenic older adults. (B) Cumming estimation plots showing mean diﬀerences of the log-transformed coherence area plotted in the upper axes separately for both sarcopenic (SARC, darkish color) and healthy control older adults (CTRL, light color). Each mean diﬀerence is represented by dots and plotted on the (C) lower axes as a bootstrap sampling distribution while the ends of the vertical error bars denote the 95% Figure 3. Results relative to the log-transformed coherence area (sum of coherence above signiﬁcant conﬁdence limits) between the EEG Cz sensor and the muscles located in the upper part of the lower limb: Vastus Lateralis (Cz VL) Vastus Medialis (Cz VM) Biceps Femoris (Cz BF) and Rectus Femoris Figure 3. Results relative to the log-transformed coherence area (sum of coherence above signiﬁcant conﬁdence limits) between the EEG Cz sensor and the muscles located in the upper part of the lower limb: Vastus Lateralis (Cz-VL), Vastus Medialis (Cz-VM), Biceps Femoris (Cz-BF) and Rectus Femoris (Cz-RF). (A) shows the ROC curve with the respective accuracy (i.e., AUC estimate) depicted within the plot, precision (i.e., 1-speciﬁcity) shown on the x-axis while sensitivity presented in the y-axis. The dotted diagonal line depicts the 50% chance of diﬀerentiating between sarcopenic and non-sarcopenic older adults. (B) Cumming estimation plots showing mean diﬀerences of the log-transformed coherence area plotted in the upper axes separately for both sarcopenic (SARC, darkish color) and healthy control older adults (CTRL, light color). Each mean diﬀerence is represented by dots and plotted on the (C) lower axes as a bootstrap sampling distribution, while the ends of the vertical error bars denote the 95% conﬁdence intervals. 12 of 22 12 of 22 J. Clin. Med. 3. Results 2020, 9, 720 Figure 4. Results relative to the log-transformed coherence area (sum of coherence above significant confidence limits) between the EEG Cz sensor and the muscles located in the lower part of the lower limb: Tibialis Anterior (Cz-TA), Gastrocnemius Lateralis (Cz-GL), Gastrocnemius Medialis (Cz- GM)and Soleus (Cz-SOL). (A) shows the ROC curve with the respective accuracy (i.e., AUC estimate) depicted within the plot, precision (i.e., 1-specificity) shown on the x-axis while sensitivity presented in the y-axis. The dotted diagonal line depicts the 50% chance of differentitating between sarcopenic and non-sarcopenic older adults. (B) Cumming estimation plots showing mean differences of the log- transformed coherence area plotted in the upper axes separately for both sarcopenic (SARC, darkish color) and healthy control older adults (CTRL, lightish color). Each mean difference is represented by dots and plotted on the (C) lower axes as a bootstrap sampling distribution, while the ends of the vertical error bars denote the 95% confidence intervals. 4. Discussion Figure 4. Results relative to the log-transformed coherence area (sum of coherence above signiﬁcant conﬁdence limits) between the EEG Cz sensor and the muscles located in the lower part of the lower limb: Tibialis Anterior (Cz-TA), Gastrocnemius Lateralis (Cz-GL), Gastrocnemius Medialis (Cz-GM) and Soleus (Cz-SOL). (A) shows the ROC curve with the respective accuracy (i.e., AUC estimate) depicted within the plot, precision (i.e., 1-speciﬁcity) shown on the x-axis while sensitivity presented in the y-axis. The dotted diagonal line depicts the 50% chance of diﬀerentitating between sarcopenic and non-sarcopenic older adults. (B) Cumming estimation plots showing mean diﬀerences of the log-transformed coherence area plotted in the upper axes separately for both sarcopenic (SARC, darkish color) and healthy control older adults (CTRL, lightish color). Each mean diﬀerence is represented by dots and plotted on the (C) lower axes as a bootstrap sampling distribution, while the ends of the vertical error bars denote the 95% conﬁdence intervals. Figure 4. Results relative to the log-transformed coherence area (sum of coherence above significant confidence limits) between the EEG Cz sensor and the muscles located in the lower part of the lower limb: Tibialis Anterior (Cz-TA), Gastrocnemius Lateralis (Cz-GL), Gastrocnemius Medialis (Cz- GM)and Soleus (Cz-SOL). (A) shows the ROC curve with the respective accuracy (i.e., AUC estimate) depicted within the plot, precision (i.e., 1-specificity) shown on the x-axis while sensitivity presented in the y-axis. 3. Results The dotted diagonal line depicts the 50% chance of differentitating between sarcopenic and non-sarcopenic older adults. (B) Cumming estimation plots showing mean differences of the log- transformed coherence area plotted in the upper axes separately for both sarcopenic (SARC, darkish color) and healthy control older adults (CTRL, lightish color). Each mean difference is represented by dots and plotted on the (C) lower axes as a bootstrap sampling distribution, while the ends of the vertical error bars denote the 95% confidence intervals. 4. Discussion Figure 4. Results relative to the log-transformed coherence area (sum of coherence above signiﬁcant conﬁdence limits) between the EEG Cz sensor and the muscles located in the lower part of the lower limb: Tibialis Anterior (Cz-TA), Gastrocnemius Lateralis (Cz-GL), Gastrocnemius Medialis (Cz-GM) and Soleus (Cz-SOL). (A) shows the ROC curve with the respective accuracy (i.e., AUC estimate) depicted within the plot, precision (i.e., 1-speciﬁcity) shown on the x-axis while sensitivity presented in the y-axis. The dotted diagonal line depicts the 50% chance of diﬀerentitating between sarcopenic and non-sarcopenic older adults. (B) Cumming estimation plots showing mean diﬀerences of the log-transformed coherence area plotted in the upper axes separately for both sarcopenic (SARC, darkish color) and healthy control older adults (CTRL, lightish color). Each mean diﬀerence is represented by dots and plotted on the (C) lower axes as a bootstrap sampling distribution, while the ends of the vertical error bars denote the 95% conﬁdence intervals. J. Clin. Med. 2020, 9, 720 13 of 22 Table 4. Descriptive Statistics of the logarithmically transformed sum of coherence above signiﬁcant conﬁdence limits reported as mean (standard deviation) and results of the Receiver Operating Characteristic (ROC) curve analysis as well as DABEST comparisons with respective eﬀect sizes. 4. Discussion Corticomuscular coherence represents an eﬀective mechanism of corticospinal interaction and central drive to skeletal muscle [19–23] and it can be assessed during locomotor tasks such as walking [28,29,35], where it has been observed to diﬀerently modulate in older compared to younger adults [24–26,48,50]. Measures of central drive to skeletal muscle (e.g., corticomuscular coherence) showed to represent putative biomarkers able to distinguish several neurological and neuromuscular diseases, such as stroke [51], motor neuron disease [46], and dystonia [52]. The quality of walking has been recognized as an important biomarker of mortality and fall risk in aging [84]. The aim of this study was to perform screening of sarcopenia in community-dwelling older adults and then comparing sarcopenic with non-sarcopenic older adults using corticospinal control of locomotion by means of corticomuscular coherence between motor scalp electrocortical activity and skeletal muscles electrical activity of the lower limbs recorded during gait. The results show, to the best of our knowledge for the ﬁrst time, that corticomuscular coherence between EEG Cz sensor and EMG electrodes located either on the Vastus Medialis or Biceps Femoris muscles may serve to diﬀerentiate the presence of sarcopenia with high accuracy, precision and sensitivity. The ROC results of our explorative study are supported by the observed additional results showing that CMC relative to these muscles are signiﬁcantly diﬀerent coupled with a large magnitude of eﬀect when comparing sarcopenic and healthy control older adults, as shown by the Data Analysis with Bootstrapped ESTimation (DABEST) analysis. CMC is a linear measure indicating higher synchronization between motor cortical areas and skeletal muscles as it increases. This has been shown for example by observing motor learning induced increase of CMC estimates in healthy adults [85,86], but also in central drive to ankle dorsiﬂexors that is enhanced in older adults following a training period with exergames [80]. Both these ﬁndings are hinting towards an amelioration of the corticospinal interaction. However, one may think that an increasing value of CMC would indicate a better functioning or communicating neuromuscular system while a reduced CMC value would be pointing toward the opposite. In the available literature using similar assessment approaches this was, however, not always the case. CMC may lead to reduced CMC values in diseased populations; e.g., in stroke patients [51,87–91] and in Parkinson’s Disease patients [92]. 3. Results Log (Sum of Coherence) Sensitivity 1-Speciﬁcity Cut-Oﬀ AUC (95.0%CI) SE z p-Value Hedge’s (95.0%CI) p-Value Control Sarcopenic Cz-TA −6.3 (1.7) −5.3 (1.5) 0.71 0.75 −5.4 (0.46) 0.68 (0.38–0.98) 0.15 1.17 0.242 0.57 (−0.53–1.52) 0.272 Cz-GL −6.1 (1.5) −6.2 (1.4) 1.00 0.14 −7.8/−7.3/−3.5 (0.14) b 0.45 (0.11–0.79) 0.17 −0.29 0.768 −0.02 (−1.20–1.04) 0.798 Cz-GM −6.6 (1.2) −5.5 (1.7) 0.82 0.67 −6.1 (0.48) 0.70 (0.45 – 0.95) 0.13 1.54 0.123 0.66 (−0.28–1.61) 0.149 Cz-SOL −5.5 (1.2) −4.9 (2.1) 0.63 0.71 −3.8 (0.44) 0.57 (0.24–0.90) 0.17 0.43 0.669 0.35 (−0.75–1.42) 0.685 Cz-VL −5.6 (1.3) −5.1 (2.2) 0.44 1.00 −4.9 (0.50) 0.61 (0.31–0.91) 0.15 0.72 0.473 0.2 (−0.7–1.3) 0.517 Cz-VM −6.9 (1.2) −3.7 (1.6) 1.00 0.89 −5.2 (0.89) 0.98 (0.92–1.04) a 0.03 15.20 <0.001 2.2 (1.3–3.1) 0.005 Cz-BF −7.5 (2.1) −4.5 (1.7) 1.00 0.70 −6.2 (0.70) 0.86 (0.68–1.03) a 0.09 3.97 <0.001 1.5 (0.7–2.2) 0.010 Cz-RF −7.2 (1.4) −5.7 (3.0) 0.50 1.00 −4.4 (0.34) 0.70 (0.40–0.99) 0.15 1.32 0.187 0.6 (−0.6–1.6) 0.224 TA = Tibialis Anterior, GL = Gastrocnemius Lateralis, GM = Gastrocnemius Medialis, SOL = Soleus, VL = Vastus Lateralis, VM = Vastus Medialis, BF = Biceps Femoris, RF = Rectus Femoris. a upper conﬁdence limit can exceed the value of 1.0 in some case, due to the nonparametric estimation method used here of DeLong (1988). b easyROC calculation of optimal cut-oﬀpoints yielded to some multiple cut-oﬀpoints in some cases. In our analysis this was the case only for this parameter, which was, however, not signiﬁcant. Table 4. Descriptive Statistics of the logarithmically transformed sum of coherence above signiﬁcant conﬁdence limits reported as mean (standard deviation) and esults of the Receiver Operating Characteristic (ROC) curve analysis as well as DABEST comparisons with respective eﬀect sizes. ialis Anterior, GL = Gastrocnemius Lateralis, GM = Gastrocnemius Medialis, SOL = Soleus, VL = Vastus Lateralis, VM = Vastus Medialis, BF = Biceps Femoris, RF = Rectus a upper conﬁdence limit can exceed the value of 1.0 in some case, due to the nonparametric estimation method used here of DeLong (1988). b easyROC calculation of optimal oints yielded to some multiple cut-oﬀpoints in some cases. In our analysis this was the case only for this parameter, which was, however, not signiﬁcant. J. Clin. Med. 2020, 9, 720 14 of 22 14 of 22 4. Discussion Interestingly, and seemingly in contrast to some of the ﬁndings reported in literature, in our population, aﬀected by sarcopenia, we found CMC values to be signiﬁcantly larger in comparison to matched individuals without sarcopenia. This relative increased value was, furthermore, able to distinguish sarcopenic from non-sarcopenic older adults when determined from the vastus medialis and biceps femoris muscles. A possible explanation would be that sarcopenic older adults supposedly exhibit an impaired pyramidal system and a worsening of their brain-muscle communication. In this context an increase of CMC in sarcopenic older adults would be a reaction of the neuromuscular system on the sarcopenia muscle condition, that is trying to counteract the muscle weakness by increasing the synchronization eﬀorts between motor cortical areas and skeletal muscles. This would be in accordance with ﬁndings reported for stroke patients [51,87–91]. Clearly this reasoning represents all but a hypothesis that should be further investigated in longitudinal studies. Some researchers hypothesize that the observed change in motor output may be due to a decline in dopaminergic output from the basal ganglia which, in turn, leads to a need to invest more cognitive resources into movements [93,94]. Other researchers hint towards the possibility of neuromuscular junctions’ dysfunction as a possible root cause with neurological origin for sarcopenia [95]. Moreover, although it has been shown that gait is driven by eﬀerent input from motor cortical areas [28,29], disentangling any potential and additional modulations of eﬀerent and aﬀerent contributions to brain-muscle connectivity [35,96] might be important in order to gain a more complete picture of the neurophysiological integrity underlying sarcopenia. For instance, future investigations might consider whether a reduced aﬀerent contribution from a progressively wasted muscle to sensorimotor areas (i.e., as in sarcopenia) might be responsible for the increased CMC observed in this study, for example as a potential compensatory mechanism. Muscle weakness is known to place elders at an increased risk of mobility limitations and mortality [12] with an incidence of 10–25% among older adults above 65 years old [6,13] and up to 50% in those aged above 80 [13]. 15 of 22 J. Clin. Med. 2020, 9, 720 Similar percentages account for the presence of knee osteoarthritis (KOA) [97–101]. Thus, KOA are usually older adults resembling muscle disuse and weakness that closely resemble the sarcopenia condition. 4. Discussion Interestingly, it has been found that Vastus Medialis (VM) functionality (e.g., motor unit recruitment) diﬀerently modulates in KOA patients, potentially due to a compensatory mechanism in response to KOA [102]. That is, the VM structural changes observed in KOA patients are believed to be linked to neurogenic muscle atrophy [103]. Our explorative ﬁndings get support from a clinical perspective when we consider that aging associated muscle weakness underlies diverse mechanisms and cannot solely be explained by muscle atrophy [104]. The descending drive from the motor cortex declines with age, together with the overall ability to maximally activate a muscle with the nervous system, and this in sum, contributes to the decreases in voluntary contraction of muscles [18]. Voluntary activation, deﬁned as “the level of voluntary drive during an eﬀort” [105,106] is especially diminished in older-old individuals [107–109] and up to one third of losses in force production capacity may be explained by voluntary activation [110]. A recent study on cancer cachexia, which clinically presents with muscle atrophy and associated motor deﬁcits, found that impaired neural respiratory drive was a signiﬁcant contributor to respiratory muscles insuﬃciency [111]. Based on research ﬁndings and theoretical reasoning this has led to questioning of the conceptualizing of sarcopenia as primarily being a condition tied to the muscular system. An emerging view is that sarcopenia should be rather seen as tied to neurological factors [112]. The focus on the disease as being tied to the muscular system may also explain contradictory ﬁndings of non-pharmacological interventions used so far to prevent or control sarcopenia. Exercise programs show to be highly variable regarding type and mode of exercise oﬀered [113] and result in low quality of evidence when summarized in a systematic review and meta-analysis [114]. Strength training for example, an exercise mode often used to treat sarcopenia, may not always be eﬀective because it is not able to overcome the age-related blunted eﬀerent neural drive plasticity [115]. This is independent from gains in force-generating capacity of the muscles seen when young and older master athletes are compared. Both groups may exhibit superior maximum strength and rapid muscle force production, however, the magnitude of the eﬀerent neural drive in active older adults is substantial lower compared to that of younger individuals [109,116]. Furthermore, using a composite sarcopenia phenotype applying two diﬀerent widely accepted deﬁnitions for sarcopenia does not associate with independent aging [117]. 4. Discussion This implies that training programs should be developed that explicitly target neural structures such as training programs that use motor-cognitive approaches through step-training exergames [80,118–120] that also eﬀect on leg muscles strength [121,122]. However, our ﬁndings should be considered with caution and several limitations of our study should be mentioned. Clearly the sample size is a limitation of the present study, although the large eﬀect size found in CMC of VA and BF softens this argument. In a very recent study CMC assessed during gait revealed poor reliability within a test-retest framework [76]. However, only Tibialis Anterior (TA) has been considered in this previous work and it remains, therefore, unclear whether other muscles show diﬀerent reliability patterns. Thus, further research is warranted based on these ﬁrst ﬁndings from our exploratory trial. We suggest that such future research should also focus on test-retest reliability of CMC measures taken during locomotor tasks such as during walking. Another limitation may be the fact that it is not possible to guarantee that EEG data has been completely denoised from movement-related artifacts. However, it should be taken in consideration that completely denoising EEG data is almost impossible and that, for that reason, we adopted extensive care to remove artifacts from the EEG data with several precautions, such as not including data segments subsequent of heel strikes, which might have excessively contaminated EEG data by artifacts due to the impact of the foot with the ground. We also applied ASR and multi-modal AMICA algorithms as well as machine learning techniques for detecting artifactual/cerebral ICs components (i.e., ICLabel), and, in addition, adopted the strategy of transferring ICA weights and spheres to a more conventional non-ASR cleaned dataset. Both data segments (i.e., heel strikes multiplied by number of tapers used in spectral analysis) inequality between participants but also within participants (between left and right side) as well as 16 of 22 J. Clin. Med. 2020, 9, 720 16 of 22 inter-participants diﬀerences in the number of cerebral components detected by ICLabel can both represent a further limitation of the present study. 4. Discussion However the average number of brain components detected by ICLabel were in line with the suggested range of brain components that can be reliably detected from Indipendent Component Analysis (ICA) of EEG data [73] and we estimated signiﬁcant CMC at individual level using conﬁdence limits based on the number of segments respective to each participant and speciﬁc to each side. The latter was a precaution in the coherence estimation due to the unequal number of segments between subjects but also within subjects but between left and right sides. A further limitation of this study is not having considered potential diﬀerent results relative to the well-known diﬀerent possible causes for Sarcopenia, and in particular primary (i.e., due to the aging process) and secondary Sarcopenia (i.e., due to muscle disuse, malnutrition or inﬂammation) [123]. However, this represents a further aspect for future studies wanting to explore additional features of corticospinal control of gait in relation to sarcopenia. 5. Conclusions This preliminary exploratory investigation showed that corticospinal control of locomotion can represent a novel determinant in sarcopenia diagnosis, where corticomuscular coherence between EEG Cz sensor and Vastus Medialis or Biceps Femoris EMG sensors is able to distinguish between sarcopenic and non-sarcopenic older adults with high accuracy, precision and sensitivity. Moreover, our ﬁndings shed light on possible implications for future interventions either with pre-habilitation or re-habilitation purposes for the prevention and/or treatment of sarcopenia, which might employ EEG-EMG coherence, and in speciﬁc using Vastus Medialis or Biceps Femoris muscles which are shown to be signiﬁcantly diﬀerent with large magnitude of eﬀect in sarcopenia, as an important parameter of the progression of the disease and/or of the potential recovery from this age-related muscle disorder. Author Contributions: F.G. developed the research question, conceptualization, methodology and design of the study, while E.D.d.B., A.K.-L. and P.M. acted as methodological council. F.G. conducted data acquisition, analysis, and interpretation of the results with editing and improvement by E.D.d.B., A.K.-L., P.M. and K.D.B., F.G. produced a ﬁrst original version of the manuscript, while E.D.d.B., A.K.-L., P.M. and K.D.B. revised and edited the manuscript to bring it to its current version. All authors have read and approved the ﬁnal manuscript. Funding: This research has been supported by a scientiﬁc research grant from the association AILA-OIL (Lugano, Switzerland) awarded to F.G. in 2019 and by the AO Foundation (Davos, Switzerland) and the ETH Zurich Foundation (Zurich, Switzerland) with the AO Research Opportunity Award in Health Sciences and Technology awarded to F.G. in 2018. Acknowledgments: We would like to thank both AILA-OIL, AO Foundation and ETH Zurich Foundation for having supported the present study. We would like to thank the association ATTE (Bellinzona, Switzerland) for the great help in informing community-dwelling older adults about this research project. Moreover, we would like to thank all the participants of the present study. Furthermore, we would like to thank for assisting in the preparation and execution of this research project the following supervised Master students: Luca Bizzozero and Gianluca Di Deo. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Cruz-Jentoft, A.J.; Baeyens, J.P.; Bauer, J.M.; Boirie, Y.; Cederholm, T.; Landi, F.; Martin, F.C.; Michel, J.P.; Rolland, Y.; Schneider, S.M.; et al. Sarcopenia: European consensus on deﬁnition and diagnosis: Report of the European Working Group on Sarcopenia in Older People. Age Ageing. 2010, 39, 412–423. [CrossRef] p y p J [ ] 2. Falcon, L.J.; Harris-Love, M.O. Sarcopenia and the New ICD-10-CM Code: Screening, Staging, and Diagnosis Considerations. Fed. Pract. 2017, 34, 24–32. 5. 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https://openalex.org/W2984725288	http://d-scholarship.pitt.edu/41092/2/antibodies-08-00053.pdf	English	null	Engineering a Novel Antibody-Peptide Bispecific Fusion Protein Against MERS-CoV	Antibodies	2,019	cc-by	8,225	antibodies Article Engineering a Novel Antibody-Peptide Bispeciﬁc Fusion Protein Against MERS-CoV Article Received: 22 August 2019; Accepted: 23 October 2019; Published: 4 November 2019 Abstract: In recent years, tremendous eﬀorts have been made in the engineering of bispeciﬁc or multi-speciﬁc antibody-based therapeutics by combining two or more functional antigen-recognizing elements into a single construct. However, to the best of our knowledge there has been no reported cases of eﬀective antiviral antibody-peptide bispeciﬁc fusion proteins. We previously developed potent fully human monoclonal antibodies and inhibitory peptides against Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a novel coronavirus that causes severe acute respiratory illness with high mortality. Here, we describe the generation of antibody-peptide bispeciﬁc fusion proteins, each of which contains an anti-MERS-CoV single-chain antibody m336 (or normal human IgG1 CH3 domain as a control) linked with, or without, a MERS-CoV fusion inhibitory peptide HR2P. We found that one of these fusion proteins, designated as m336 diabody-pep, exhibited more potent inhibitory activity than the antibody or the peptide alone against pseudotyped MERS-CoV infection and MERS-CoV S protein-mediated cell-cell fusion, suggesting its potential to be developed as an eﬀective bispeciﬁc immunotherapeutic for clinical use. Keywords: MERS-CoV; mAbs; polypeptides; bispeciﬁc; immunotherapeutics antibodies Engineering a Novel Antibody-Peptide Bispeciﬁc Fusion Protein Against MERS-CoV Lili Wang 1,2, Jiyan Xu 1, Yu Kong 1, Ruiying Liang 3, Wei Li 4, Jinyao Li 5 , Jun Lu 6 , Dimiter S. Dimitrov 4, Fei Yu 3,* , Yanling Wu 1,* and Tianlei Ying 1 Lili Wang 1,2, Jiyan Xu 1, Yu Kong 1, Ruiying Liang 3, Wei Li 4, Jinyao Li 5 , Jun Lu 6 , Dimiter S. Dimitrov 4, Fei Yu 3,* , Yanling Wu 1,* and Tianlei Ying 1 1 MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; liliwang@fudan.edu.cn (L.W.); 13301050116@fudan.edu.cn (J.X.); kongyu@fudan.edu.cn (Y.K.); tlying@fudan.edu.cn (T.Y.) 1 MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; liliwang@fudan.edu.cn (L.W.); 13301050116@fudan.edu.cn (J.X.); kongyu@fudan.edu.cn (Y.K.); tlying@fudan.edu.cn (T.Y.) 2 Research Center of Chinese Jujube, Hebei Agricultural University, Baoding 071001, China 3 College of Life and Science, Hebei Agricultural University, Baoding 071001, China; ruiyingliang@outlook.com 4 Center for Antibody Therapeutics, University of Pittsburgh Medical School, Pittsburgh, PA 15261, USA LIWEI171@pitt.edu (W.L.); dsd116@pitt.edu (D.S.D.) 5 Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science a Technology, Xinjiang University, Urumqi 830046, China; ljyxju@xju.edu.cn gy j g y q jy j j 6 School of Science, and School of Interprofessional Health Studies, Faculty of Health & Environmental Sciences, Auckland University of Technology, Auckland 1142, New Zealand; jun.lu@aut.ac.nz * C d h f@h b d (F Y) li @f d d (YW ) * Correspondence: shmyf@hebau.edu.cn (F.Y.); yanlingwu@fudan.edu.cn (Y.W.); Tel.: +86-21-5423-7761 (F.Y.); +86-21-5423-7761 (Y.W.) www.mdpi.com/journal/antibodies 1. Introduction The antibody-based therapeutic modalities have shown clinical success in the treatment of many diseases [1–4]. In recent years, tremendous eﬀorts have been made in the engineering of bispeciﬁc or multi-speciﬁc antibodies by combining two or more functional antigen-recognizing elements into a single construct [5,6]. Such novel antibodies, or antibody-based fusion proteins, could be particularly beneﬁcial for the treatment of viral infections, which typically require potent and multi-functional therapeutics to prevent the frequent incidence of viral escape mutants [7]. For instance, we previously engineered a bispeciﬁc and multivalent anti-HIV-1 fusion protein, by incorporating the HIV-1 neutralizing antibody and the engineered single-domain CD4 into a single antibody-like molecule, Antibodies 2019, 8, 53; doi:10.3390/antib8040053 www.mdpi.com/journal/antibodies 2 of 11 Antibodies 2019, 8, 53 and found that it was able to neutralize all tested HIV-1 isolates, mediate potent antibody-dependent cellular cytotoxicity (ADCC) against HIV-1-infected cells, and eﬀectively suppress HIV-1 or SHIV infection in humanized mice and chronically infected macaques [8–10]. Notably, in addition to antibody-based therapeutics, the polypeptides-based fusion inhibitors represent another type of eﬀective antivirals, that could inhibit the entry of viruses by inhibiting virus-mediated cell-cell fusion [11]. However, due to the vast diﬀerences in activity, bioavailability, and biophysical properties between polypeptides and monoclonal antibodies, there has been no reported case of antibody-peptide bispeciﬁc fusion protein that is able to eﬀectively neutralize and inhibit cell-cell fusion mediated by viruses. y The Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus ﬁrst isolated in September 2012 from a patient in Saudi Arabia [12,13]. It causes SARS-like symptoms, including fever, cough, shortness of breath, and can lead to respiratory or renal failure [14,15]. Bats are natural reservoirs of MERS-CoV, but it is predominantly transmitted via dromedary camels and humans [16–23]. At the end of May 2019, 27 countries have reported 2442 laboratory-conﬁrmed cases of MERS-CoV infections with at least 842 related deaths since September 2012 (http://www.who.int/emergencies/mers-cov/en/). The eﬀective therapeutics and vaccines are urgently needed, considering the possibility of evolution and pandemic potential of MERS-CoV [24–27]. p p Like SARS-CoV, MERS-CoV is an enveloped virus using its spike (S) protein to enter target cells. The S protein can be cleaved into two subunits, S1 and S2 whereby the S1 subunit binds to the cellular receptor DPP4 and S2 subunit mediates membrane fusion [28–32]. Therefore, both S1 and S2 subunits could be targets for the development of prophylactic and therapeutic agents against MERS-CoV infection [33]. 1. Introduction In previous studies, by screening a large phage-displayed antibody Fab library, we have identiﬁed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of the MERS-CoV S protein S1 subunit. Among these antibodies, the mAbs m336 showed the most potent virus neutralization activity at low nanomolar concentrations [34–37]. Further structural study indicated that the binding epitope of m336 on MERS-CoV almost completely overlapped with the viral receptor-binding site, revealing the mechanism for its high neutralizing potency [38]. Meanwhile, fusion inhibitory peptides derived from heptad repeat 2 domain (HR2) of MERS-CoV S protein S1 subunit can inhibit the formation of six helix bundles (6-HB), which are required for fusion of the virus with its target cell [39,40]. Two of the peptides, P1 and HR2P, were reported to interact with heptad repeat 1 domain (HR1) of S protein S2 subunit, to form a 6-HB complex and block viral fusion and replication [40]. It was recently found that the combination of m336 antibody and HR2P peptide exhibited potent synergism in inhibiting MERS-CoV S protein-mediated cell-cell fusion and pseudovirus infections [41]. However, the peptide was mixed with antibody at a molar concentration ratio of 10,000:1, and no synergic eﬀect was observed when mixing the peptide with antibody at a ratio of 1:1, probably due to the signiﬁcant diﬀerence between the peptide and antibody in their bioactivities and biophysical properties. 2.3. MERS-CoV S Protein Binding The experiments were performed using the ProteOn XRP36 system (Bio-Rad, Hercules, CA, USA) to measure the binding kinetics of m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep to MERS-CoV S protein (amino acid 18–725). The S protein was immobilized on a ProteOn GLM biosensor chip using standard amine coupling chemistry (300 nM in 10 mM sodium acetate buﬀer, pH 5.0), and ~3000 resonance units were immobilized. The surface of the sensor chip was activated with 200 mM 1-ethyl-3-dimethyl aminopropylcarbodiimide hydrochloride and 50 mM N-hydroxysulfosuccinimide. m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep were prepared in PBS, pH 7.4, containing 0.005% Tween-20 (PBS-T) and injected (50 µL/min for 120 s, 1:3 dilution from 200 nM). The dissociation phase was followed for 600 s and chip surfaces were regenerated by injecting 10 mM glycine HCl, pH2.0, 100 µL/min for 18 s. Data were analyzed using ProteOn Manager 3.1 software and ﬁtted to the 1:1 interaction model [42]. 2.1. Gene Construction Four constructs, m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep were generated. The m336 scFv was generated by linking the m336 variable region heavy and variable region light chains with a 15 amino acids (G4S)3 linker. Fusion protein m336 scFv-pep was prepared by linking m336 scFv with MERS-CoV-derived HR2P peptide (LTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL) using another (G4S)3 linker as the spacer. Fusion protein m336 diabody-pep was generated by replacing the (G4S)3 linker between VH and VL of m336 scFv-pep with a 5 amino acids GGGGS linker, so that two VHs and VLs can pair together to form the dimeric diabody. The CH3-pep was also generated by fusing HR2P peptide with IgG1 CH3 domain. Coding fragments were synthesized by Genewiz Biotech 3 of 11 Antibodies 2019, 8, 53 Co., and inserted between two SﬁI restriction sites in the pComb3x vector with Flag-tag (DYKDDDDK) and His6-tag on C-termini. The sequences of the constructs were veriﬁed by direct DNA sequencing. Co., and inserted between two SﬁI restriction sites in the pComb3x vector with Flag-tag (DYKDDDDK) and His6-tag on C-termini. The sequences of the constructs were veriﬁed by direct DNA sequencing. 2.4. MERS-CoV Neutralization Assay MERS-CoV neutralization assay was performed as previously described [43,44]. Brieﬂy, 293T cells in 10 cm2 dishes were transiently co-transfected with a pcDNA3.1-MERS-CoV-S plasmid and a PNL4-3.luc.RE plasmid encoding an Env-defective luciferase-expressing HIV-1genome. After 48 h post-transfection, the produced pseudovirus was harvested from the supernatant, and ﬁltered through 0.45 µm sterilized membrane. The MERS-CoV pseudovirus was incubated with four inhibitors at 37 ◦C for 30 min, and then pseudovirus and inhibitors were added to DPP4-expressing Huh-7 cells (104/well) preplated in 96 well tissue culture plates for 6 h. After 12 h, fresh medium was added to the plates and incubated for another 48 h. Cells were lysed with lysis reagent (Promega, Madison, WI, USA) and lysates were transferred into 96-well Costar ﬂat-bottom luminometer plates (Corning, Corning, NY, USA). Luciferase substrate was added and the readings were recorded with an Ultra 384 Microplate Reader (Tecan, Männedorf, Switzerland). 2.2. Protein Expression and Puriﬁcation All four constructs were expressed as soluble proteins in E. coli and puriﬁed on Ni-NTA column. Brieﬂy, the expression vectors were transformed into E. coli strain HB2151 competent cells. A single fresh colony was inoculated into 3 mL of 2YT medium containing 100 µg/mL ampicillin and incubated at 37 ◦C, 250 rpm overnight. The incubated culture was transferred to 200 mL of fresh 2YT medium with 100 µg/mL ampicillin for large-scale protein production and 4–6 h growth at 37 ◦C. Then, 1 mM isopropyl-β-d-thiogalactoside (IPTG) was applied to induce protein overexpression, and the cells were grown overnight at 30 ◦C before harvesting by centrifugation. Protein fragments were harvested from the bacterial cell pellet and the precipitate was re-suspended with 50 mL PBS with 0.5 M NaCl. Then, 0.2 mL 1 M polymyxin B was added to lyse bacteria and the sample was rotated 30 min at room temperature, 250 rpm. The cultures were centrifuged at 12,000 rpm for 15 min at 4 ◦C. Supernatant was collected and loaded onto a Ni-NTA Superﬂow (Qiagen, Redwood City, CA, USA). After the impurities were removed, target proteins were eluted with elution buﬀer (250 mM imidazole in PBS). The proteins were resolved with SDS-PAGE and the purity was estimated as >90%. The protein purity was further conﬁrmed by size exclusion chromatography using an FPLC AKTA system (GE Healthcare, Chicago, IL, USA) with a Superdex 200 10/300 GL column (GE Healthcare). The protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare). 3.1. Generation of Anti-MERS-CoV Fusion Proteins diabody with two VHs and VLs. A dimeric d b li ki HR2P id d I G1 CH To generate the bispeciﬁc antibody-peptide inhibitor, the MERS-CoV S2-derived peptide HR2P was fused to m336 scFv using a ﬂexible (G4S)3 linker. Furthermore, a dimeric format, namely m336 diabody-pep, was also generated by replacing the (G4S)3 linker between variable region heavy (VH) and variable region light (VL) of m336 scFv with a 5 amino acids GGGGS linker. The shorter linker can result in the pairing of VH and VL from two diﬀerent chains and thus the formation of dimeric diabody with two VHs and VLs. A dimeric peptide-only control, namely CH3-pep, was also generated by linking HR2P peptide and IgG1 CH3 domain with the ﬂexible (G4S)3 linker. This fusion protein possesses two peptides and comparable molecular weight compared to other constructs (Figure 1A). generated by linking HR2P peptide and IgG1 CH3 domain with the flexible (G4S)3 linker. This fusion protein possesses two peptides and comparable molecular weight compared to other constructs (Figure 1A). The four proteins, m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep were soluble expressed in E. coli cells with high efficiency. Purified proteins were obtained with yields of 15–30 mg/L bacterial culture. The m336 diabody-pep migrated as a monomer under the denaturing conditions of the SDS-PAGE, and thus displayed similar molecular weight (~30 kDa) with m336 scFv and m336 scFv-pep (Figure 1B). All proteins were monomeric as demonstrated by size exclusion chromatography (data not shown) Figure 1. Construction and characterization of anti-MERS-CoV inhibitors. (A). Schematic structure of proteins m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep. (B). SDS-PAGE of the anti- MERS-CoV inhibitors. Figure 1. Construction and characterization of anti-MERS-CoV inhibitors. (A) Schematic structure of proteins m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep. (B) SDS-PAGE of the anti-MERS-CoV inhibitors. Figure 1. Construction and characterization of anti-MERS-CoV inhibitors. (A). Schematic structure of proteins m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep. (B). SDS-PAGE of the anti- MERS-CoV inhibitors. Figure 1. Construction and characterization of anti-MERS-CoV inhibitors. (A) Schematic structure of proteins m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep. (B) SDS-PAGE of the anti-MERS-CoV inhibitors. 3.2. Interactions between Fusion Proteins and MERS-CoV S Protein To measure the binding kinetics of the fusion proteins with MERS-CoV S protein, the surface plasmon resonance (SPR) assay was performed by using a ProteOn system SPR biosensor instrument (Figure 2). 2.5. Cell-Cell Fusion Assay To further compare the inhibitory eﬀects of fusion proteins, the cell-cell fusion assay was performed as MERS-CoV S protein, which was expressed on the cell surface, can mediate cell fusion 4 of 11 diluted d Huh-7 Antibodies 2019, 8, 53 plates at 37 °C f m336 scFv m336 with neighboring cells [43]. First, 293T cells were transiently transfected with pAA-IRES-MERS-EGFP (293T/MERS/EGFP, eﬀector cells) encoding the MERS-CoV proteins or pAA-IRES-EGFP (293T/EGFP) as a negative control. Cells were cultured in DMEM containing 10% FBS at 37 ◦C for 48 h. Preplated Huh-7 cells (104, target cells), which express the DPP4 MERS-CoV receptor, were cultured in 96 well plates at 37 ◦C for 5 h. After addition of 293T/MERS/EGFP or 293T/EGFP cells with 2-fold diluted m336 scFv, m336 scFv-pep, m336 diabody-pep, or CH3-pep from 2.5 µg/mL, the mixture and Huh-7 cells (target cells) were further cultures at 37 ◦C for 4 h, and fused or unfused cells were visualized under an inverted ﬂuorescent microscope (Nikon Eclipse Ti-S100, Nikon, Tokyo, Japan) [45]. ( g ) , under an inverted fluorescent microscope (Nikon Eclipse Ti-S100, Nikon, Tokyo, Japan) [45]. 3. Results 3.1. Generation of Anti-MERS-CoV Fusion Proteins To generate the bispecific antibody-peptide inhibitor, the MERS-CoV S2-derived peptide HR2P was fused to m336 scFv using a flexible (G4S)3 linker. Furthermore, a dimeric format, namely m336 diabody-pep, was also generated by replacing the (G4S)3 linker between variable region heavy (VH) 3.1. Generation of Anti-MERS-CoV Fusion Proteins diabody with two VHs and VLs. A dimeric d b li ki HR2P id d I G1 CH We have previously demonstrated that m336 bound specifically to MERS-CoV S protein, The four proteins, m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep were soluble expressed in E. coli cells with high efficiency. Purified proteins were obtained with yields of 15–30 mg/L bacterial culture. The m336 diabody-pep migrated as a monomer under the denaturing conditions of the SDS-PAGE, and thus displayed similar molecular weight (~30 kDa) with m336 scFv and m336 scFv-pep (Figure 1B). All proteins were monomeric as demonstrated by size exclusion chromatography (data not shown). but not other unrelated proteins [35]. As shown in Table 1, all thr scFv, m336 scFv-pep, and m336 diabody-pep) exhibited potent b 3.2. Interactions between Fusion Proteins and MERS-CoV S Protein p g y T bl 1 Bi di ki i l i f f i i MERS C V S i Table 1. Binding kinetics analysis of fusion proteins to MERS-CoV S protein. p g y Table 1. Binding kinetics analysis of fusion proteins to MERS-CoV S protein. Fusion Proteins kon (M−1s−1) koff (s−1) KD (M) CH3-pep 5.99 × 104 6.56 × 104 1.09 m336 scFv 4.66 × 105 3.80 × 10−4 8.14 × 10−1 m336 scFv-pep 7.47 × 105 1.20 × 10−4 1.60 × 10−1 m336 diabody-pep 1.14 × 106 1.23 × 10−3 1.08 × 10− Table 1. Binding kinetics analysis of fusion proteins to MERS-CoV S protein. Fusion Proteins kon (M−1s−1) koﬀ(s−1) KD (M) CH3-pep 5.99 × 104 6.56 × 104 1.09 m336 scFv 4.66 × 105 3.80 × 10−4 8.14 × 10−10 m336 scFv-pep 7.47 × 105 1.20 × 10−4 1.60 × 10−10 m336 diabody-pep 1.14 × 106 1.23 × 10−3 1.08 × 10−9 3.3. Neutralization of MERS-CoV Pseudovirus Infection y p p li i f E S C d i I f i 3.3. Neutralization of MERS-CoV Pseudovirus Infection 3.3. Neutralization of MERS-CoV Pseudovirus Infection Next, we measured the neutralization activity of m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep at graded concentrations against pseudotyped MERS-CoV infection. As shown in Figure 3, m336-scFv, m336 scFv-pep and m336 diabody-pep exhibited potent neutralization activity. Among them, m336 scFv-pep exhibited the most potent neutralization capability, with 50% inhibitory concentration (IC50) of 0.21 ± 0.06 nM. Although, displaying comparable IC50 (m336 diabody-pep, 0.25 ± 0.07 nM; m336 scFv, 0.69 ± 0.03 nM), interestingly, m336 diabody-pep inhibited the infection more potent than that of m336 scFv at low protein concentrations (<0.03 nM). In a previous study, IgG1 m336 displayed exceptional neutralization against pseudotyped MERS-CoV infection with IC50 of 0 03 M [35] The o t ol fu io otei CH3 e ould ot i hibit the i fe tio Next, we measured the neutralization activity of m336 scFv, m336 scFv-pep, m336 diabody-pep, and CH3-pep at graded concentrations against pseudotyped MERS-CoV infection. As shown in Figure 3, m336-scFv, m336 scFv-pep and m336 diabody-pep exhibited potent neutralization activity. Among them, m336 scFv-pep exhibited the most potent neutralization capability, with 50% inhibitory concentration (IC50) of 0.21 ± 0.06 nM. but not other unrelated proteins [35]. As shown in Table 1, all thr scFv, m336 scFv-pep, and m336 diabody-pep) exhibited potent b 3.2. Interactions between Fusion Proteins and MERS-CoV S Protein binding pattern. The equilibrium dissociation constant (KD) of m336 scFv for S protein was 0.8 nM with on-rate (kon) of 4.7 × 105 M−1s−1 and off-rate (koff) of 3.8 × 10−4 s−1. The m336 diabody-pep displayed similar binding kinetics to that of m336 scFv (kon 1.1 × 106 M−1s−1, koff 1.2 × 10−3 s−1, KD 1.1 nM). The m336 To measure the binding kinetics of the fusion proteins with MERS-CoV S protein, the surface plasmon resonance (SPR) assay was performed by using a ProteOn system SPR biosensor instrument (Figure 2). We have previously demonstrated that m336 bound speciﬁcally to MERS-CoV S protein, but not other unrelated proteins [35]. As shown in Table 1, all three m336-based fusion proteins 5 of 11 Antibodies 2019, 8, 53 (m336 scFv, m336 scFv-pep, and m336 diabody-pep) exhibited potent binding to S protein with very similar binding pattern. The equilibrium dissociation constant (KD) of m336 scFv for S protein was 0.8 nM with on-rate (kon) of 4.7 × 105 M−1s−1 and oﬀ-rate (koﬀ) of 3.8 × 10−4 s−1. The m336 diabody-pep displayed similar binding kinetics to that of m336 scFv (kon 1.1 × 106 M−1s−1, koﬀ1.2 × 10−3 s−1, KD 1.1 nM). The m336 scFv-pep had slightly more potent binding aﬃnity (KD 0.2 nM) with slower dissociation rate constant (koﬀ1.2 × 10−4 s−1), compared to the other two fusion proteins. In contrast, the CH3-peptide displayed much lower aﬃnity, compared to the antibody m336-based fusion proteins (KD 1.1 M). Antibodies 2019, 8, x FOR PEER REVIEW 5 of 11 scFv-pep had slightly more potent binding affinity (KD 0.2 nM) with slower dissociation rate constant (koff 1.2 × 10−4 s−1), compared to the other two fusion proteins. In contrast, the CH3-peptide displayed much lower affinity compared to the antibody m336-based fusion proteins (KD 1 1 M) Figure 2. Interactions between m336 scFv (A), m336 scFv-pep (B), and m336 diabody-pep (C) with MERS-CoV S protein measured using the ProteOn XRP36 SPR system Figure 2. Interactions between m336 scFv (A), m336 scFv-pep (B), and m336 diabody-pep (C) with MERS-CoV S protein measured using the ProteOn XRP36 SPR system. Figure 2. Interactions between m336 scFv (A), m336 scFv-pep (B), and m336 diabody-pep (C) with MERS CoV S protein measured using the ProteOn XRP36 SPR system Figure 2. Interactions between m336 scFv (A), m336 scFv-pep (B), and m336 diabody-pep (C) with MERS-CoV S protein measured using the ProteOn XRP36 SPR system. 3 4 Inhibition of MERS S Protein Mediated Cell Cell Fusion 3.4. Inhibition of MERS S Protein-Mediated Cell-Cell Fusion measured, and percent neutralization was calculated. 3 4 Inhibition of MERS S Protein Mediated Cell Cell Fusion 3.4. Inhibition of MERS S Protein-Mediated Cell-Cell Fusion measured, and percent neutralization was calculated. 3.4. Inhibition of MERS S Protein Mediated Cell Cell Fusion We used a well-established MERS-CoV S protein-mediated cell-cell fusion assay to determine whether the fusion proteins have the ability to inhibit MERS-CoV fusion with the target cells [40,46]. In such assay, the 293T cells that express MERS-CoV S protein and EGFP were used as the effector cells, and Huh-7 cells that express DPP4 receptor were used as the target cells. The CH3-pep showed no appreciable activity at a concentration of 83 nM, which is consistent with the previous report that the HR2P peptide did not have significant inhibitory effect at concentrations lower than 100 nM [40]. Notably, we found that m336 diabody-pep was able to block the fusion between 293T/MERS/EGFP cells and Huh-7 cells at a concentration as low as 0.5 nM (Figure 4). The calculated IC50 for m336 diabody-pep was 0.81 ± 0.02 nM. As shown in Figure 4, the m336 diabody-pep was significantly more potent than m336 scFv in inhibiting cell-cell fusion. The m336 scFv-pep also showed evidently more potent inhibitory activity than m336 scFv at low concentrations, with slightly lower IC50 (m336 scFv- pep, 5.25 ± 0.03 nM; m336 scFv, 7.76 ± 0.02 nM). These results indicate that antibody-peptide bispecific fusion proteins, especially the m336 diabody-pep, have the potential to be developed as potent MERS-CoV inhibitors that could, not only neutralize MERS-CoV infection, but also inhibit MERS- CoV S protein mediated cell cell fusion We used a well-established MERS-CoV S protein-mediated cell-cell fusion assay to determine whether the fusion proteins have the ability to inhibit MERS-CoV fusion with the target cells [40,46]. In such assay, the 293T cells that express MERS-CoV S protein and EGFP were used as the eﬀector cells, and Huh-7 cells that express DPP4 receptor were used as the target cells. The CH3-pep showed no appreciable activity at a concentration of 83 nM, which is consistent with the previous report that the HR2P peptide did not have signiﬁcant inhibitory eﬀect at concentrations lower than 100 nM [40]. Notably, we found that m336 diabody-pep was able to block the fusion between 293T/MERS/EGFP cells and Huh-7 cells at a concentration as low as 0.5 nM (Figure 4). The calculated IC50 for m336 diabody-pep was 0.81 ± 0.02 nM. 3 4 Inhibition of MERS S Protein Mediated Cell Cell Fusion 3.4. Inhibition of MERS S Protein-Mediated Cell-Cell Fusion measured, and percent neutralization was calculated. As shown in Figure 4, the m336 diabody-pep was signiﬁcantly more potent than m336 scFv in inhibiting cell-cell fusion. The m336 scFv-pep also showed evidently more potent inhibitory activity than m336 scFv at low concentrations, with slightly lower IC50 (m336 scFv-pep, 5.25 ± 0.03 nM; m336 scFv, 7.76 ± 0.02 nM). These results indicate that antibody-peptide bispeciﬁc fusion proteins, especially the m336 diabody-pep, have the potential to be developed as potent MERS-CoV inhibitors that could, not only neutralize MERS-CoV infection, but also inhibit MERS-CoV S protein-mediated cell-cell fusion. 3.4. Inhibition of MERS S Protein-Mediated Cell-Cell Fusion We used a well-established MERS-CoV S protein-mediated cell-cell fusion assay to determine whether the fusion proteins have the ability to inhibit MERS-CoV fusion with the target cells [40,46]. In such assay, the 293T cells that express MERS-CoV S protein and EGFP were used as the effector cells, and Huh-7 cells that express DPP4 receptor were used as the target cells. The CH3-pep showed no appreciable activity at a concentration of 83 nM, which is consistent with the previous report that the HR2P peptide did not have significant inhibitory effect at concentrations lower than 100 nM [40]. Notably, we found that m336 diabody-pep was able to block the fusion between 293T/MERS/EGFP cells and Huh-7 cells at a concentration as low as 0.5 nM (Figure 4). The calculated IC50 for m336 diabody-pep was 0.81 ± 0.02 nM. As shown in Figure 4, the m336 diabody-pep was significantly more potent than m336 scFv in inhibiting cell-cell fusion. The m336 scFv-pep also showed evidently more potent inhibitory activity than m336 scFv at low concentrations, with slightly lower IC50 (m336 scFv- pep, 5.25 ± 0.03 nM; m336 scFv, 7.76 ± 0.02 nM). These results indicate that antibody-peptide bispecific fusion proteins, especially the m336 diabody-pep, have the potential to be developed as potent MERS-CoV inhibitors that could, not only neutralize MERS-CoV infection, but also inhibit MERS- CoV S protein-mediated cell-cell fusion Figure 4. Inhibitory activity of anti-MERS-CoV inhibitors on MERS-CoV S protein-mediated cell-cell fusion. The number of 293T/MERS/EGFP cells fused or unfused with Huh-7 cells were counted, and h f i hibi i l l d Figure 4. Inhibitory activity of anti-MERS-CoV inhibitors on MERS-CoV S protein-mediated cell-cell fusion. The number of 293T/MERS/EGFP cells fused or unfused with Huh-7 cells were counted, and the percentage of inhibition was calculated. Figure 4. but not other unrelated proteins [35]. As shown in Table 1, all thr scFv, m336 scFv-pep, and m336 diabody-pep) exhibited potent b 3.2. Interactions between Fusion Proteins and MERS-CoV S Protein Although, displaying comparable IC50 (m336 diabody-pep, 0.25 ± 0.07 nM; m336 scFv, 0.69 ± 0.03 nM), interestingly, m336 diabody-pep inhibited the infection more potent than that of m336 scFv at low protein concentrations (<0.03 nM). In a previous study, IgG1 m336 displayed exceptional neutralization against pseudotyped MERS-CoV infection with IC50 of 0.03 nM [35]. The control fusion protein CH3-pep could not inhibit the infection. 6 of 11 6 of 11 Antibodies 2019, 8, 53 , , ntibodies 2019, 8, x FOR PEER REVIEW 6 of 1 Figure 3. Potent neutralization of MERS-CoV by anti-MERS-CoV inhibitors. Pseudotyped virus was incubated with proteins before infection of DPP4-expressing Huh-7 cells. Luciferase activities were measured, and percent neutralization was calculated. Figure 3. Potent neutralization of MERS-CoV by anti-MERS-CoV inhibitors. Pseudotyped virus was incubated with proteins before infection of DPP4-expressing Huh-7 cells. Luciferase activities were measured, and percent neutralization was calculated. Antibodies 2019, 8, x FOR PEER REVIEW 6 of 11 Figure 3. Potent neutralization of MERS-CoV by anti-MERS-CoV inhibitors. Pseudotyped virus was incubated with proteins before infection of DPP4-expressing Huh-7 cells. Luciferase activities were , , odies 2019, 8, x FOR PEER REVIEW 6 o Figure 3. Potent neutralization of MERS-CoV by anti-MERS-CoV inhibitors. Pseudotyped virus was incubated with proteins before infection of DPP4-expressing Huh-7 cells. Luciferase activities were measured, and percent neutralization was calculated. Figure 3. Potent neutralization of MERS-CoV by anti-MERS-CoV inhibitors. Pseudotyped virus was incubated with proteins before infection of DPP4-expressing Huh-7 cells. Luciferase activities were measured, and percent neutralization was calculated. Figure 3. Potent neutralization of MERS-CoV by anti-MERS-CoV inhibitors. Pseudotyped virus was incubated with proteins before infection of DPP4-expressing Huh-7 cells. Luciferase activities were 4. Discussion Monoclonal antibodies represent one of the most promising immunotherapeutic agents for the treatment of cancers and infectious diseases [7,47–52]. Previously, we used RBD of MERS-CoV S protein to screen a non-immune phage-displayed Fab library and identiﬁed a highly potent human neutralizing mAb m336 and a panel of other mAbs against MERS-CoV [34,36,37]. Jiang et al. also used MERS-CoV S RBD to screen a non-immune yeast-displayed scFv library and identiﬁed two potent MERS-CoV neutralizing mAbs, MERS-4 and MERS-27 [53]. Tang et al. used a full-length S protein to screen a non-immune phage-display scFv library and identiﬁed a potent MERS-CoV neutralizing mAb 3B11 [54]. All these reported mAbs inhibit the binding of virus to cellular reporter DPP4 by recognizing the diﬀerent epitopes of RBD, thus having the potential to be developed as anti-MERS-CoV therapeutics. However, further engineering is still needed to address the concerns in using such mAbs in clinics, e.g., the high production costs, etc. In this study, we generated two bispeciﬁc anti-MERS-CoV fusion proteins, m336 scFv-pep and m336 diabody-pep. Compared with the antibody or peptide inhibitors that block only one site of the spike protein, the bispeciﬁc inhibitors showed greater potency. The m336 scFv-pep showed the highest binding aﬃnity to MERS-CoV S protein, as well as the most potent neutralizing activity, while the m336 diabody-pep was the most potent in blocking MERS-CoV S-mediated cell-cell fusion. These results conﬁrmed the improved potency of bispeciﬁc antibody-peptide inhibitors compared to antibody or peptide alone. The m336-scFv interferes with viral attachment to the DPP4 receptor on human cells [35], while the HR2P peptide inhibits the formation of the 6-helix-bundle fusion core and thus interrupts viral fusion with the target cell membrane [40]. It is probably that the antibody-peptide fusion proteins, which are composed of both antiviral components, linked by ﬂexible (G4S)3 linker, can act at both steps during viral infection. p g Interestingly, the m336 diabody-pep was found to potently block MERS-CoV S-mediated cell-cell fusion even at concentrations lower than 0.5 nM. This phenomenon may be attributed to the diﬀerence in steric hindrance of the fusion proteins. Compared to the m336 scFv-pep, the m336 diabody-pep has a much larger (twice) molecular weight and size, and thus, inhibits the viral fusion to target cells by competitive inhibition and steric hindrance, leading to the much greater inhibitory activity than that of m336 scFv-pep. 3 4 Inhibition of MERS S Protein Mediated Cell Cell Fusion 3.4. Inhibition of MERS S Protein-Mediated Cell-Cell Fusion measured, and percent neutralization was calculated. Inhibitory activity of anti-MERS-CoV inhibitors on MERS-CoV S protein-mediated cell-cell fusion. The number of 293T/MERS/EGFP cells fused or unfused with Huh-7 cells were counted, and the percentage of inhibition was calculated. Figure 4. Inhibitory activity of anti-MERS-CoV inhibitors on MERS-CoV S protein-mediated cell-cell fusion. The number of 293T/MERS/EGFP cells fused or unfused with Huh-7 cells were counted, and Figure 4. Inhibitory activity of anti-MERS-CoV inhibitors on MERS-CoV S protein-mediated cell-cell fusion. The number of 293T/MERS/EGFP cells fused or unfused with Huh-7 cells were counted, and the percentage of inhibition was calculated. Figure 4. Inhibitory activity of anti-MERS-CoV inhibitors on MERS-CoV S protein-mediated cell-cell fusion. The number of 293T/MERS/EGFP cells fused or unfused with Huh-7 cells were counted, and the percentage of inhibition was calculated. Antibodies 2019, 8, 53 7 of 11 7 of 11 4. Discussion Although, having largely improved potency in inhibiting cell-cell fusion, m336 diabody-pep did not show evidently increased neutralization activity compared to m336 scFv. Further investigations are required to explore the mechanism for the disparate performance of m336 diabody-pep in diﬀerent assays. As shown in Figures 3 and 4, the IC50 of m336 diabody-pep was 0.25 ± 0.07 nM in the neutralization assay, and 0.81 ± 0.02 nM in the cell-cell fusion assay. Importantly, m336 diabody-pep was much more potent than m336 scFv at concentrations higher than 5 nM in the cell-cell fusion assay. At these concentrations, all the fusion proteins can result in high percentage of inhibition (>70%) in the neutralization assay, and therefore, may explain the little diﬀerence among diﬀerence groups. All the m336-based fusion proteins could be easily produced in E. coli in large amount and low production cost. Their sizes are larger than m336 scFv or HR2P peptides, suggesting that they would possess longer in vivo half-life. Still, their half-life would be shorter than full-size IgG due to the lack of Fc region. Therefore, their prophylactic and therapeutic eﬃcacy against MERS-CoV should be assessed carefully using diﬀerent approaches and doses in vivo. However, the RBD of MERS-CoV speciﬁcally targets on human DPP4 [29], and most small animal models are not susceptible to MERS-CoV infection [55], which pose a signiﬁcant barrier to the development of anti-MERS-CoV inhibitors. Fortunately, researchers have constructed several animal models recently that simulate the morbidity and mortality of human infections, of which nonhuman primates (NHP) models and human DPP4-expressing mouse model are considered to be ideal candidates [56] and the latter is promising to be utilized in our following studies. Antibodies 2019, 8, 53 8 of 11 5. Conclusions In conclusion, our study indicated that the bispeciﬁc inhibitors have increased the eﬃcacy against MERS-CoV, compared to the neutralizing antibody or polypeptide alone. Such inhibitors with the advantage of multiple biologics have the potential to be further developed as eﬀective prophylactic and therapeutic agents, and may ﬁnd wide application in treating viral diseases. Author Contributions: L.W., Y.W., F.Y., and T.Y. conceived and designed the experiments; L.W., J.X., Y.K., R.L., W.L., J.L. (Jinyao Li), J.L. (Jun Lu), and D.S.D. performed the experiments; L.W., J.X., and Y.W. analyzed the data; L.W., F.Y., and T.Y. wrote the paper. All authors discussed the results and contributed to the ﬁnal manuscript. Funding: This work was supported by grants from the National Natural Science Foundation of China (81601761, 31570936, 81630090, 81822027 and 81501735), the grant from Chinese Academy of Medical Sciences (2019PT350002), Fudan’s Undergraduate Research Opportunities Program (15088), Starting Grant from Hebei Agricultural University (YJ201843), National Fundamental Fund of Training Basic Science (J1210041-1629), Program for Talents Introduction from studying overseas of Hebei Province (CN201707), Program for Youth Talent of Higher Learning Institutions of Hebei Province (BJ2018045), National Megaprojects of China for Major Infectious Diseases (2018ZX10301403), and the New Zealand Ministry of Education’s New Zealand-China Tripartite Partnership Fund. Acknowledgments: We are grateful to Shibo Jiang, Lu Lu and Cong Wang at Fudan University for providing cell-cell fusion system. Conﬂicts of Interest: The authors declare no conﬂicts of interest. 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https://openalex.org/W4235786598	https://www.qeios.com/read/B1TOOX/pdf	English	null	Contamination	Definitions	2,020	cc-by	50	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Contamination National Cancer Institute National Cancer Institute Qeios ID: B1TOOX · https://doi.org/10.32388/B1TOOX Source National Cancer Institute. Contamination. NCI Thesaurus. Code C68768. The presence of any substance or organism that makes a preparation impure. Qeios ID: B1TOOX · https://doi.org/10.32388/B1TOOX 1/1
https://openalex.org/W3013335580	https://europepmc.org/articles/pmc7177904?pdf=render	English	null	Detection of Rare Objects by Flow Cytometry: Imaging, Cell Sorting, and Deep Learning Approaches	International journal of molecular sciences	2,020	cc-by	43,989	Received: 9 December 2019; Accepted: 25 March 2020; Published: 27 March 2020 Abstract: Flow cytometry nowadays is among the main working instruments in modern biology paving the way for clinics to provide early, quick, and reliable diagnostics of many blood-related diseases. The major problem for clinical applications is the detection of rare pathogenic objects in patient blood. These objects can be circulating tumor cells, very rare during the early stages of cancer development, various microorganisms and parasites in the blood during acute blood infections. All of these rare diagnostic objects can be detected and identiﬁed very rapidly to save a patient’s life. This review outlines the main techniques of visualization of rare objects in the blood ﬂow, methods for extraction of such objects from the blood ﬂow for further investigations and new approaches to identify the objects automatically with the modern deep learning methods. Keywords: imaging ﬂow cytometry; cell sorting; deep learning; circulating tumor cells; cell labeling; liquid biopsy; ﬂow cytometry data analysis Detection of Rare Objects by Flow Cytometry: Imaging, Cell Sorting, and Deep Learning Approaches Denis V. Voronin 1,2,* , Anastasiia A. Kozlova 1 , Roman A. Verkhovskii 1,3 , Alexey V. Ermakov 1,4, Mikhail A. Makarkin 1, Olga A. Inozemtseva 1 and Daniil N. Bratashov 1 Denis V. Voronin 1,2,* , Anastasiia A. Kozlova 1 , Roman A. Verkhovskii 1,3 , Alexey V. Ermakov 1,4, Mikhail A. Makarkin 1, Olga A. Inozemtseva 1 and Daniil N. Bratashov 1 Laboratory of Biomedical Photoacoustics, Saratov State University, 410012 Saratov, Russia; anastasia.kozlova245@yandex.ru (A.A.K.); r.a.verhovskiy@mail.ru (R.A.V.); ermakov.ssu@gmail.com (A.V.E.); makmih50@gmail.com (M.A.M.); inozemtsevaoa@mail.ru (O.A.I.); bratashovdn@info.sgu.ru (D.N.B.) 1 Laboratory of Biomedical Photoacoustics, Saratov State University, 410012 Saratov, Russia; anastasia.kozlova245@yandex.ru (A.A.K.); r.a.verhovskiy@mail.ru (R.A.V.); ermakov.ssu@gmail.com (A.V.E.); makmih50@gmail.com (M.A.M.); inozemtsevaoa@mail.ru (O.A.I.); bratashovdn@info.sgu.ru (D.N.B.) 2 D t t f Ph i l d C ll id Ch i t N ti l U i it f Oil d G (G bki U i it ) g ( ) ( ) g ( ) 2 Department of Physical and Colloid Chemistry, National University of Oil and Gas (Gubkin University), 119991 Moscow, Russia 2 Department of Physical and Colloid Chemistry, National University of Oil and Gas (Gubkin University 119991 Moscow, Russia 3 School of Urbanistics, Civil Engineering and Architecture, Yuri Gagarin State Technical University of Saratov 410054 Saratov, Russia 3 School of Urbanistics, Civil Engineering and Architecture, Yuri Gagarin State Technical University of Saratov, 410054 Saratov, Russia 4 Department of Biomedical Engineering, I. M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia * Correspondence: denis.v.voronin@gmail.com; Tel.: +7-917-303-7673 * Correspondence: denis.v.voronin@gmail.com; Tel.: +7-917-303-7673 International Journal of Molecular Sciences International Journal of Molecular Sciences Int. J. Mol. Sci. 2020, 21, 2323; doi:10.3390/ijms21072323 1. Introduction The problem of detection and extraction of rare objects from the blood ﬂow arises in a number of situations. This includes the search for the very rare circulating tumor cells (CTCs) at early stages of cancer development by liquid biopsy [1,2], the detection of microorganisms during acute blood infections to determine its strain very rapidly [3], early detection of malaria parasites including in vivo [4,5] and other pathogenic states that impose high risks to human life and well-being. In addition to the detection and extraction of such rare objects, a lot of developments are targeted on eliminating it from the blood ﬂow by a sort of blood ﬁltering. The current state of the art in this ﬁeld is deﬁned by the progress in cell imaging and sorting techniques, sample enrichment, and separation along with the new approaches for automatization of data analysis based on machine learning and deep learning methods. Here we provide an overview of diﬀerent techniques designed to detect very rare objects in the blood ﬂow, sort it out or ﬁlter from the bloodstream and extract for further investigation. According to the statistics of the World Health Organization for 2016, around 71% of the overall 57 million deaths are caused by noncommunicable diseases, including cardiovascular disease—31% and cancer—16% [6]. During 2016, 216 million cases of malaria were detected [7]. The promising Int. J. Mol. Sci. 2020, 21, 2323; doi:10.3390/ijms21072323 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2323 2 of 60 approach to diagnose these diseases is the detection of untypical objects in blood and lymph samples. However, there are two signiﬁcant challenges: the ﬁrst one is the rarity of untypical blood elements [8] and the second one is the small volume of the sample [9] that has to be subjected to detailed analysis. The last generation of the ﬂow cytometry systems with the possibility of object visualization allows verifying received data but has additional restrictions. For example, the object size is limited by the resolution of the cytometer optical system, and there are ﬂow speed limits based on the sensitivity of detection scheme, laminarity of object ﬂow and required quality of the image. However, there is no doubt that a solution to the current main medical issues is related to the detailed analysis of single cells, which requires their separation from ordinary objects. 2. Flow Cytometry Hardware The optical detection system is the main part of the ﬂow cytometer that deﬁne the overall system performance and provide the quality of data (high signal-to-noise ratio, high sensitivity, good repeatability) at a reasonable processing speed. Typically, a ﬂow cytometry system consists of three main parts: illumination subsystem, usually including one or multiple lasers of diﬀerent wavelengths; ﬁne-tuned optics, comprising dichroic band-pass and cut-oﬀﬁlters; and detection system, usually based on high-sensitivity photomultiplier tubes (PMTs) or camera for imaging systems. 1. Introduction Currently, liquid biopsy is one of the most informative and broadly described analyses in medicine. A number of diseases could be diagnosed by detecting of untypical blood objects—emboli in the lymph or blood. For example, CTCs are the prognostic factor of diﬀerent cancer types [10–16], which can be detected at early stages. Some species of Protozoa that parasitize in human (e.g., Babesia microti, B. divergens [17], Trypanosoma cruzi [18], Plasmodium falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi [19], T. brucei [20]) and cause infectious diseases like babesiosis [17], Chagas disease [18], malaria [19], African trypanosomiasis [20], etc. can be detected in whole blood. Bloodstream infections [21] also can be diagnosed by the detection of microorganisms in the blood [22]. Theoretically, other emboli types such as a fat embolism [23] and blood clots [24] can be found by the analysis of a blood sample as well. Generally, there are two opposite approaches for the selection of target cell subpopulations from the entire population. The positive selection implicates the direct isolation of target objects from a general population. Oppositely, the negative selection means the exclusion of all objects except for the target [25]. Both of these methods have advantages and disadvantages. However, the negative approach is more eﬃcient for untypical object analysis in lymph or blood due to the exclusion of all objects except for embolus. The signiﬁcant step towards the isolation of rare blood circulating objects was the invention of the Fluorescence Activated Cell Sorter (FACS) by Bonner, Sweet, Hulett, Herzenberg et al. in the 60s of the last century [26]. Development of new ﬂuorophores and methods of labeling diﬀerent cell structures allowed for sorting cells according to many features and selection of small subpopulations and even single cells [27]. Currently, there are a number of methods based on the physical and biological properties of cells, allowing their sorting. Here we review the modern methods and approaches used for ﬂow cytometer design, cell labeling, their viability evaluation, and cell sorting along with other methods to separate cell subpopulations and the automatic approaches for following data analysis based on machine learning and deep learning methods. 2.1. Illumination Subsystem Lasers are the excitation light sources for virtually every modern ﬂow cytometer. They should provide stable, monochromatic, coherent light for both forward- and side scatter channels of detection as well as to excite various ﬂuorescent probes containing in cells to identify them and to investigate their morphology, cell cycle state, etc. [28] Although the ﬁrst cytometers were based on lamp sources like mercury lamps, with the technology development they were replaced by the lasers due to their higher stability and the ability to produce highly coherent light. Int. J. Mol. Sci. 2020, 21, 2323 3 of 60 About 40 years have gone since the creation of the ﬁrst 488 nm laser, nevertheless, blue-green argon-ion lasers are still the most frequently used because of the high variety of ﬂuorescent labels excited at this wavelength: ﬂuorescein, acridine, and their derivatives, cell viability dyes Calcein AM and propidium iodide, etc. [29] However, with the development of cytometry, the number of new ﬂuorochromes increased, which caused further production of lasers with diﬀerent wavelengths, from ultraviolet to infrared. Currently, the excitation of almost full UV–visible spectrum is provided by the combination of earlier gas sources and modern solid-state lasers [30]. Nevertheless, the combination of only three of them (ultraviolet, 488 nm, and red diode) in one ﬂow cytometer could provide theoretically the ability to analyze up to 17 existing ﬂuorescent labels and could also give access to ﬂuorochromes previously unavailable on usual instruments. The employment of additional lasers, in turn, can increase the number of simultaneously measured parameters, so advanced ﬂow cytometers support the introduction of up to 10 lasers with diﬀerent wavelengths to maximize sensitivity and allow tuning of excitation conditions to the precise experiments. 2.1.1. Laser Separation The choice of the laser for each cytometer is limited by a number of technical parameters that should be taken into account. First, the two or more excitation sources used in one ﬂow cytometer must be separated to allow the interrogation of cells and stream by multiple lasers. In this case spatially and temporally separated laser beams could be used: each laser focuses on its own point of the stream when appropriate collection pinholes are aligned to the particular part of the ﬂow channel. This scheme can include seven or more lasers and collection channels simultaneously. Another separation way is used in commercial cytometers like Accuri™/Accuri™Plus (BD Biosciences, San Jose, CA, USA) and Guava®easyCyte™(Luminex, Austin, TX, USA). There are several collinear lasers, temporally separated to avoid overlapping of the ﬂuorochrome emission and the laser excitation light at the same wavelength, for example, in a combination of PE-Cy5 dye and red HeNe laser. Here the subsequent picosecond activation of each laser is used with a synchronized simultaneous activation of the corresponding detector. 2.1.2. Laser Type There are two main groups of lasers integrated into ﬂow cytometers, which are the earlier generation of gas lasers (water-cooled and then air-cooled) and modern solid-state lasers. The ﬁrst produced 488 nm laser had the argon-ion active medium and water cooling. Since it could produce only a single wavelength excitation, more advanced cell sorters were equipped by krypton-ion lasers, which were able to generate high power signals up to several hundred mW at a number of various wavelengths. Further, the water-cooled lasers were replaced by air-cooled, which, however, have a lack of high-power output signal (10–30 mW). One of them, helium-neon (HeNe), took its place in many ﬂow cytometers by generating an important red 633 nm laser line along with less powerful green (543 nm), yellow (592 nm), and orange (612 nm) lines. After the development of modern solid-state light sources: laser diodes and diode-pumped solid-state (DPSS) lasers they have mostly replaced the gas lasers. Direct diodes use a semiconductor heterostructure to directly produce a laser line when DPSS source pumps a solid-state nonlinear medium by an infrared laser and generates output wavelength by doubling, tripling and quadrupling of the initial one. 2.1.3. Beam Shape and Quality Beam shape, quality, and laser noise level are critical parameters for virtually any ﬂow cytometer. There are two types of laser beam proﬁles: a single-mode having a single circular or elliptical spot and a multi-mode providing multiple spots along one optical axis. Typically ﬂow cytometers require a single-mode beam with a circular Gaussian transverse electronic mode (TEM00) characteristic. Some ﬂow cytometers can be equipped with optics allowing for an additional reshaping of the beam into an Int. J. Mol. Sci. 2020, 21, 2323 4 of 60 elliptical one. A single spot is easily focused on the stream while mixing of prisms and cylindrical lenses can provide a ﬂat-top proﬁle with sharper edges of the beam. This mode is excellently generated by gas lasers and DPSS laser sources. The multi-mode beam generating by a direct diode is more powerful but has a complex beam proﬁle with several peak patterns. In this form, it can not be used in ﬂow cytometry as complex beam handling may cause large power losses. However, the beam proﬁle can be signiﬁcantly improved if passed through cylindrical lenses or anamorphic prism pairs. Laser noise level refers to the degree of its stability in the time interval the cell passes through the exposed region. It should not exceed 0.5% peak-to-peak signal amplitude and can vary from 20 kHz to 20 MHz with respect to many factors, including the laser medium, power supply, electronics, and laser self-regulation [30]. Although gas lasers and diodes have self-regulating light control mode, for the DPSS and some diode sources the temperature control is a critical issue and can inﬂuence the resulting laser noise. It also limits the applications of the particular laser because of the decrease in resolution with noise growth. However, modern low-noise ﬂow cytometers allow us to carry out successfully precise measurements like DNA analysis and immunophenotyping. One more important parameter is laser collimation, corresponding to the degree of divergence of the laser beam after it leaves the output. Currently available commercial sources integrated into ﬂow cytometers provide a well-collimated beam even several meters from the output that is suﬃcient for their further focusing and shaping. 2.1.4. Laser Delivery The delivery of laser beam to the stream and light intersect region can occur in two ways: in free space, using prisms, dichroic mirrors, and long-pass ﬁlters to merge and steer multiple beams and focus them on the stream; and in single-mode ﬁber optics. Using ﬁber does not require any additional expensive optic elements, provides alignment stability and is generally safer in comparison to free space sources. Any defects in the beam proﬁle can be ﬁxed by cutting the edges of spatial distribution, however, this leads to losses in the resulting power. The FACSAria™(BD Biosciences) cytometer uses a ﬁber system for laser delivery. However, there are some disadvantages including signiﬁcant power losses for UV and violet laser wavelengths and the expensive ﬁber materials, degrading with time. Therefore, modern ﬂow cytometers like Amnis® ImageStream®X Mark II (Luminex) and CytoFLEX® (Beckman Coulter, Brea, CA, USA) system still include free-space laser delivery. Additionally, high-quality damage-resistant coated dichroic mirrors are required to work in the UV range. 2.1.5. Laser Coherence The spectral width of the line that laser emits is a critical parameter as well. This value does not exceed 1 nm for highly coherent lasers, although the light, produced by the laser diodes, has the main intensive part at one wavelength and slightly less intensive background at lower and higher wavelengths. For the application of these lasers, modern cytometers are equipped with additional narrow width notch ﬁlters that make laser line essentially monochromatic. 2.2. Optical Arrangement Although, the image resolution in currently available commercial cytometers is diﬀraction-limited, there are some new developing techniques to overcome this limit [33]. itation source and detection beam path. Diffusion of light at small angles (forward scatte annel, FSC), where the light is collected between 0.5–8° to the optical axis, is used to determine l size [31]. At the side scatter channel (SSC), the light is collected orthogonally to the optical d represents a combination of the diffusion, reflection, and refraction components that are rela cell morphology, granulation, and other structural properties. A high numerical aperture objec s collects either emitted fluorescence, scattered and transmitted light from the cells in a spec ge of 420-800 nm [32], and then relayed it to the spectral decomposition part. This part consist tack of five dichroic long-pass filters with progressively longer cut-off wavelength placed in fr a backing mirror. Each of them reflects a certain wavelength band towards a specific detecto rts of a camera sensor. As a result, signals from each cell are separated into several sub-ima ghtfield, side scatter and the set of fluorescent images, corresponding to the different spec nds. This assembly generates simultaneously six spectral fluorescent images of each cell ows by the second objective lens, focusing this light at the time delay integration (TDI) cam d forming images in a row on the detector. Latest ImageStream®X (Luminex) systems support meras simultaneously, providing up to 12 images per object at a 60x magnification, with fraction-limited resolution of an image. The lasers are staffed correspondingly to suppress ss-talk between two cameras. Although, the image resolution in currently available commer ometers is diffraction-limited, there are some new developing techniques to overcome this li ]. Figure 1. The optical system of an imaging flow cytometer. Figure 1. The optical system of an imaging ﬂow cytometer. igure 1. The optical system of an imaging flow cytomete Figure 1. The optical system of an imaging ﬂow cytometer. Another optical arrangement is realized in the opensource imaging cytometer SPI [34]. T sign makes extensive use of the existing SPIM structured light imaging system [35,36] and a w cell with the appropriate fluidic system based on the syringe pump. 2.2. Optical Arrangement Since modern cytometers provide both brightﬁeld and ﬂuorescence imaging of ﬂowing objects, they provide identiﬁcation of the single cell according to the distribution of ﬂuorescent markers and the cell morphology. However, there are several optical schemes for visualization of ﬂowing objects in real-time. The ﬁrst kind of imaging optical system is realized in the Amnis® (Luminex) imaging ﬂow cytometer, which was designed to enhance the ﬂuorescent sensitivity in comparison to ﬂuorescent microscopes and provide high-speed operation comparable with conventional ﬂow cytometers (up to several thousands of objects per second). The basic scheme of the optical system is presented in Int. J. Mol. Sci. 2020, 21, 2323 gure 1. The light sheet i ovides scattered light (fo 5 of 60 tion reg th diffe Figure 1. The light sheet illumination of cells ﬂowing one-by-one through the illumination region provides scattered light (forward and side channel) and the set of ﬂuorescent channels with diﬀerent excitation source and detection beam path. Diﬀusion of light at small angles (forward scattered channel, FSC), where the light is collected between 0.5–8◦to the optical axis, is used to determine the cell size [31]. At the side scatter channel (SSC), the light is collected orthogonally to the optical axis and represents a combination of the diﬀusion, reﬂection, and refraction components that are related to cell morphology, granulation, and other structural properties. A high numerical aperture objective lens collects either emitted ﬂuorescence, scattered and transmitted light from the cells in a spectral range of 420-800 nm [32], and then relayed it to the spectral decomposition part. This part consists of a stack of ﬁve dichroic long-pass ﬁlters with progressively longer cut-oﬀwavelength placed in front of a backing mirror. Each of them reﬂects a certain wavelength band towards a speciﬁc detector or parts of a camera sensor. As a result, signals from each cell are separated into several sub-images: brightﬁeld, side scatter and the set of ﬂuorescent images, corresponding to the diﬀerent spectral bands. This assembly generates simultaneously six spectral ﬂuorescent images of each cell and follows by the second objective lens, focusing this light at the time delay integration (TDI) camera and forming images in a row on the detector. Latest ImageStream®X (Luminex) systems support two cameras simultaneously, providing up to 12 images per object at a 60x magniﬁcation, with the diﬀraction-limited resolution of an image. The lasers are staﬀed correspondingly to suppress the cross-talk between two cameras. 2.3. Camera Most ﬂow cytometers employ imaging systems based on charged coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) sensors, which have a number of diﬀerences [37]. Additionally, point detectors such as photomultiplier tube (PMT) or avalanche photodiode can be employed. Diﬀerent sensor types solve the trade-oﬀbetween temporal resolution and spatial resolution. In comparison to point detector systems, camera-based ﬂow cytometers oﬀer direct imaging of single cells along with the information on morphology and texture at a single-cell level. The main challenge in imaging systems is the minimization of optical blur occurred due to the high rate of cell movement [38]. CCD sensors are typically composed of a two-dimensional array of metal-oxide-semiconductor CCD cells where every diode provides a charge build-up during image capture. Each photodiode serves as a discrete sensor that corresponds to the pixels of the ﬁnal image. Therefore, the size of the imaged object is deﬁned by the size of the photodiode. The CCD cameras are limited by the sequential transfer time, which determines the rate of the image acquisition as all pixel charges must be transferred before a new image can be captured. In a number of commercial imaging ﬂow cytometers (such as Amnis® (Luminex), IN Cell Analyzers (GE Healthcare Life Sciences, Little Chalfont, UK), ImageXpress® (Molecular Devices, San Jose, CA, USA) CCD sensors accompanied by wide-ﬁeld illumination. Alternatively, ImageStream® (Luminex) and FlowSight® (Luminex) cytometers employ high-speed time delay integration CCD cameras [39,40]. This type of CCD sensor was designed to image ﬂuorescent objects moving along one axis, which is provided by using multiple rows of sensors pixels that shift charge synchronously with the movement of an object along the same axis. This approach allows increasing the signal from each object by several orders of magnitude (from the microsecond scale to the millisecond scale) without an increase of noise, thus leading to high sensitivity of the system. Similar to CCD, CMOS sensors also employ a two-dimensional array of photodetectors to perform photoelectric conversion of detected signals. The main diﬀerence comparing with CCD is CMOS sensors amplify the signal at each sensor, followed by the storage of the overall information. This approach signiﬁcantly reduces the time of single image acquisition. Additionally, each pixel acts as an individual ampliﬁer increasing the pixel-to-pixel variability. Further, in 2016 Fairchild Imaging improved the design of the CMOS sensors resulting in low read noise and high quantum eﬃciency (up to 82%). 2.2. Optical Arrangement The optical system, in e, consists of a laser collimation scheme forming a light-sheet either by a Bessel beam and scann with the galvo mirror or by static arrangement where the light-sheet is formed by pair of cylindr s and objective lens. Then the usual microscopic tube is placed perpendicular to the light-sh ne that consists of another objective lens with better resolution/higher numerical apertu Another optical arrangement is realized in the opensource imaging cytometer SPI [34]. This design makes extensive use of the existing SPIM structured light imaging system [35,36] and adds ﬂow cell with the appropriate ﬂuidic system based on the syringe pump. The optical system, in this case, consists of a laser collimation scheme forming a light-sheet either by a Bessel beam and scanning it with the galvo mirror or by static arrangement where the light-sheet is formed by pair of cylindrical lens and objective lens. Then the usual microscopic tube is placed perpendicular to the light-sheet plane that consists of another objective lens with better resolution/higher numerical aperture, intermediate camera tube lens, and scientiﬁc camera. Int. J. Mol. Sci. 2020, 21, 2323 6 of 60 2.3. Camera The new design was named scientiﬁc CMOS (sCMOS) [41]. The choice between CCD and sCMOS cameras depends on the particular task and should take into account the pixel size, frame rate, quantum eﬃciency, spectral response, dynamic range, and noise of the sensor. In general, sCMOS cameras are characterized by higher frame rate, enhanced dynamic range, and larger ﬁeld of view (up to 4–16 times larger area), while CCD provides higher sensitivity and uniformity over the sensor, which is important for super-resolution and multiphoton applications [42]. Modern electron-multiplying CCD (EM-CCD) sensors have a better low-light sensitivity and increased dynamic range in comparison to CCD, but they have a larger pixel size [43]. The sCMOS sensors also oﬀer an option to improve imaging quality. For instance, the sCMOS sensor can operate in a global shutter mode (all pixels are measured simultaneously) and rolling shutter mode (sensor is read line by line and an only single line is active) [44]. Using a rolling shutter can improve contrast and increase image quality, however, it can suﬀer from image artifacts when measuring high-speed moving objects. Both CCD and CMOS detectors may be combined with light-sheet illumination to employ optical sectioning to reduce photo-induced stress of the biological samples. The light-sheet approach uses illumination made perpendicularly to the axis of observation [45]. The excited ﬂuorescence from the light sheet is then projected onto an imaging sensor [46]. The main advantage of light-sheet is the relatively low irradiation of ﬂuorescent molecules during the image acquisition in combination with the good resolution and image contrast that makes it very popular in imaging in vivo, measurement of rapidly moving and changing objects with the minimum damage [47,48]. On the other hand, the employment of camera-based systems may be limited by the weak ﬂuorescent signal and the high speed of cell ﬂow. To overcome this, a number of PMT-based detectors Int. J. Mol. Sci. 2020, 21, 2323 7 of 60 are employed. PMTs are equipped with a light-sensitive photocathode that converts light into photoelectrons, which are further ampliﬁed by acceleration by dynodes voltage [49,50]. Although the quantum eﬃciency of PMTs is not high (about 20%), they provide suﬃcient ampliﬁcation (up to thousands of times) of the detected signal. 2.3. Camera To date, multiple discrete PMT detectors are employed in most of confocal laser scanning microscopes (CLSM) to detect ﬂuorescent signal from multiple ﬂuorescent probes (up to ﬁve simultaneously) [51] and in imaging ﬂow cytometers, such as Acumen® (LabTech, Melbourn, UK), iGeneration instruments (CompuCyte, Westwood, MA, USA), and iCyte® (ThorLabs, Newton, NJ, USA). These systems employ optical time-stretch imaging in order to capture fast objects in real-time at the rate of tens of MHz. PMTs oﬀer higher sensitivity and higher dynamic range due to electron multiplication and gain adjustment within the sensor. PMT-based ﬂow cytometers also demonstrate higher bandwidth with reduced noise in comparison to CCD and sCMOS sensors, which helps to increase throughput. However, PMT does not provide spatial information, which limits the number of its applications. Some systems combine PMTs with high-speed microscopy laser scanning techniques in order to provide full information from the sample. For instance, Cellomics® ArrayScan® VTI (Thermo Scientiﬁc, Waltham, MA, USA), and Operetta® (Perkin Elmer, Waltham, MA, USA) commercial ﬂow cytometers employ CLSM schemes. In conventional CLSM, single-pixel detectors or PMTs are employed to collect the signal. A specimen is scanned pixel by pixel over the surface by the focused spot to build an entire image [51]. Additionally, the so-called multi-ﬁeld of view imaging ﬂow cytometer (MIFC) method can be applied to provide imaging of multiple channels simultaneously, which signiﬁcantly increases throughput [52]. Mckenna et al. developed a microﬂuidic ﬂow cytometer containing 384 parallel channels with one-dimensional multicolor confocal ﬂuorescence imaging to provide a collection of information from rare-cell samples [53]. A combination of point detector with an optoelectronic image-encoding/decoding time-stretching method allows for reaching high image quality and increased throughput [54]. Mapping images into the radio frequency spectrum using conventional cameras provided increased readout methods comparing to usual methods [55]. The ﬂow cytometry system based on precisely-deﬁned focal spot and a line scan CMOS sensor was ﬁrst developed by Heng in 2011 and allowed the parallel collection of multiple ﬂuorescence channels with high resolution but low throughput (1000 cells/s) [56]. 3.1. Fluorescent Labeling Fluorescent labeling is probably the most common and well-developed technique to distinguish one type of cell from others. Currently, a great number of ﬂuorophores with diﬀerent spectral properties are widely used in microbiology, cell biology, and medicine. Modern ﬂuorescent dyes are used to label speciﬁc structures on the surface and inside the cell and to estimate the rate of metabolic processes. In addition, vector-encoded ﬂuorescent proteins are also used for cell labeling. 3. Cell Labeling The most straightforward and reasonable approach to distinguish one type of cell from another one is the direct labeling of cells with an appropriate and well detectable label. At the moment, there is a number of methods used for cell labeling, based on which the separation of target cells from the bulk can be performed. Either ﬂuorescent, magnetic, or acoustic properties of various tags are successfully applied for cell sorting. This section highlights the basic currently used cell markers. 3.1.1. Fluorescent Label Conjugated Antibodies A variety of fluorescent labels to conjugate with antibodies is available, including small-molecule organic dyes, quantum dots, fluo e e t otei oly e dye a d ta de oly e dye Thus, the strategy of cell labeling is based on the fact that speciﬁc cell populations express deﬁned surface markers that are unambiguously identiﬁed by the corresponding antibodies conjugated with a ﬂuorescent label. The main beneﬁt of this approach is the possibility of direct cell labeling in vivo by the injection to the blood circulation [63–65]. A variety of ﬂuorescent labels to conjugate with antibodies is available, including small-molecule organic dyes, quantum dots, ﬂuorescent proteins, polymer dyes, and tandem polymer dyes. fluorescent proteins, polymer dyes, and tandem polymer dyes. Small-molecule organic dyes are the simplest and most reasonable choice to conjugate to antibodies. They are commonly used due to relatively good photostability and ease of preparation. Indeed, the protein conjugation protocols are well established, and there are commercially available conjugation kits allowing to attach the most common dyes to the desired antibody as well as already conjugated antibodies that may be purchased from suppliers. There are several widely used small- molecule organic dyes. Among them, probably the most popular are derivatives of fluorescein, h d i d i d Small-molecule organic dyes are the simplest and most reasonable choice to conjugate to antibodies. They are commonly used due to relatively good photostability and ease of preparation. Indeed, the protein conjugation protocols are well established, and there are commercially available conjugation kits allowing to attach the most common dyes to the desired antibody as well as already conjugated antibodies that may be purchased from suppliers. There are several widely used small-molecule organic dyes. Among them, probably the most popular are derivatives of ﬂuorescein, rhodamine, and cyanine dyes. rhodamine, and cyanine dyes. Fluorescein and its derivatives exhibit fluorescence due to a multi-ring π-conjugated aromatic xanthene structure [66]. The absorption maximum of the dye is in the region of 488–495 nm and, thus, can be effectively excited by the argon 488 nm laser. The maximum of emission spectra is typically between 512 and 525 nm with a relatively high quantum yield [67,68]. The fluorescein derivatives are usually prepared by the substitution of either fifth or sixth carbon of the lowest ring. Fluorescein-5- isothiocyanate (FITC) is the most popular fluorescent label ever created. 3.1.1. Fluorescent Label Conjugated Antibodies Attachment of ﬂuorescent labels to cell populations of interest via antibodies is widely employed in cytometry and cell imaging analysis. Generally, antibodies are immunoglobulins with a similar structure (Figure 2) including heavy (H) and light (L) chains linked to each other by disulﬁde bonds and are composed of the variable (V) and constant (C) regions [57,58]. Polymeric immunoglobulins have an additional J chain responsible for polymerization [57,59]. Antibodies are able to bind with a variety of antigens due to V regions, which is also known as fragment antibody binding (Fab) regions, Int. J. Mol. Sci. 2020, 21, 2323 I t J M l S i 2019 20 FOR 8 of 60 8 of 60 at the ends of H and L chains [57,58]. Antigen-antibody interactions depend on the three-dimensional structure of the Fab region determined by the amino acid sequence and their charge [60]. The aﬃnity of antigen-antibody binding is driven by entropic (hydrophobic) and enthalpic (Van der Waals, hydrogen bonding, salt bridge) eﬀects, or by entropy-enthalpy compensation [61]. Additionally, to describe the mechanisms of antigen-antibody interaction two main theories (“Key-Lock” and “Induced Fit”) based on a conformational change of proteins were derived [61,62]. at the ends of H and L chains [57,58]. Antigen-antibody interactions depend on the three-dimensional structure of the Fab region determined by the amino acid sequence and their charge [60]. The affinity of antigen-antibody binding is driven by entropic (hydrophobic) and enthalpic (Van der Waals, hydrogen bonding, salt bridge) effects, or by entropy-enthalpy compensation [61]. Additionally, to describe the mechanisms of antigen-antibody interaction two main theories (“Key-Lock” and “Induced Fit”) based on a conformational change of proteins were derived [61,62]. Figure 2. Sketch of an antibody structure. Figure 2. Sketch of an antibody structure. Figure 2 Sketch of an antibody structure Figure 2. Sketch of an antibody structure. Thus, the strategy of cell labeling is based on the fact that specific cell populations express defined surface markers that are unambiguously identified by the corresponding antibodies conjugated with a fluorescent label. The main benefit of this approach is the possibility of direct cell labeling in vivo by the injection to the blood circulation [63-65]. 3.1.1. Fluorescent Label Conjugated Antibodies It contains sulfonate groups in a third and ﬁfth position of the lowest ring that can form stable sulfonamide bonds. Texas Red has the maximum absorption at 589 nm and the maximum of emission at 615 nm, along with a relatively high quantum yield. Since the maximum of absorption and emission are shifted to the red region, Texas Red perfectly suits the dual-mode ﬂuorescent imaging combining with ﬂuorescein dyes. Conventional cyanine dyes generally consist of two cationic ring structures, including nitrogen atoms, and one of them must carry a positive charge while the rings are linked with an unsaturated polymethine chain [78]. The ring structure may vary from single rings containing ﬁve or six atoms in the cycle to complex conjugated structures consisting of several aromatic rings. The length of the polymethine chain also varies and may include from one to seven carbon atoms indicating diﬀerent subfamilies of cyanine dyes with various spectral properties. Therefore, Cy3 dyes usually absorb and emit light at 500 nm band, whereas Cy5 and Cy7 dyes have maximums of absorption and emission at 600 nm and 700 nm bands respectively. An additional modiﬁcation of the ring structure allows for ﬁne-tuning of spectral properties by shifting the spectral maximums to the blue or red region. However, the most attractive cyanine dyes are those with the spectral maximum of emission in the near-infrared (NIR) region due to the great suitability for bioimaging applications. Thus, the structure of the dyes keeps being optimized to design the dyes with the enhanced properties. For instance, Li et al. designed a NIR frequency upconversion Cy7-NO2 probe for the detection of a nitroreductase enzyme based on Cy7 dye [79]. Ge and Tian reported on the ﬂuorescent probe based on cyanine dye coupled with Zn-Dpa recognition units to monitor p-tau protein in a single neuron [80]. In order to make cyanine dyes suitable for proteins conjugation, the sulfonated groups are introduced to their structure. Generally, commercially available cyanine dyes have from two to four sulfonated groups for the optimal conjugation in aqueous solutions. Quantum dots (QDs) are nanosized semiconductor crystals that emit photoluminescent light due to electron-hole recombination [81]. QDs have several advantages comparing to conventional small-molecule ﬂuorophores. First, QDs combine high quantum yield (0.1–0.9) with a large molar extinction coeﬃcient (105–107 M−1cm−1) that results in a bright luminescence [82]. 3.1.1. Fluorescent Label Conjugated Antibodies The conjugation of a protein molecule with the FITC label is driven by the formation of isothiourea linkage between the protein primary amine group and the FITC isothiocyanate group [69]. Recently, Chaganti et al. described the modified protein FITC labeling technique employing tandem affinity purification tags at the N- and Fluorescein and its derivatives exhibit ﬂuorescence due to a multi-ring π-conjugated aromatic xanthene structure [66]. The absorption maximum of the dye is in the region of 488–495 nm and, thus, can be eﬀectively excited by the argon 488 nm laser. The maximum of emission spectra is typically between 512 and 525 nm with a relatively high quantum yield [67,68]. The ﬂuorescein derivatives are usually prepared by the substitution of either ﬁfth or sixth carbon of the lowest ring. Fluorescein-5-isothiocyanate (FITC) is the most popular ﬂuorescent label ever created. The conjugation of a protein molecule with the FITC label is driven by the formation of isothiourea linkage between the protein primary amine group and the FITC isothiocyanate group [69]. Recently, Chaganti et al. described the modiﬁed protein FITC labeling technique employing tandem aﬃnity puriﬁcation tags at Int. J. Mol. Sci. 2020, 21, 2323 9 of 60 the N- and C-termini of the target protein [70]. Currently, FITC is widely used for labeling various entities of biological nature, such as peptides [71,72], antibodies [73,74] and, polysaccharides [75]. Rhodamine structure is similar to that of ﬂuorescein except for the oxygen atoms in the outer rings are substituted by the nitrogen. All rhodamine-based labels are derivatives of this primary structure. The most widely used modiﬁcation for biomolecule labeling and further ﬂuorescent imaging is tetramethylrhodamine-5-isothiocyanate (TRITC) [76]. Comparing to the initial rhodamine molecule, TRITC has two methyl groups attached to each nitrogen atom on the outer rings and ﬁfth carbon atom at the lowest ring-substituted to isothiocyanate group. The TRITC molecule has the spectral maximum of absorbance at 545 nm, and the emission maximum at 570 nm. The mechanism of TRITC conjugation with protein molecules is the same as for FITC due to the isothiocyanate functional group [69]. In comparison with FITC, TRITC has a smaller quantum yield, however, it is more photostable, and its ﬂuorescence is less aﬀected by an external medium. Additionally, the TRITC emission band is better for ﬂuorescent imaging in vivo or in bioﬂuids. Texas Red (sulforhodamine 101) is another rhodamine derivative widely used for ﬂuorescent protein labeling [77]. 3.1.1. Fluorescent Label Conjugated Antibodies QDs have a broad absorption band and narrow symmetric ﬂuorescence spectra (full width at a half-maximum of about 25–35 nm) shifted from the absorption peak to 100 nm [82]. This allows to avoid spectral overlapping during multiplex detection experiments and thus less compensation is required between QD channels. The spectral maximum (“color”) of emitted light may be adjusted by the size and material of QD crystals [83]. Finally, QDs are much more photostable and have longer lifetimes of the excited state that makes them more suitable for dynamics tracking experiments [84]. Typically, QDs are referred to as I−VI, III−V, and IV−VI binary compounds and their core/shell structures [85]; however, these compositions have biocompatibility issues. With this regard, in recent years, the QDs prepared of carbon materials attract much attention [86,87]. Int. J. Mol. Sci. 2020, 21, 2323 10 of 60 However, the main diﬀerence between QDs and organic dyes is in protein conjugation. Small-molecule ﬂuorophores typically have single reactive group coupling with speciﬁc sites on a protein molecule [88]. The general principle is to mix the excess of the dye with the target molecules. As the size of ﬂuorescent labels is comparable with the size of amino acid residues, the target protein structure generally remained the same, preserving the speciﬁcity and avidity of the antibody. Unlike the small-molecule dyes, QDs have a much larger size and a considerable surface area that can be modiﬁed by the biomolecules at many diﬀerent sites as well as with irregular orientation of the antibodies on the QD surface, which aﬀects biological activity [89]. This makes challenging the conjugation of QDs with antibodies regarding eﬀective cell labeling and sorting. However, there are two strategies of QD controllable conjugation. These are covalent conjugation methods and self-assembly based techniques. The covalent conjugation implies the formation of covalent bonds between the target antibody and QD surface ligands. Although conventional covalent conjugation does not allow for the control over the number of conjugated proteins and their orientation, the recent advances in developing of chemoselective ligation [90,91] and bio-orthogonal [92] reactions have resulted in eﬀective and controllable conjugation of QDs and biomolecules [93,94]. On the other hand, self-assembly based conjugation employs speciﬁc high-aﬃnity interactions without the formation of covalent bonds. The typical example of this speciﬁc recognition is tightly binding between biotin (vitamin B7) and avidin-like proteins that are known as the strongest non-covalent bond in nature [95]. 3.1.1. Fluorescent Label Conjugated Antibodies The basic strategy for conjugation is a modiﬁcation of the QD surface with avidin-family protein that binds with a biotinylated molecule of interest [96]. To date, there are commercially available kits to attach biotin to any biomolecule as well as QDs modiﬁed with streptavidin for the following speciﬁc binding. However, conjugation via biotin-avidin binding is limited in control over conjugation valence (i.e., the number of molecules attached to a QD) [97] and orientation of antibodies. Currently, one of the emerging self-assembly strategies for QD bioconjugation providing the best overall control is the exploiting of polyhistidine tag [98]. Polyhistidine is a sequence in a protein molecule consisting of six histidine residues with an aﬃnity for divalent metal cations. Polyhistidine tag is typically bonded with a molecule of interest and then coupled to metal atoms on the QD surface. The advantage of polyhistidine self-assembly is the fact that it does not require additional QD surface modiﬁcation, employs well-established protocols and provides reasonable control over resulted stoichiometry [99]. Currently, the preparation of more sophisticated polyhistidine tags is carried out providing less steric hindrance during assembly with QDs and better binding aﬃnity with biomolecules [100]. Fluorescent phycobiliproteins were considered as probes for immunoﬂuorescent experiments since the 1980s [101]. These naturally arisen ﬂuorophores are a part of the photosynthetic complex of certain cyanobacteria and algae. The main function of phycobiliproteins is to absorb and transfer the light energy to adjacent chlorophyll molecules for further utilization in the cell life cycle. Thus, they are naturally designed to have a large quantum yield (up to 0.98) and molar extinction coeﬃcient (2.4 × 106 M−1cm−1 at 545 nm for B-Phycoerythrin) that are far beyond of those of small-molecule dyes [69]. Typically, phycobiliproteins contain multiple chromophoric bilin groups that are linear tetrapyrroles covalently bonded to apoprotein (Figure 3). Depending on the number and organization of bilin groups, there are four main types of pigments in phycobiliprotein structure: yellow-colored phycourobilin, red-colored phycoerythrobilin, blue-colored phycocyanobilin, and purple phycobiliviolin [102]. The resulted protein spectral properties are deﬁned by the relative content of each pigment in the protein structure [103]. For instance, the two main forms of phycoerythrin employed as ﬂuorescent labels, which are B-Phycoerythrin and R-Phycoerythrin, contain 34 bilin groups and have a broad absorption band from green to yellow region of the visible spectrum with multiple peaks and maximum of emission at 575–578 nm. 3.1.1. Fluorescent Label Conjugated Antibodies This makes them perfect for bioimaging as their ﬂuorescent signal is detected in a red and near-infrared region along with variable excitation sources. Phycobiliproteins can be conjugated with antibodies, avidin, biotin, and other biomolecules [104–106] without losing their ﬂuorescent properties via common heterofunctional crosslinkers, including N-succinimidyl 11 of 60 iments Int. J. Mol. Sci. 2020, 21, 2323 binding affinity with bio Fluorescent phycob 3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB) [69]. The crosslinkers react with amine groups of phycobiliproteins producing activated sites for the coupling of sulfhydryl-containing molecules. The targeted biomolecule can be modiﬁed with thiol groups to complete the conjugation. However, despite their outstanding brightness, one should consider that phycobiliproteins are susceptible to photobleaching and are not well suitable for long-term dynamics monitoring. certain cyanobacteria and algae. The main function of phycobiliproteins is to absorb and transfer the light energy to adjacent chlorophyll molecules for further utilization in the cell life cycle. Thus, they are naturally designed to have a large quantum yield (up to 0.98) and molar extinction coefficient (2.4 × 106 M−1cm−1 at 545 nm for B-Phycoerythrin) that are far beyond of those of small-molecule dyes [69]. Typically, phycobiliproteins contain multiple chromophoric bilin groups that are linear tetrapyrroles covalently bonded to apoprotein (Figure 3). Figure 3. Chemical structure of phycoerythrin bilin chromophore. Figure 3. Chemical structure of phycoerythrin bilin chromophore. Recent advances in polymer chemistry and the development of organic optoelectronic systems gave rise probably for the most sophisticated type of ﬂuorescent labels that are engineered polymer dyes. Analogous to phycobiliproteins, polymer dyes consist of a number of optical subunits that are able collectively to absorb light and respond to quenching. Structurally, polymer dyes are specially designed π-conjugated polymers with multiple π-orbitals that can absorb and emit light due to electron delocalization similar to small-molecule organic dyes [107]. However, unlike small-molecule dyes, the absorption and emission of light by polymer dye molecule co-occurs in many sites over the polymer chain. This multiple impact results in relatively high quantum yield (up to 0.65) and a molar extinction coeﬃcient comparable to that of QDs and phycobiliproteins (about 106 M−1cm−1). Furthermore, the length and structure of the polymer chain can be modiﬁed to adjust the spectral properties of the resulted dye. 3.1.2. Cell Tracking Dyes Fluorescent cell labeling can be performed by using ﬂuorescent cell tracking dyes. These dyes allow for analyzing cell cycle, migration, proliferation, permeabilization of the membrane, etc. by staining diﬀerent components of the cell. Furthermore, the dyes being incorporated into the live cells allow for their distinguishing without aﬀecting cell metabolic activity. Coupling of tracking dyes with antibodies provides the additional possibility to carry out a phenotype analysis due to the formation of the dye-antibody combination. Depending on the aﬃnity, ﬂuorescent dyes are divided into the three main groups that are nucleic acid, cytoplasmic, and membrane-bound dyes. Nucleic acid dyes typically bind with DNA and/or RNA. They are generally used to quantify DNA and to observe cells distribution during the cell cycle (Propidium Iodide, 7-Aminoactinomycin D or 7-AAD, DyeCycle Violet, 4′,6-Diamidino-2-phenylindole or DAPI, Hoescht 33342), to estimate cell viability (PI, DAPI), to isolate chromosomes for sorting (Hoescht 33342, Chromomycin A3), to quantify apoptotic cell fractional DNA content (Acridine Orange or AO, 7-AAD), etc. [109,110] These dyes are frequently used for cell migration and tracking analysis due to their high retention. Nevertheless, DNA-binding dyes are not applicable for proliferation analysis because the content (and intensity) of the dye decreases after each cell cycle [111]. A large family of ﬂuorochromes developed for bioimaging is prepared from “acridine derivatives”. The chemical structure of ﬂuorochromes from the acridine family is performed by several aromatic rings forming xanthene that produces a planar conﬁguration responsible for the binding to the speciﬁc target. Physico-chemical behavior is driven by the three-dimensional conformation of the target site. Other interactions, such as hydrogen and Van der Waals bonds, can enhance or reduce binding mode depending on the reaction conditions. The most widespread and frequently used ﬂuorochrome is Acridine Orange (AO). This dye is appreciated due to its metachromatic nature: according to the microenvironment, AO can vary the emission spectrum. It emits green ﬂuorescence when bound to double-stranded DNA and red ﬂuorescence when bound to single-stranded DNA or RNA. The excitation/emission maximum wavelengths are 460/650 nm for RNA-binding and 500/526 nm for DNA-binding analysis. This fascinating property provides its usage for diﬀerentiation of double- and single-stranded conﬁgurations that are in detail described in the literature devoted to cell kinetics, damage, and apoptosis. The AO dye can be suﬃciently excited by a 488 nm blue line of an argon laser. 3.1.1. Fluorescent Label Conjugated Antibodies Additionally, polymer dyes are designed to be very photostable and absorb light only of a speciﬁc wavelength (i.e., to have a narrow excitation band) that makes them suitable for multiplex and long-term detection experiments [108]. Polymer dyes can be conjugated with antibodies via conventional conjugation protocols as they are pre-synthesized with deﬁned speciﬁc binding groups providing well-controllable coupling considering both the number of conjugated labels and location of binding sites. The example of polymer dye is Brilliant Violet BV421 ﬂuorochrome. Additionally, phycobiliproteins and polymer dyes can be modiﬁed with other ﬂuorophores to create dyes with modulated spectral properties employing ﬂuorescence resonance energy transfer (FRET). The ﬂuorophores are combined to form donor-acceptor couples so that the emission band of one ﬂuorophore overlaps with the absorption band of another one. This results in extremely bright tandem dye structures with enhanced Stokes shift up 300 nm that can be eﬀectively excited by two laser sources. Formation of tandem dye structures may be employed to shift the emission of the initial ﬂuorophore to the red or near-infrared band, which is beneﬁcial for bioimaging applications, or in case of low antigen density. However, one should consider that tandem dyes are quite unstable and have rather variable energy transfer eﬃciency. The examples of tandem dyes are the conjugates of B-Phycoerythrin and Allophycocyanin phycobiliproteins; R-Phycoerythrin and Cy-5 dye; B-Phycoerythrin and Texas Red dye; the family of Brilliant Violet dyes based on BV 421 polymer core. Int. J. Mol. Sci. 2020, 21, 2323 12 of 60 12 of 60 3.1.2. Cell Tracking Dyes Thus, it is employed for cell cycle analysis analogous to DAPI. The mechanism of staining is similar to DAPI, as well as the excitation/emission wavelengths (361/497 nm, respectively). Propidium Iodide (PI) and Ethidium Bromide (EB) are the two main dyes of intercalating phenanthridine dye family to perform analysis of proliferation and cell viability. As they are not able to permeate the intact membrane of live cells, they are frequently used to diﬀerentiate dead cells in a population. Thus, staining with these dyes requires to ﬁx the cells or to permeabilize them for further DNA binding. As an acridine derivative, PI can bind both with double-stranded DNA and with double-stranded RNA chains due to interaction with amines within a cell to determine if the cell membrane is intact. The EB reacts in the same way. Furthermore, PI and EB have a speciﬁc spectral feature: they are almost non-ﬂuorescent as free molecules in aqueous solution but provide highly increased quantum eﬃciency when intercalated into nucleic acids. The free molecules excited by the adequate light tend to dissipate the energy to the surrounding polar water molecules. In turn, the intercalated molecules can only disperse the energy by the light emission. The excitation maximum of PI is 535 nm, which is appropriate for the 488 nm argon laser line, with the spectral maximum of emission at 617 nm. Although the large variety of new DNA-aﬃnitive dyes that have been developed in the last years, PI is still the most commonly used for cell cycle analysis and DNA/RNA detection. y y y y Cytoplasmic dyes have been widely used for cell migration and proliferation studies due to their longer retention in comparison with nucleic acid binding dyes [112–115]. However, they exhibit a higher level of cytotoxicity to cells as they bind to cytoplasmic proteins and therefore aﬀect the cellular enzyme functions [116]. The most prevalent cytoplasmic dyes are ﬂuorescein-based carboxyﬂuorescein succinimidyl ester (CFSE), calcein acetoxymethyl (calcein-AM), calcein violet acetoxymethyl (calcein violet AM), and resazurin-based alamarBlue, and CellTiter-Blue. The dyes of the ﬂuorescein group can easily penetrate the intact membrane of live cells due to their hydrophobic properties. Afterward, the dye molecules are hydrolyzed by intracellular esterases that leads to suﬃcient enhance of the intrinsic ﬂuorescence. Concerning calcein AM, after its transport into the cell, the esterases remove two acetoxymethyl groups, and non-ﬂuorescent molecule converts to a green-ﬂuorescent calcein. 3.1.2. Cell Tracking Dyes 7-Aminoactinomycin D or 7-AAD is the ﬂuorochrome that belongs to the group of dyes forming a complex with a guanine base (in the presence of magnesium ions). It selectively binds to guanine-cytosine regions of DNA and provides an explicit banding pattern in polytene chromosomes and chromatin for chromosome banding studies. This family also includes mithramycin, chromomycin, and olivomycin dyes with similar chemical behaviors. The absorption maximum is 546 nm, but 7-AAD is eﬀectively visualized by a 488 nm ﬂow cytometer laser line. This ﬂuorescent intercalator performs spectral shift upon binding with DNA and emits light in the far-red region, having an emission maximum at 647 nm. It makes nucleic acid stain useful for multicolor ﬂuorescence microscopy and ﬂow cytometry. 7-AAD is used for cell cycle analysis in bioimaging, but it encounters some obstacles during its utilization. As well as it cannot penetrate the intact cell membrane, it is generally excluded from live cells and proposed for apoptotic cell death study. Nevertheless, in the case of ﬁxed and permeabilized cells, the 7-AAD is applicable. Another group of DNA-binding ﬂuorochromes is presented by diamino-phenylindole derivatives; one of those is 4′,6-diamidino-2-phenylindole (DAPI). DAPI binds to adenine-thymine regions of DNA. This nuclear counterstain has the absorption maximum in the ultraviolet (UV) region and provides blue-ﬂuorescent emission at 470 nm. Although the spectral maximum of absorption is at 350 nm, DAPI can be excited by a 405 nm laser line while exhibits high quantum yield. The spectral properties make DAPI applicable for multicolor ﬂow cytometry experiments, especially in combination with green (FITC, GFP) and red (Rhodamine, Texas Red) ﬂuorophores. In bioimaging, DAPI is in common use for counting cells, estimation of apoptosis and cell viability, sorting cells based on DNA content due to high aﬃnity for DNA, and as nuclear segmentation tool in imaging analysis. Generally, this Int. J. Mol. Sci. 2020, 21, 2323 13 of 60 13 of 60 ﬂuorochrome can stain ﬁxed cells because of its impermanence, but at higher concentrations, it is possible to use DAPI for live cells. However, one should take into account that DAPI has a wide emission range (96 nm at a half-maximum) and may bind non-speciﬁcally when the staining period is longer than in the approved protocol. One more popular UV-excited live cell stain is Hoechst 33342. The dye can permeate the intact cellular membrane and bind to DNA even of live cells without their further damaging. 3.1.2. Cell Tracking Dyes The intensity of the resulted overall ﬂuorescent signal depends on the esterase activity that is directly proportional to the number of viable cells. In turn, the dead cells lack the active esterases that prevent a calcein-AM conversion to the ﬂuorescent form. The spectral maximum of calcein extinction is at 495 nm, and therefore, this dye can be eﬀectively excited by a ﬂow cytometry setup equipped with 488 nm argon laser. The spectral maximum of emission corresponds to 515 nm. Generally, calcein is exploited for a variety of studies, including cell adhesion, chemotaxis, drug resistance, cell viability, apoptosis, and cytotoxicity. Resazurin-based dyes, for example, AlamarBlue, are the type of cytoplasmic ﬂuorochromes, in which ﬂuorescent properties are caused by the reaction of chemical reduction. Once trapped by the live cell, resazurin reduces by mitochondria and converts to the ﬂuorescent form called resoruﬁn. Thus, the AlamarBlue dye has a 560 nm emission wavelength maximum with another band at 590 nm in the red region. High ﬂuorescence signal allows us to use it in ﬂow cytometry studies for cell viability, cytotoxicity and proliferation analysis. However, it should be noted that AlamarBlue ﬂuorescence may overlap with the ﬂuorescence regions of other compounds being tested. Additionally, it can provide a minor toxic eﬀect on the stained cells. Membrane-inserting ﬂuorochromes are represented by lipophilic dyes (dialkylcarbocyanine derivatives) that diﬀuse laterally within the cellular plasma membrane resulting in the entire cell staining at dye optimal concentrations. Dye molecules localize at the membrane surface anchoring to Int. J. Mol. Sci. 2020, 21, 2323 14 of 60 14 of 60 it by lipophilic “tails”. Carbocyanines have short alkyl tails that attached to the imine nitrogens; thus, they can be used both as membrane-potential sensors and as organelle stains for mitochondria and the endoplasmic reticulum. Those with longer alkyl tails provide long-term labeling of cells. In addition, this type of dye is likely to be less toxic than the previous (nucleic acid and cytoplasm-binding) as well as it possesses longer retention [117]. The main dialkylcarbocyanine dyes are 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-1-propenyl]-, perchlorate (DiO,) and 1,1’-dioctadecyl-3,3,3’3’-tetramethylindocarbocyanine perchlorate (DiI). The ﬂuorescence of these dyes is greatly enhanced after incorporation into membranes or binding to lipophilic biomolecules weakly ﬂuorescent in water, such as proteins. Along with it, dialkylcarbocyanine derivatives perform high molar extinction coeﬃcients, polarity-dependent ﬂuorescence, and short excited-state lifetime (~1 nanosecond). 3.1.2. Cell Tracking Dyes This is an advantage for ﬂow cytometry applications because it allows for more excitation/emission cycles during ﬂow transit. Additionally, dialkylcarbocyanines are quite photostable and provide a suitable imaging tool for ﬂow cytometry study of live cells due to the distinct ﬂuorescence colors. In particular, DiO emits green light (abs. 484 nm/em. 501 nm), DiI emits orange light (abs. 549 nm/em. 565 nm), DiD emits red light (abs. 644 nm/em. 663 nm), and ﬁnally, DiR emits deep red light (abs. 748 nm/em. 780 nm). Among them, DiO can be eﬀectively excited by 488 nm argon laser, and its ﬂuorescence spectrum is comparable with that of FITC, whereas the 633 nm He-Ne laser is convenient for DiD. TRICT and DiI can be visualized by the same optical ﬁlter sets due to their excitation/emission wavelengths similarity. Lipophilic carbocyanines as membrane-bound counterstains have been appreciated for their use to visualize membrane fusion and cell permeabilization, for cell migration and proliferation studies [116,118]. 3.1.3. Fluorescent Proteins One more approach to distinguish cells of interest is to make the cells ﬂuorescent themselves without external labeling that might potentially aﬀect their behavior. This is achievable through the modern molecular genetic methods allowing for the transfer of ﬂuorescent protein (FP) genes into the cells. The FP labeling for bioimaging is one of the most widespread applications of FPs. The general strategy for cell labeling is to include the nucleotide sequence of FP responsible for ﬂuorescence in the gene tailor the gene body of the labeled cell protein depending on its structure. In some cases, a ﬂexible Gly-rich linker between the FP and the labeled protein is used in order to prevent potential steric conﬂicts [119]. The most signiﬁcant advantage of FPs is the possibility to monitor the dynamics of the desired cell population in extremely long-term experiments as all next cell generations preserve bright ﬂuorescence, even after multiple divisions. Additionally, FPs are suitable to study the mobility of proteins inside a cell [120], for visualization of intracellular structures [121], and as markers to highlight a promoter activity in gene engineering and cell biology [122]. Furthermore, the FPs that can shift their spectral maximum of emission in time, thereby allowing them to determine their expression retrospectively, are of particular interest [123]. The FPs with DNA and RNA binding domains are used for real-time labeling and tracking of mRNA [124], DNA, and various structures associated with DNA [125–128]. The creation of cell lines expressing a certain amount of several ﬂuorescent proteins allows for carrying out multicolor cell labeling with more than 100 shades. This technique is applied in immunology, neurobiology, and transplantology [121]. Currently, several types of FPs are available with diﬀerent spectral properties determined by variations of their amino acid sequences [129] and, therefore, their structure (Figure 4). Generally, FPs are classiﬁed according to their spectral maximum of emission on the blue (BFPs; 440–470 nm), cyan (CFPs; 471–500 nm), green (GFPs; 501–520 nm), yellow (YFPs; 521–550 nm), orange (OFPs; 551–575 nm), red (RFPs: 576–610 nm), and far-red (FRFPs; 611–660 nm) FPs [130]. However, despite the variety of “colors”, the blue, cyan, and yellow emitting FPs are mutated allelic forms of GFP, which was ﬁrst isolated from the jellyﬁsh Aequorea Victoria [131]. GFP contains a ﬂuorophore group that includes 15 of 60 15 of 60 Int. J. Mol. Sci. 3.1.3. Fluorescent Proteins The RFP has broad excitation and emission spectra with the corresponding maximums at 558 nd 583 nm [143]. Variations in the amino acid sequence of RFP, for instance, the mutation of lysine o methionine at location 83, is shifting its spectral maximum of emission to 602 nm [144]. After iscovery of DsRed, many derivatives of this protein, such as a DsRed2 [145], DsRed-express [146], DsRed-Monomer [147], mCherry [148] were obtained. In addition, RFP may be obtained from other ources; for example, HcRed1 was derived from the sea anemone Heteractis crispa [148]. RFPs emitting light in yellow, orange, red, and far-red regions were isolated from some species f Anthozoa class. The ﬁrst RFP was isolated from Discosoma striata and called DsRed [142]. The ﬂuorophore of the DsRed protein includes glutamine, tyrosine, and glycine, located at 66, 67, and 8 sites. The RFP has broad excitation and emission spectra with the corresponding maximums at 58 and 583 nm [143]. Variations in the amino acid sequence of RFP, for instance, the mutation of ysine to methionine at location 83, is shifting its spectral maximum of emission to 602 nm [144]. After discovery of DsRed, many derivatives of this protein, such as a DsRed2 [145], DsRed-express [146], DsRed-Monomer [147], mCherry [148] were obtained. In addition, RFP may be obtained from other ources; for example, HcRed1 was derived from the sea anemone Heteractis crispa [148]. RFPs emitting light in yellow, orange, red, and far-red regions were isolated from some species of Anthozoa class. The first RFP was isolated from Discosoma striata and called DsRed [142]. The fluorophore of the DsRed protein includes glutamine, tyrosine, and glycine, located at 66, 67, and 68 sites. The RFP has broad excitation and emission spectra with the corresponding maximums at 558 and 583 nm [143]. Variations in the amino acid sequence of RFP, for instance, the mutation of lysine to methionine at location 83, is shifting its spectral maximum of emission to 602 nm [144]. After discovery of DsRed, many derivatives of this protein, such as a DsRed2 [145], DsRed-express [146], DsRed-Monomer [147], mCherry [148] were obtained. In addition, RFP may be obtained from other sources; for example, HcRed1 was derived from the sea anemone Heteractis crispa [148]. RFPs emitting light in yellow, orange, red, and far-red regions were isolated from some species of Anthozoa class. The ﬁrst RFP was isolated from Discosoma striata and called DsRed [142]. 3.1.3. Fluorescent Proteins 2020, 21, 2323 the sequence of three amino acids: the serine, tyrosine, and glycine located at 65, 66, and 67 sites. This group forms the imidazoline-5-one heterocyclic nitrogen ring by reacting between carboxyl carbon of serine and the amino nitrogen of glycine and subsequent conjugation with tyrosine [131,132]. The initial GFP originally isolated from the Aequorea Victoria has major and minor excitation peaks at 395 and 475 nm with emission peaks at 503 and 508 nm, respectively, and a quantum yield of 0.79 [133]. Additionally, there are several Aequorea protein derivatives with modiﬁed properties, such as eGFP [134], Superfolder GFP [135], YFP [136], TagCFP [137]. Furthermore, some GFPs were obtained from other organisms. These are Amazing green (from stony coral, Galaxeidae) [138], dendGFP (from octocoral Dendronephthya sp.) [139], TurboGFP (from Pontellina plumata Copepoda) [140]. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 15 of 60 such as eGFP [134], Superfolder GFP [135], YFP [136], TagCFP [137]. Furthermore, some GFPs were obtained from other organisms. These are Amazing green (from stony coral, Galaxeidae) [138], dendGFP (from octocoral Dendronephthya sp.) [139], TurboGFP (from Pontellina plumata Copepoda) [140]. Figure 4. (a) Structural schemes of different chromophore groups (atom color coding: grey—carbon, red—oxygen, blue—nitrogen, yellow—sulfur; R/R1/R2 symbolize protein rests); (b) excitation and emission spectra of different fluorophores. Reprinted with permission from [141]. Copyright 2009, John Wiley and Sons. Figure 4. (a) Structural schemes of diﬀerent chromophore groups (atom color coding: grey—carbon, red—oxygen, blue—nitrogen, yellow—sulfur; R/R1/R2 symbolize protein rests); (b) excitation and emission spectra of diﬀerent ﬂuorophores. Reprinted with permission from [141]. Copyright 2009, John Wiley and Sons. Figure 4. (a) Structural schemes of different chromophore groups (atom color coding: grey—carbon, red—oxygen, blue—nitrogen, yellow—sulfur; R/R1/R2 symbolize protein rests); (b) excitation and emission spectra of different fluorophores. Reprinted with permission from [141]. Copyright 2009, John Wiley and Sons. Figure 4. (a) Structural schemes of diﬀerent chromophore groups (atom color coding: grey—carbon, red—oxygen, blue—nitrogen, yellow—sulfur; R/R1/R2 symbolize protein rests); (b) excitation and emission spectra of diﬀerent ﬂuorophores. Reprinted with permission from [141]. Copyright 2009, John Wiley and Sons. RFPs emitting light in yellow, orange, red, and far-red regions were isolated from some species f Anthozoa class. The first RFP was isolated from Discosoma striata and called DsRed [142]. The luorophore of the DsRed protein includes glutamine, tyrosine, and glycine, located at 66, 67, and 68 ites. 3.2. Labeling by Magnetic Beads The application of the external magnetic ﬁeld is another reliable way of highly selective cell manipulation by the external force [155]. This is achievable since biological materials have a very low magnetic susceptibility, and thus, the cells labeled with magnetic particles or that having the intrinsic magnetic properties can be eﬀectively isolated without the interference with the surrounding medium and objects [156]. Therefore, the essential prerequisite of the magnetic sorting is a high magnetic response of the isolated objects (or its absence in case of the negative sorting). If the cells of interest do not possess the high magnetic susceptibility itself (for instance, as the red blood cells), one should consider to label them with an appropriate magnetic tag. Generally, depending on the magnetic susceptibility, bulk materials can be classiﬁed as diamagnetics, paramagnetics, which have no magnetic order, and magnetically ordered materials like ferromagnetics [157,158]. The diamagnetics do not have magnetic dipoles in the absence of an external magnetic ﬁeld due to the closed-shell electronic structure of the atoms [159]. These materials possess a very low magnetic susceptibility and a negative magnetization response that means the material magnetization is directed oppositely to the applied magnetic ﬁeld. Ordinary, all biological and organic materials exhibit diamagnetic behavior. Conversely, the paramagnetic materials contain unpaired electrons in their atom-shell and therefore have randomly oriented magnetic dipoles that can be aligned with magnetic ﬁeld lines [159]. This results in a positive magnetization response that means their magnetization vector is collinear to that of the applied magnetic ﬁeld. However, the magnitude of their magnetic susceptibility is still very low [160]. Both paramagnetics and diamagnetics demonstrate the absence of spontaneous magnetization without an external magnetic ﬁeld. On the other hand, ferromagnetic materials reveal intrinsic magnetization induced by the electronic shell structure of their atoms and the crystal lattice type. In these materials, the short-distance exchange coupling between the electrons prevails over the thermal disorientation that results in the collective interaction of their magnetic moments (spins) leading to the long-range magnetic ordering of the entire material volume at the macroscale. As a result, the ferromagnetics have a very high magnetic susceptibility and a positive magnetization response [160]. However, the intrinsic bulk magnetization substantially increases the magnetostatic energy of the material. In order to minimize the overall internal energy, the magnetic domains are formed. 3.1.3. Fluorescent Proteins The ﬂuorophore of the DsRed protein includes glutamine, tyrosine, and glycine, located at 66, 67, and 68 sites. The RFP has broad excitation and emission spectra with the corresponding maximums at 558 and 583 nm [143]. Variations in the amino acid sequence of RFP, for instance, the mutation of lysine to methionine at location 83, is shifting its spectral maximum of emission to 602 nm [144]. After discovery of DsRed, many derivatives of this protein, such as a DsRed2 [145], DsRed-express [146], DsRed-Monomer [147], mCherry [148] were obtained. In addition, RFP may be obtained from other sources; for example, HcRed1 was derived from the sea anemone Heteractis crispa [148]. sources; for example, HcRed1 was derived from the sea anemone Heteractis crispa [148]. Recently, a family of photoactivatable fluorescent proteins (PAFP) attracts much attention due to their ability to change the fluorescent properties under treatment by light energy [149,150]. These include groups of reversible and irreversible PAFP [130]. Reversible PAFP can change their fluorescence properties through conversable alterations of their chromophore conformation [151,152]. Proteins of this group were obtained from the corals of the Pectiniidae family [153]. A group of irreversible PAFP is divided into two classes depending on the switching mechanism. The first one Recently, a family of photoactivatable ﬂuorescent proteins (PAFP) attracts much attention due to their ability to change the ﬂuorescent properties under treatment by light energy [149,150]. These include groups of reversible and irreversible PAFP [130]. Reversible PAFP can change their ﬂuorescence properties through conversable alterations of their chromophore conformation [151,152]. Proteins of this group were obtained from the corals of the Pectiniidae family [153]. A group of irreversible PAFP is divided into two classes depending on the switching mechanism. The ﬁrst one is oxidative Int. J. Mol. Sci. 2020, 21, 2323 16 of 60 16 of 60 decarboxylation that is the carboxylate group that forms a weakly ﬂuorescent neutral form of the chromophore is removed under the irradiation [154]. Another possible switching mechanism is the β-elimination of the peptide bond between alpha nitrogen and alpha carbon of two amino acids participating in the formation of two diﬀerent conformations of the ﬂuorophore [130]. 3.2. Labeling by Magnetic Beads The domains are groups of atoms where the collective magnetization vector is oriented along the easy magnetization axis and speciﬁcally polarized relative to the magnetization vector of the other domains. The magnetic domains in the bulk ferromagnetics along with a magnetic crystalline anisotropy are the main origins of the magnetic hysteresis that can be interpreted as a delay in magnetization response when the magnetic ﬁeld is applied. The delay is related to the additional consumption of magnetic ﬁeld energy for the movement of domain walls and overcoming the anisotropy barrier to align the magnetization vector along the magnetic ﬁeld direction. However, the formation of magnetic domains is restricted by the material volume. To put in other words, there is a particular size threshold for a ferromagnetic particle, below which to support a single-domain volume magnetization is more energetically favorable rather than to form the domain walls [161]. This is called a single-domain state. With the further reduction of the single-domain particle size, the magnetic anisotropy energy of the particle becomes comparable with the energy of thermal magnetization ﬂuctuations. Thus, under a certain ambient temperature, the magnetization vector can quickly ﬂip over the magnetic anisotropy barrier separating two equivalent easy directions of magnetization and change its orientation to the opposite one [161]. That results in zero time-averaged or particle ensemble-averaged magnetization in the absence of the applied magnetic ﬁeld. Controversially, when the magnetic ﬁeld is turned on, the single-domain particle exhibits a typical paramagnetic Int. J. Mol. Sci. 2020, 21, 2323 Sci. 2019, 20, x FOR PEER 17 of 60 17 of 60 behavior. It shows the positive magnetization response without magnetic hysteresis along with the tremendous magnetic susceptibility inherent to ferromagnetics. In this case, the particle is named superparamagnetic. The single-domain transition threshold varies for various materials but typically is about 1–50 nm. This perfectly suits for conjugation with the antibodies and immuno-responsive cell labeling. Thus, the combination of these properties makes the single-domain superparamagnetic nanoparticles the best and reasonable choice for the tags for magnetic cell manipulations. ect labeling of cells of interest by conjugation of the magnetic particle with the corre [162]. Another possible option for magnetically driven cell sorting is related to the absorption capacities of various cells (endocytosis) [163]. 3.3. Labeling by Negative Acoustic Contrast Particles Standing acoustic wave is an array of alte formed in resonance conditions [1 Cell sorting can be performed as well by standing acoustic waves, produced inside microﬂuidic devices. Standing acoustic wave is an array of alternate nodes and antinodes of surrounding media pressure, formed in resonance conditions [165]. Depending on the object’s density and compressibility, cells are distributed in the pressure node and antinode regions. In the isotonic solutions, such as a physiological solution and phosphate-buﬀered saline, cells typically exhibit positive acoustic contrast, which leads to their distribution in pressure nodes (Figure 5a) [166,167]. Some polymeric materials, such as elastomers, oppositely have negative acoustic contrast [168]. This means that objects made from elastomers in the standing wave conditions are placed in pressure antinodes (Figure 5a). Therefore, bio-functionalized particles made from these polymeric compounds (negative acoustic contrast particles—NACPs), can be used for cell sorting by the displacement of positive acoustic contrast cells linked with particles in pressure antinodes (Figure 5b) [166,167]. Russom’s group in 2017 had demonstrated the possibility of colon carcinoma cell separation by microﬂuidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method [169]. o ed i eso a ce co di io s [ 65] epe di g o e objec s de sibility, cells are distributed in the pressure node and antinode regions. In the , such as a physiological solution and phosphate-buffered saline, cells typicall acoustic contrast, which leads to their distribution in pressure nodes (Figure 5a) lymeric materials, such as elastomers, oppositely have negative acoustic contrast [ hat objects made from elastomers in the standing wave conditions are placed in s (Figure 5a). Therefore, bio-functionalized particles made from these polymeric com e acoustic contrast particles—NACPs), can be used for cell sorting by the displac acoustic contrast cells linked with particles in pressure antinodes (Figure 5b) s group in 2017 had demonstrated the possibility of colon carcinoma cell sepa idic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method [169]. 5. Distribution of: (a) elastomeric particles and cells and (b) cells binded with elas s in nodes and antinodes of the acoustic standing wave. Reprinted with permissi Copyright 2014, American Chemical Society. Figure 5. Distribution of: (a) elastomeric particles and cells and (b) cells binded with elastomeric particles in nodes and antinodes of the acoustic standing wave. Reprinted with permission from [166]. Copyright 2014, American Chemical Society. Distribution of: (a) elastomeric particles and cells and (b) cells binded with ela in nodes and antinodes of the acoustic standing wave. 3.2. Labeling by Magnetic Beads This allows for effective s cytes with low absorption capacity from macrophages with high absorption cap ent, a label-free magnetic separation may be considered as a third labeling appro Generally, there are three main approaches to cell labeling for magnetic separation. The first one is the direct labeling of cells of interest by conjugation of the magnetic particle with the corresponding antibody [162]. Another possible option for magnetically driven cell sorting is related to the different internal absorption capacities of various cells (endocytosis) [163]. This allows for effective separation of monocytes with low absorption capacity from macrophages with high absorption capacity. To some extent, a label-free magnetic separation may be considered as a third labeling approach. The label-free separation is effective to sort the cells with a natural magnetic response from the non-magnetic ones [164]. , g p y g pp e separation is effective to sort the cells with a natural magnetic response from c ones [164]. ing by Negative Acoustic Contrast Particles sorting can be performed as well by standing acoustic waves, produced inside mic Standing acoustic wave is an array of alternate nodes and antinodes of surroundin 4. The Fluidic System for Sample Preparation, Flow Cytometry Measurement, and Cell Sorting 4. The Fluidic System for Sample Preparation, Flow Cytometry Measurement, and Cell Sorting he Fluidic System for Sample Preparation, Flow Cytometry Measurement, and Cell Sorting 4.1. Sample Enrichment by Target Cells 4.1. Sample Enrichment by Target Cells Diagnostics of diseases by the detection of an embolus in the whole blood still is a diﬃcult task. One of the major challenges is the rarity of untypical objects, which are prognostic or diagnostic factors for some diseases. For example, the presence of circulating tumor cells (CTCs) is a prognostic factor for a number of cancers [10–16]. However, the detection frequency of these objects in relatively small blood volume is extremely low. For instance, the number of CTCs may vary from 0 to 23.6 cells per 7.5 mL of whole blood sample depending on the cancer type, stage, medication, and operative treatment [170]. Thus, the enrichment of the analyzed blood volume by target cells is a necessary prerequisite for reliable diagnostics. There are a number of methods, allowing for sample enrichment, such as red blood cell lysis, density gradient centrifugation, and cell ﬁltration. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 18 of 60 treatment [170]. Thus, the enrichment of the analyzed blood volume by target cells is a necessary prerequisite for reliable diagnostics. There are a number of methods, allowing for sample enrichment, 4.1.1. Red Blood Cell Lysis such as red blood cell lys The red blood cell (RBC) lysis method is one of the commonly used approaches for sample enrichment by target cells [171,172]. This method allows for eﬀective elimination of RBCs, the concentration of which in the blood is 4.5 × 106 cells/µL, thus signiﬁcantly simplify observing of rear objects in a blood sample. The enrichment is based on soft osmotic lysis of RBC by mixing cells with cold water [173] or specialized RBC lysis buﬀer [171]. The lysis is performed in soft conditions to prevent signiﬁcant damage to target cells and is timely stopped by adjusting the salt concentration. However, this method has some limitations. For example, it eliminates only RBCs and does not allow to exclude platelets from the sample. With this regard, the RBC lysis method is often used in combination with other methods to receive the appropriate results. 4.1.1. Red Blood Cell Lysis The red blood cell (RBC) lysis method is one of the commonly used approaches for sample enrichment by target cells [171,172]. This method allows for effective elimination of RBCs, the concentration of which in the blood is 4.5 × 106 cells/µL, thus significantly simplify observing of rear objects in a blood sample. The enrichment is based on soft osmotic lysis of RBC by mixing cells with cold water [173] or specialized RBC lysis buffer [171]. The lysis is performed in soft conditions to prevent significant damage to target cells and is timely stopped by adjusting the salt concentration. However, this method has some limitations. For example, it eliminates only RBCs and does not allow to exclude platelets from the sample. With this regard, the RBC lysis method is often used in combination with other methods to receive the appropriate results. 3.3. Labeling by Negative Acoustic Contrast Particles Standing acoustic wave is an array of alte formed in resonance conditions [1 Reprinted with permissi opyright 2014, American Chemical Society. Figure 5. Distribution of: (a) elastomeric particles and cells and (b) cells binded with elastomeric particles in nodes and antinodes of the acoustic standing wave. Reprinted with permission from [166]. Copyright 2014, American Chemical Society. Int. J. Mol. Sci. 2020, 21, 2323 18 of 60 18 of 60 4.1.3. Cell Filtration Cell ﬁltration is applicable if there is a signiﬁcant distinction in the size of the target and bulk cells. A great amount of data shows that CTCs are typically larger than the rest of the blood cells that allows for employing ﬁltration for sample enrichment (Figure 7) [180,181]. The small size emboli, such as bacteria, also can be selected by ﬁltration for bacteremia diagnostics [182]. To perform cell ﬁltration, microﬂuidic devices are equipped with integrated microﬁlter [183], membrane microﬁlter [184] along with other variations of devices [182,185] based on this method are used. Sci. 2019, 20, x FOR PEER REVIEW 7. SEM images of (a) commercial membrane filter; (b) microfabricated parylene m c) parylenemembrane filter with cells captured without SEM fixation treatment, ne membrane filter with cells captured after SEM fixation procedure. Reprin Figure 7. SEM images of (a) commercial membrane ﬁlter; (b) microfabricated parylene membrane ﬁlter; (c) parylenemembrane ﬁlter with cells captured without SEM ﬁxation treatment, and (d) parylene membrane ﬁlter with cells captured after SEM ﬁxation procedure. Reprinted with permission from [184]. Copyright 2007, Elsevier. SEM images of (a) commercial membrane filter; (b) microfabricated parylene m parylenemembrane filter with cells captured without SEM fixation treatment membrane filter with cells captured after SEM fixation procedure. Reprin Figure 7. SEM images of (a) commercial membrane ﬁlter; (b) microfabricated parylene membrane ﬁlter; (c) parylenemembrane ﬁlter with cells captured without SEM ﬁxation treatment, and (d) parylene membrane ﬁlter with cells captured after SEM ﬁxation procedure. Reprinted with permission from [184]. Copyright 2007, Elsevier. 4.1.2. Density Gradient Centrifugation 4.1.2. Density Gradient Centrifugation Another way is the sample fractionation by the density gradient centrifugation method. This method is based on the distribution of objects with diﬀerent density in density gradient media. This method is widely used to separate macromolecules, viruses, cell organelles, and diﬀerent cell subpopulations [174]. There are a number of commercial kits based on organic, inorganic compounds, and nanoscale silica particles. For example, Ficoll™[175], Histopaque® [176], OncoQuick® [177], Percoll® [178] are successfully used for the isolation of mononuclear and cancer cells from a whole blood sample. The principle of the isolation is shown in Figure 6 as in the case of the OncoQuick separation kit. Additionally, this method is used to select bacterial cells from the sample for bacteremia diagnostics [179]. The employment of several media with diﬀerent density allows us to fractionate the blood sample accurately and prevent fraction contamination by RBC and platelets. Another way is the sample fractionation by the density gradient centrifugation method. This method is based on the distribution of objects with different density in density gradient media. This method is widely used to separate macromolecules, viruses, cell organelles, and different cell subpopulations [174]. There are a number of commercial kits based on organic, inorganic compounds, and nanoscale silica particles. For example, Ficoll™ [175], Histopaque® [176], OncoQuick® [177], Percoll® [178] are successfully used for the isolation of mononuclear and cancer cells from a whole blood sample. The principle of the isolation is shown in Figure 6 as in the case of the OncoQuick separation kit. Additionally, this method is used to select bacterial cells from the sample for bacteremia diagnostics [179]. The employment of several media with different density allows us to fractionate the blood sample accurately and prevent fraction contamination by RBC and platelets. Figure 6. Cell separation by gradient centrifugation method with OncoQuick separation kit. Reprinted with permission from [180]. Copyright 2007, Elsevier. Figure 6. Cell separation by gradient centrifugation method with OncoQuick separation kit. Reprinted with permission from [180]. Copyright 2007, Elsevier. Figure 6. Cell separation by gradient centrifugation method with OncoQuick separation kit. Reprinted with permission from [180]. Copyright 2007, Elsevier. Figure 6. Cell separation by gradient centrifugation method with OncoQuick separation kit. Reprinted with permission from [180]. Copyright 2007, Elsevier. Int. J. Mol. Sci. 2020, 21, 2323 19 of 60 19 of 60 4.2.2. Acoustic Focusing The acoustic focusing is based on the eﬀect of acoustic pressure described by Kundt and Lehmann in 1874. However, only in 2008, the feasibility of acoustic focusing in ﬂow cytometry was described by Kaduchak et al. [187] and a year later, for the ﬁrst time, acoustic focusing was introduced in ﬂow cytometry setup Attune® (ThermoFisher Scientiﬁc), [188]. Acoustic focusing is based on the redistribution of objects with diﬀerent density in the nodes and antinodes of the standing acoustic wave (Figure 8b). The method is attractive due to its cost eﬃciency and applicability for biological objects. To perform acoustic focusing, the length of the acoustic wave should be equal width of microﬂuidic channel divided by a natural number. Currently, the multinode acoustic focusing is developed for parallel ﬂow cytometry devices that allows for improving throughput comparing to conventional ﬂow cytometers [189]. 4.2.1. Hydrodynamic Focusing For the ﬁrst time, hydrodynamic focusing was described by Reynolds in 1883 [31]. It implicates the codirectional movement of two liquid streams, one of which is the sample suspension, and the second one is sheath ﬂuid. The velocity or density distinctions between the sample suspension and sheath ﬂuids results in the formation of a two-layer stable ﬂow and alignment of the objects in the middle of the channel [186]. Diﬀerent conﬁgurations of microﬂuidic devices are used to perform hydrodynamic focusing, however, ﬂow cytometry systems typically use conﬁguration based on the coaxial laminar ﬂow [31] (Figure 8a). mission from [184]. C 4.2. Sample Focusing ple Focusing carry out flow cytometry measurements, the cells must be aligned in the focal pla system of the cytometer. This can be done by hydrodynamic or acoustic nally, focusing prevents capillary blockages. Both acoustic and hydrodynamic fo commercial flow cytometers (Figure 8), although the latter one is more wid To carry out ﬂow cytometry measurements, the cells must be aligned in the focal plane of the optical system of the cytometer. This can be done by hydrodynamic or acoustic focusing. Additionally, focusing prevents capillary blockages. Both acoustic and hydrodynamic focusing is used in commercial ﬂow cytometers (Figure 8), although the latter one is more widespread. Moreover, the ﬂow cytometry systems with visualization option (e.g., Amnis® ImageStream® Mark II system) also use hydrodynamic focusing for ﬂuorescent and brightﬁeld imaging. The laser tweezers technique can be used to manipulate single objects in a ﬂowing stream as well, yet it is not used for focusing in ﬂow cytometry setups. 20 of 60 20 of 60 Int. J. Mol. Sci. 2020, 21, 2323 Figure 8. (a) Hydrodynamic and (b) acoustic focusing in a microﬂuidic channel. Figure 8. (a) Hydrodynamic and (b) acoustic focusing in a microﬂuidic channel. 4.3. Sorting Isolation of speciﬁc cell types from bulk heterogeneous biological samples (i.e., blood) is a ﬁrst and necessary step for many biomedical applications. Eﬀective cell sorting signiﬁcantly improves the quality of the sample analysis. Conventional cell sorting approaches implies either the separation of antibody conjugated cells or label-free sorting. Separation of antibody conjugated cells generally exploits speciﬁc cell labeling with magnetic beads or with ﬂuorescent tags. The ﬁrst one allows us to sort cells by a magnetic ﬁeld while the second one assists in ﬂuorescence-activated cell sorting (FACS). Int. J. Mol. Sci. 2020, 21, 2323 21 of 60 On the other hand, label-free sorting allows for cell separation with respect to their size, shape, and morphology through ﬁltration, centrifugation, or sedimentation. However, modern tendencies require fast and automated techniques to analyze a large number of cells. In this perspective, microﬂuidic methods of cell sorting are expected to provide one with more sophisticated solutions to meet the modern demands in a rapid and reliable analysis. On the other hand, label-free sorting allows for cell separation with respect to their size, shape, and morphology through ﬁltration, centrifugation, or sedimentation. However, modern tendencies require fast and automated techniques to analyze a large number of cells. In this perspective, microﬂuidic methods of cell sorting are expected to provide one with more sophisticated solutions to meet the modern demands in a rapid and reliable analysis. There are several features of microﬂuidic systems that are relevant to cell sorting applications (Figure 9). First, narrow microscale channels tend to have a laminar liquid ﬂow that aligns the cells into well-ordered streamlines. Furthermore, microchannels have a ﬂow speed gradient over the cross-section area that allows for employing an additional mechanism of hydrodynamic separation that is not possible on a macro scale. The small size of microﬂuidic chips enables one to apply locally strong gradients of magnetic, electric, and acoustic ﬁelds as well as to combine multiple chips in a single sorting device. Figure 9. Various types of separation microﬂuidic mechanisms: (a) electrokinetic; (b) magnetic; and (c) acoustic separation. Figure 9. Various types of separation microﬂuidic mechanisms: (a) electrokinetic; (b) magnetic; and (c) acoustic separation. To isolate a particular cell from the carrying ﬂuid ﬂow one should apply a force to this cell. 4.3. Sorting Depending on the applied force nature, several general types of separation mechanisms in microsystems are available: mechanical separation by the direct contact with the structure of the separation system based on the cell size, shape, and morphology; employment of external ﬁeld gradient (e.g., magnetic, electric, and acoustic ﬁeld); separation by hydrodynamic force via introduction of secondary ﬂuid ﬂows. On the other hand, with respect to cell modiﬁcation approaches, one can outline the separation of cells conjugated with ﬂuorescent labels, cells conjugated with beads, and a label-free separation. Additionally, label-free separation methods can be divided into active and passive. Electrokinetic Cell Separation Electrokinetic cell separation exploits various phenomena that may be activated by an applied electric ﬁeld (Figure 9a). Typically, electrophoresis, dielectrophoresis, and electroosmosis are employed. Electrophoresis is a movement of a charged particle in a uniform electric ﬁeld under the Coulomb force. Alternatively, electroosmosis is a movement of a polarizable medium in the electric ﬁeld due Int. J. Mol. Sci. 2020, 21, 2323 electric field (Figure 9 employed Electrophore 22 of 60 osis are der the to the collective action of the Coulomb force on its molecules (Figure 10) [190]. This may be used to separate several ﬂuid streams. Coulomb force. Alternatively, electroosmosis is a movement of a polarizable medium in the electric field due to the collective action of the Coulomb force on its molecules (Figure 10) [190]. This may be used to separate several fluid streams. Figure 10. Schematic of conventional electroosmosis in a microchannel. Reprinted with permission from [190]. Copyright 2017, John Wiley and Sons. Figure 10. Schematic of conventional electroosmosis in a microchannel. Reprinted with permission from [190]. Copyright 2017, John Wiley and Sons. Figure 10. Schematic of conventional electroosmosis in a microchannel. Reprinted with permission from [190]. Copyright 2017, John Wiley and Sons. Figure 10. Schematic of conventional electroosmosis in a microchannel. Reprinted with permission from [190]. Copyright 2017, John Wiley and Sons. Finally, the application of the non-uniform electric field causes the movement of cells due to their polarization (Figure 11), which is called dielectrophoresis (DEP) [191]. If the cells are more polarizable then the medium, they will move to the regions with the highest electric field strength (positive DEP—pDEP) and vice versa, in case the cells are less polarizable, they will move to the regions with the lowest electric field strength (negative DEP–nDEP). Finally, the application of the non-uniform electric ﬁeld causes the movement of cells due to their polarization (Figure 11), which is called dielectrophoresis (DEP) [191]. If the cells are more polarizable then the medium, they will move to the regions with the highest electric ﬁeld strength (positive DEP—pDEP) and vice versa, in case the cells are less polarizable, they will move to the regions with the lowest electric ﬁeld strength (negative DEP–nDEP). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 22 of 60 Figure 11. Scheme of the particle polarization process and dielectrophoretic response: left—positive DEP, right—negative DEP. Reprinted with permission from [191]. Copyright 2018, AIP Publishing. Figure 11. Electrokinetic Cell Separation Scheme of the particle polarization process and dielectrophoretic response: left—positive DEP, right—negative DEP. Reprinted with permission from [191]. Copyright 2018, AIP Publishing. Figure 11. Scheme of the particle polarization process and dielectrophoretic response: left—positive DEP, right—negative DEP. Reprinted with permission from [191]. Copyright 2018, AIP Publishing. Figure 11. Scheme of the particle polarization process and dielectrophoretic response: left—positive DEP, right—negative DEP. Reprinted with permission from [191]. Copyright 2018, AIP Publishing. One of the first techniques employing an electric field for cell separation was FACS [26]. At its simplest, the cells of interest are labeled with a fluorescent tag via a complementary antibody. Afterward, the FACS device analyzes the fluorescent signal from the cell streamline. Additionally, FACS devices can analyze the light side- and forward scattered by the cells in order to get the information on their morphology and size. The conventional FACS devices are equipped with a One of the ﬁrst techniques employing an electric ﬁeld for cell separation was FACS [26]. At its simplest, the cells of interest are labeled with a ﬂuorescent tag via a complementary antibody. Afterward, the FACS device analyzes the ﬂuorescent signal from the cell streamline. Additionally, FACS devices can analyze the light side- and forward scattered by the cells in order to get the information on their morphology and size. The conventional FACS devices are equipped with a vibration stream Int. J. Mol. Sci. 2020, 21, 2323 23 of 60 channel to form the liquid droplets containing the cells at the outlet and a ring charger to charge the droplets with the cells of interest if the ﬂuorescence was detected. The charged droplets are isolated in a separate streamline by the oppositely charged electrode and collected [192]. However, the necessity to form water droplets is the signiﬁcant drawback of conventional FACS systems that was successfully overcome by the introduction of microﬂuidic devices. At resent years, DEP and its modiﬁcations have become the most commonly used method of electrokinetic cell separation in microﬂuidic platforms [193]. Generally, the DEP force acting on the cell is determined by the cell volume and dielectric properties along with the permittivity of the medium and the strength of the applied ﬁeld [194]. Thus, the essential beneﬁt of DEP is the possibility of both label-free and immunoaﬃnity cell isolation depending on their size and dielectric properties regardless of the initial cell surface charge. Electrokinetic Cell Separation For instance, in the last few years, various DEP setups were shown to be eﬀective for the isolation of circulating tumor cells (CTCs), which are known to have a neutral charge [195] from the healthy blood cells. Bulfoni et al. demonstrated DEP isolation of epithelial-like and mesenchymal-like breast tumor cells from the blood samples obtained from the clinical patients with a commercially available DEPArray system [196]. Kim et al. used DEP for eﬀective isolation of separate cancer cells with a very high eﬃciency followed by an intracellular enzymatic assay at the single-cell level [197]. Later, the same group proposed a microﬂuidic DEP setup that allows for precise localization of rare CTCs in the desired area along with a reduction of the sample volume for further molecular analysis [198]. Alazzam et al. fabricated the DEP device for continuous separation of CTCs, from a heterogeneous mixture of cancer and healthy blood cells employing one-sidewall displacement of the electrodes in the microchannel [199]. The resulted non-uniform electric ﬁeld acted on the MDA-MB-231 human breast cancer cells with pDEP force whereas the nDEP force acted on the healthy cells leading to almost 100% sorting eﬃciency (Figure 12a). Li et al. demonstrated continuous high-throughput selective capture of CTCs by DEP at arrays of wireless bipolar electrodes [200]. Noticeable feature of this setup is an easy increase of separation area that can signiﬁcantly improve the volume rate of processing samples. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 23 of Figure 12. (a) Example of dielectrophoretic cell separation in the microfluidic device. Reprinted with permission from [199]. Copyright 2017, John Wiley and Sons. (b) Schematic of contactless dielectrophoretic (cDEP) device with the electromagnetic forces acting in the microfluidic channel. Reprinted with permission from [201]. Copyright 2010, the Royal Society of Chemistry. Figure 12. (a) Example of dielectrophoretic cell separation in the microﬂuidic device. Reprinted with permission from [199]. Copyright 2017, John Wiley and Sons. (b) Schematic of contactless dielectrophoretic (cDEP) device with the electromagnetic forces acting in the microﬂuidic channel. Reprinted with permission from [201]. Copyright 2010, the Royal Society of Chemistry. ure 12. (a) Example of dielectrophoretic cell separation in the microfluidic device. Reprinted wit mission from [199]. Copyright 2017, John Wiley and Sons. (b) Schematic of contactles lectrophoretic (cDEP) device with the electromagnetic forces acting in the microfluidic channe printed with permission from [201]. Copyright 2010, the Royal Society of Chemistry. Figure 12. Electrokinetic Cell Separation (a) Example of dielectrophoretic cell separation in the microﬂuidic device. Reprinted with permission from [199]. Copyright 2017, John Wiley and Sons. (b) Schematic of contactless dielectrophoretic (cDEP) device with the electromagnetic forces acting in the microﬂuidic channel. Reprinted with permission from [201]. Copyright 2010, the Royal Society of Chemistry. Another important DEP application is the isolation of stem cells. It is currently attracting mu ention due to its high potential in clinical applications and regenerative medicine. Sun et orted on microfluidic DEP device with self-assembled ionic liquid electrodes for continuous c aration [202]. The device was successfully employed for the separation of human adipose-deriv m cells (ADSC) from MDA-MB-231 human breast cancer cells via pDEP with an accuracy of abo %. Yoshioka et al. employed pDEP for the isolation of human bone marrow-derived mesenchym m cells (UE7T-13) [203]. Moreover, the important result of their study was that pDEP effects 7T-13 cell gene expression of the surface differentiation marker due to mechanical deformatio sed by separation. Additionally, recent studies demonstrated that DEP could be employed for t Another important DEP application is the isolation of stem cells. It is currently attracting much attention due to its high potential in clinical applications and regenerative medicine. Sun et al. reported on microﬂuidic DEP device with self-assembled ionic liquid electrodes for continuous cell separation [202]. The device was successfully employed for the separation of human adipose-derived stem cells (ADSC) from MDA-MB-231 human breast cancer cells via pDEP with an accuracy of about 85%. Yoshioka et al. employed pDEP for the isolation of human bone marrow-derived mesenchymal stem cells (UE7T-13) [203]. Moreover, the important result of their study was that pDEP eﬀects on UE7T-13 cell gene expression of the surface diﬀerentiation marker due to mechanical deformations caused by separation. Additionally, recent studies demonstrated that DEP could be employed for the separation of heterogeneous populations of stem cells with respect to their biological fate. For instance, neural stem Int. J. Mol. Sci. 2020, 21, 2323 24 of 60 24 of 60 and progenitor cell (NSPCs) heterogeneous populations contain both neuron and astrocyte progenitors that give rise to neurons and astrocytes respectively. The separation of neuron and astrocyte progenitors is crucial to improve the purity of transplanted stem cells in clinical therapy. Adams et al. Electrokinetic Cell Separation demonstrated that these subpopulations could be eﬀectively separated by the alternate current (AC) DEP due to the diﬀerent electrophysiological properties of the cell membranes (whole-cell membrane capacitance) revealing at a certain stage of the cell diﬀerentiation [204]. Previously, El-Badawy et al. examined the electrophysiological properties of adipose stem cells (ASCs) and bone-marrow mesenchymal stromal cells (BM-MSCs) with AC DEP [205]. They found out the diﬀerences in cell traveling and rotation speed that potentially may be exploited for cell separation. p p y y p p The realization of conventional DEP implies a fabrication of metal electrodes in the microﬂuidic channel that is potentially harmful to the contacting cells. This leads to the development of DEP modiﬁcations to avoid the direct contact of the analyzed cells with the metal electrode surface. For instance, in insulator-based DEP (iDEP), the DEP force originates due to the polarization of dielectric structures placed into the microﬂuidic channel by applying an external electric ﬁeld [206]. The metal electrodes are typically placed in the inlet and outlet ports of the device and do not contact with the microchannel interior. First, iDEP was shown to be eﬀective in the separation of live and dead bacteria E. coli [207] and later on the separation of diﬀerent types of live bacteria [208]. As a more recent example, Lewpiriyawong et al. demonstrated the iDEP setup run by the DC-biased AC electric ﬁeld [209]. The AC controls iDEP force for the cell isolation whereas the DC ﬁeld generates electroosmotic motion of the liquid through the channel. The combination of AC and DC electric ﬁelds was shown to be eﬀective for the cell capturing at the liquid ﬂow rate up 5 mL/min. Further development of DEP approaches reveals the contactless dielectrophoresis (cDEP) technique. The main idea of cDEP is an exploiting of microchannels ﬁlled with high-conductive liquids as so-called liquid electrodes (see Figure 12b). Generally, the liquid electrodes are separated from the main sample-carrying microchannel with an insulating membrane minimizing the contact between the electrodes and analyzing cells [210]. This results in a signiﬁcant reduction of contamination of biologicals samples, electro-induced heating, electrochemical eﬀects, and bubbles formation. The inhomogeneity of the intrinsic electric ﬁeld required for DEP separation is achieved through the corresponding geometry of the side liquid-electrode channels and placing of the dielectric elements inside the sample-channel. Shaﬁee et al. Electrokinetic Cell Separation ﬁrst reported on a successful selective separation of live human leukemia cells from dead ones by cDEP basing on their dielectric properties [201]. Later, Henslee et al. ﬁrst demonstrated the ability of cDEP isolation of the target MDA-MB-231 human breast cancer cells from the heterogeneous cell mixture [211]. The same group employed cDEP to analyze the area-speciﬁc membrane capacitance of red blood cells, macrophages, breast cancer, and leukemia cells [212], which is an important prerequisite for the AC electrokinetic separation. More recently, Hanson and Vargis described the cDEP setup combined with Raman spectrometer for cell isolation and identiﬁcation on a single device [213]. Rahmani et al. proposed a ﬂow microﬂuidic cDEP device for continuous bioparticles enrichment employing platinum electrodes separated from the sample-channel with PDMS layer [214]. The employment of metal electrodes instead of liquid ones allows for eﬀective separation with much lower operating voltage due to higher conductivity of the metal compared to the ionic liquid. However, the production of micro-scale electrodes for microﬂuidic systems remains quite challenging and requires delicate and complex fabrication processes. To overcome this issue, an optically induced DEP (oDEP) approach was introduced. In oDEP devices, a photoconductive layer on a plate electrode is used to induce the non-uniform electric ﬁeld inside the microchannel. When the photoconductive layer is illuminated with light passed through a deﬁned pattern, the light-exposed areas become much conductive than the dark areas and therefore form “virtual electrodes” inducing the non-uniform electric ﬁeld [215]. This allows for avoiding the fabrication of patterned microelectrode structures and makes the separation more adjustable. Chiou et al. ﬁrst demonstrated the potential of oDEP to be exploited for cell manipulation with the technique permitting a high-resolution patterning Int. J. Mol. Sci. 2020, 21, 2323 25 of 60 of electric ﬁelds on the photoconductive surface [216]. The described device enabled a simultaneous manipulation of 15,000 particle traps with an optical intensity 105 times less than that of optical tweezers. Later on, Hwang et al. described the oDEP based technique to discriminate normal and starved abnormal oocytes [217]. In further oDEP development, Huang et al. ﬁrst characterized the operating conditions for the manipulation of CTCs of prostate cancer (PC-3) and human oral cancer (OEC-M1) and leukocytes with a minor cell aggregation [218]. More recently, Chiu et al. Electrokinetic Cell Separation described the oDEP microﬂuidic system using T-shape microchannel for CTCs isolation and operating with a light bar to manipulate with leukocytes and light circles to capture and isolate PC-3 cancer cells [219]. Further, the same group proposed four-the cascade oDEP CTCs isolation scheme employed after the conventional CTCs manipulation process [220]. This allows for applying up to four isolation conditions simultaneously and implementing a higher-resolution separation. As the most recent example, these authors proposed the microﬂuidic oDEP device for high-purity isolation of CTC clusters based on their size and preserving clusters integrity for further analysis [221]. Furthermore, oDEP was shown as a versatile tool to determine the cell electrophysiological parameters. They are not only relevant biomarkers to characterize cellular phenotype and state but also the important prerequisites for cell sorting. Liang et al. described a theoretical background to ﬁgure out the cell membrane capacitance and conductance with a shell-core polarization model [222]. Afterward, they studied the response of four diﬀerent cell types to positive and negative oDEP forces with a motion-tracking technique in liquid media of various conductivity. This non-invasive approach is advantageous to get statistically important data from the individual cells to contribute a number of biomedical applications including stem cell diﬀerentiation. Additionally, DEP may be successfully combined with other separation approaches like inertial force microﬂuidic separation [223], ﬂuid ﬂow fractionation [224], and ﬁeld-modulated electroosmotic ﬂow separation [225]. As a ﬁnal remark of this section, we refer to some recent reviews devoted to electrokinetic cell manipulations in microﬂuidic devices. Adekanmbi and Srivastava have given a deep insight review of DEP manipulations on diseased cells [226]. This includes an overview of DEP techniques and devices, the theoretical background of DEP cell separation, and DEP applications for isolation of various types of cancer cells along with malaria, anthrax, and dengue. More recently, Chan et al. have reviewed microﬂuidic platforms employing DEP with respect to a particular cancer cell type [191]. Furthermore, this review also introduces the basics of DEP theory and CTCs formation. Menachery et al. have reviewed microﬂuidic label-free methods for stem cell isolation including those based on electrical cell characteristics [227]. Li and Anand have reviewed approaches of dielectrophoretic cell manipulation combined with a single cell analysis including both on-chip and oﬀ-chip realizations [228]. Nuchtavorn et al. Electrokinetic Cell Separation have described a diﬀerent separation approaches and devices employed for microchip electrophoretic manipulations with various biomedical analytes like drugs, nucleic and amino acids, peptides, proteins, antibodies, antigens, cells, and cell components [229]. Magnetic Cell Separation Generally, QMS ar flow channel with a spherical section placed in the radial gradient of the qu field oriented from the core to the walls of the channel (Figure 13). 13. (a) Cross-sectional field map of the magnetic field of a quadrupole magnetic flow s magent; (b) axial section diagram of the separation column contained within the quad t bl R i t d ith i i f [231] C i ht 2018 J h Wil d S Figure 13. (a) Cross-sectional ﬁeld map of the magnetic ﬁeld of a quadrupole magnetic ﬂow sorting (QMS) magent; (b) axial section diagram of the separation column contained within the quadrupole magnet assembly. Reprinted with permission from [231]. Copyright 2018, John Wiley and Sons. . (a) Cross-sectional field map of the magnetic field of a quadrupole magnetic flow agent; (b) axial section diagram of the separation column contained within the qu bl R i t d ith i i f [231] C i ht 2018 J h Wil d S Figure 13. (a) Cross-sectional ﬁeld map of the magnetic ﬁeld of a quadrupole magnetic ﬂow sorting (QMS) magent; (b) axial section diagram of the separation column contained within the quadrupole magnet assembly. Reprinted with permission from [231]. Copyright 2018, John Wiley and Sons. net assembly. Reprinted with permission from [231]. Copyright 2018, John Wiley and Sons. The main beneﬁt of the quadrupole ﬁeld is the fact that magnetophoretic force acting on the particle is ﬁnely deﬁned by the particle position over the channel cross-section. The QMS separation concept is based on the cell magnetophoretic mobility that is the velocity of the cell per unit of the magnetic force. Passing through the channel, the cells labeled with magnetic nanoparticles are deﬂected to the channel walls, whereas less mobile unlabeled cells remain near the core rod forming two outﬂows separated by a ﬂow splitter. The inlet ﬂow of the initial cell mixture is also separated from the channel wall by the ﬂow splitter to avoid the non-labeled cells pass near the magnet and come to the positive outﬂow with the magnetically isolated cells. Magnetic Cell Separation The st rupt the uniform magnetic field between the magnet poles whereas the dense wool he distance between the cells and the magnetic field source. These two factors in l magnetophoretic force that effectively captured the labeled cells. The cells of inter with the fluorescent tag and superparamagnetic nanoparticles by streptavid owski’s group has suggested an alternative way for continuous flow magnetic se ng a quadrupole magnetic flow sorting approach (QMS) [226,227]. Generally, QMS ar flow channel with a spherical section placed in the radial gradient of the qua field oriented from the core to the walls of the channel (Figure 13). structures creating local inhomogeneity of the magnetic ﬁeld acting as trapping or deﬂecting sites for the cells labeled with magnetic particles. This was successfully realized by Miltenyi et al., who ﬁrst proposed in 1990 a parallel cell separation technique for routine use in laboratory currently known as a conventional magnetic-activated cell separation or MACS [230]. They used a separation column ﬁlled with ferromagnetic steel wool placed between the poles of a permanent magnet. The steel wool ﬁbers disrupt the uniform magnetic ﬁeld between the magnet poles whereas the dense wool packing reduced the distance between the cells and the magnetic ﬁeld source. These two factors induced a high local magnetophoretic force that eﬀectively captured the labeled cells. The cells of interest were labeled with the ﬂuorescent tag and superparamagnetic nanoparticles by streptavidin-biotin coupling. entional magnetic-activated cell separation or MACS [230]. They used a separation h ferromagnetic steel wool placed between the poles of a permanent magnet. The st rupt the uniform magnetic field between the magnet poles whereas the dense wool he distance between the cells and the magnetic field source. These two factors in magnetophoretic force that effectively captured the labeled cells. The cells of inter with the fluorescent tag and superparamagnetic nanoparticles by streptavid owski’s group has suggested an alternative way for continuous flow magnetic se Zborowski’s group has suggested an alternative way for continuous ﬂow magnetic separation developing a quadrupole magnetic ﬂow sorting approach (QMS) [226,227]. Generally, QMS exploits an annular ﬂow channel with a spherical section placed in the radial gradient of the quadrupole magnetic ﬁeld oriented from the core to the walls of the channel (Figure 13). owski s group has suggested an alternative way for continuous flow magnetic se ng a quadrupole magnetic flow sorting approach (QMS) [226,227]. Magnetic Cell Separation The application of the external magnetic ﬁeld is one more reliable way to induce the forces responsible for highly selective cell manipulations. This is achievable since biological materials have a very low magnetic susceptibility, and thus, the cells labeled with magnetic particles or having their natural magnetic response can be eﬀectively isolated without the interference of the surrounding medium (see Figure 9b) [156]. Therefore, the essential prerequisite of the magnetic sorting is a high magnetic response of the objects to be isolated (or its absence in case of the negative sorting). If the cells of interest do not possess the high magnetic susceptibility itself (for instance, as the red blood cells), one should consider to label them with an appropriate magnetic tag. Since superparamagnetic nanoparticles have a natural magnetic polarization, a non-uniform magnetic ﬁeld (or a magnetic ﬁeld gradient) is required to move them by magnetophoretic force analogous to the dielectrophoretic separation. Thus, all magnetic separation methods employ the Int. J. Mol. Sci. 2020, 21, 2323 abeled with magneti i 1990 ll l 26 of 60 et al., w tl 26 of 60 et al., w tl structures creating local inhomogeneity of the magnetic ﬁeld acting as trapping or deﬂecting sites for the cells labeled with magnetic particles. This was successfully realized by Miltenyi et al., who ﬁrst proposed in 1990 a parallel cell separation technique for routine use in laboratory currently known as a conventional magnetic-activated cell separation or MACS [230]. They used a separation column ﬁlled with ferromagnetic steel wool placed between the poles of a permanent magnet. The steel wool ﬁbers disrupt the uniform magnetic ﬁeld between the magnet poles whereas the dense wool packing reduced the distance between the cells and the magnetic ﬁeld source. These two factors induced a high local magnetophoretic force that eﬀectively captured the labeled cells. The cells of interest were labeled with the ﬂuorescent tag and superparamagnetic nanoparticles by streptavidin-biotin coupling. Zborowski’s group has suggested an alternative way for continuous ﬂow magnetic separation developing a quadrupole magnetic ﬂow sorting approach (QMS) [226,227]. Generally, QMS exploits an annular ﬂow channel with a spherical section placed in the radial gradient of the quadrupole magnetic ﬁeld oriented from the core to the walls of the channel (Figure 13). entional magnetic-activated cell separation or MACS [230]. They used a separation h ferromagnetic steel wool placed between the poles of a permanent magnet. Magnetic Cell Separation They demonstrated eﬀective immunomagnetic labeling of MCF-7 human breast cancer, SGC 7901 human gastric cancer, Hela human cervical cancer, and PC3 human prostate cancer cell lines with magnetic composite particles along with isolation from RBCs and leucocytes. Droz et al. demonstrated an eﬀective approach for isolation of the cells expressing proteins of interest employing automated microﬂuidic setup [245]. This approach deals with some issues related to the realization of automated cell sorting like homogeneous dispersion of magnetic nanoparticles in the microchamber for eﬀective cell labeling, various rates of the protein secretion by the cells, and eﬀective isolation of monoclonal cell populations. Lee et al. successfully isolated CTCs from the blood samples of cancer patients by one-step negative enrichment of white blood cells labeled with magnetic nanoparticles [246]. They exploit a microﬂuidic multi-vortex mixing module in the microﬂuidic setup for eﬀective white blood cell labeling. An analogous approach was used by Bhagwat et al. to pre-enrich rare circulating cells from the whole blood by in-line magnetic particle-based leukocyte depletion prior to ﬂow cytometry analysis [247]. Huang et al. designed a microﬂuidic microwell device that allows for immunomagnetic single-cell trapping for further cellular analysis at a single-cell level [238]. Green et al. described a microﬂuidic magnetic sorting device that allows for the eﬀective capturing of CTCs and dividing them into subpopulations according to levels of protein surface expression [248]. The analysis of subpopulations of CTCs that exhibit diﬀerent biochemical and functional phenotypes may be useful to ﬁgure out if the CTCs acquire the metastatic potential by undergoing the epithelial-to-mesenchymal transition (EMT). An important aspect of magnetic cell sorting is the isolation of the cells with an inherent magnetic moment. There are several reasons to exploit the label-free separation of intrinsically magnetic cells An important aspect of magnetic cell sorting is the isolation of the cells with an inherent magnetic moment. There are several reasons to exploit the label-free separation of intrinsically magnetic cells rather than utilize labeling approaches. One of them is purifying biological samples from an excess of RBCs for diagnostic or therapeutic applications. For instance, isolation of RBCs form stem cells is desirable at bone marrow transplantation [249]. Additionally, it is known that blood transfusion may be associated with increased side eﬀects due to the damage of RBCs after long storage [250,251]. Another critical issue is the isolation of RBCs for further analysis. Magnetic Cell Separation To date, QMS has been successfully employed for positive selection of magnetically labeled CD34+ blood progenitor cells from blood circulation [232]; depletion of immunomagnetic labeled T-cells for clinical allogeneic bone marrow transplants [233]; separation of magnetically labeled porcine islets of Langerhans for further transplantation in diabetes treatment [234,235]; and isolation of intrinsically magnetic, deoxygenated RBCs from the whole blood [231]. Int. J. Mol. Sci. 2020, 21, 2323 27 of 60 However, modern tendencies in magnetic cell separation are mostly related to the development of microﬂuidic sorting systems. The employment of the microﬂuidics allows for the relatively simple generation of local high gradient magnetic ﬁelds in the desired area of the microchip and preparation of microﬂuidic channels of various shape and geometry by well-established microfabrication techniques. Additionally, microﬂuidic systems signiﬁcantly reduce the required volume of the analyzed sample. Novel microﬂuidic platforms for magnetic cell separation are constantly developed, numerically simulated, and tested for continuous-ﬂow separation of magnetic beads (for instance, [236–240]). Furthermore, these systems were shown to be eﬀective for the isolation of magnetically labeled cells including rare circulating cells from blood samples. As a recent example, Shi et al. successfully isolated HCT-116 human colorectal cancer CTCs selectively labeled with magnetic nanoparticles from the whole blood samples by a wavy-herringbone structured microﬂuidic device with a capture eﬃciency of 81%–95% [241]. The speciﬁc wave structure of the microchannels creates turbulence in the liquid ﬂow increasing the colliding probability with the channel walls and improving the eﬀectiveness of the cells capturing. Later, Zhang et al. employed the microﬂuidic device with a twin-layered herringbone structure for immunomagnetic isolation of Hep3B CTCs from blood samples with a capture eﬃciency of about 90% at the clinical relevant tumor cell density in blood [242]. The speciﬁc structure of the microﬂuidic channel was also exploited by Jung et al. [243] They combined magnetic separation device with slanted ridge array creating advective rotational ﬂows delivering magnetically labeled cells precisely to the area where high magnetic forces are formed. This enables us to separate the cells dispersed in whole blood at ﬂow rates up to 0.6 mL/h with an eﬃciency of about 90%. Shen and Park described a microﬂuidic magnetic separation system that can separate subpopulations of macrophage Raw 264.7 cells depending on their absorption capacity [163]. Xu et al. developed a microﬂuidic setup for high purity isolation of CTCs from the whole blood [244]. Magnetic Cell Separation Therefore, it is highly desirable to avoid additional contamination of the cells with antibody-conjugated magnetic particles. Melville et al. and Owen performed the ﬁrst experiments devoted to RBCs magnetic isolation from the whole Int. J. Mol. Sci. 2020, 21, 2323 28 of 60 28 of 60 blood conﬁrming their paramagnetic properties and proving the concept of label-free RBCs separation from the whole blood by applying high gradient magnetic ﬁeld [252,253]. Takayasu et al. described continuous ﬂow magnetic separation of RBCs from the whole venous blood in a glass tube [254]. Later on, magnetic separation of RBCs was redesigned with respect to microﬂuidic techniques (for instance, Han and Frazier [255] and Qu et al. [256]). As a more recent example, Myklatun et al. developed a setup for microﬂuidic sorting of intrinsically magnetic cells under visual control [164]. The setup was shown to be eﬀective for contamination-free isolation of intrinsically magnetic cells (magnetotactic bacteria) with the possibility of automated quantiﬁcation of the separated cells. Zborowski and Chalmers with co-workers also have studied the separation of intrinsically magnetic cells. They ﬁgured out the diﬀerence in magnetophoretic mobility of oxygenated and reduced RBCs [257], developed a theoretical model of microﬂuidic RBCs sorting [258] that was used as a basis for QMS device for continuous RBCs sorting [231]. Furthermore, the development of continuous ﬂow magnetic isolation of infected RBCs is of great importance with respect to malaria treatment. Malaria is an infectious disease caused by Plasmodium parasites aﬀecting host RBCs. During the life cycle, the parasites metabolize most of the cellular hemoglobin to hemozoin exhibiting paramagnetic properties in contrast to oxygenated hemoglobin, which is diamagnetic [259]. Moreover, recent research reported on the superparamagnetic behavior of hemozoin nanocrystals [260]. This diﬀerence in the magnetic response of healthy and parasite aﬀected RBCs can be employed for magnetic separation. Thus, a decent number of separation approaches were developed. Paul et al. ﬁrst reported on the batch separation of malaria-infected erythrocytes from whole blood using a high-gradient magnetic separation [261]. Moore et al. studied the magnetophoretic mobility of infected RBCs and demonstrated that their magnetic properties change with respect to the development stage of the parasite [262]. Further, Nam et al. developed a microﬂuidic device for continuous-ﬂow separation of malaria-infected RBCs in various stages of development [263]. Magnetic Cell Separation They reported on up to 99% eﬃciency of the separation of the late-stage infected cells and the possibility to isolate early-stage developed parasite-infected cells. As for more recent results, a number of works are performed [264–266], devoted to numerical simulation of microﬂuidic devices to magnetically isolate malaria-infected RBCs. However, a possible way to make magnetic separation to meet the demands in clinical scale blood purifying system is a building up of a new eﬃcient high-throughput mesoscale separation devices as reported by Martin et al. [267] They described a continuous ﬂow high-gradient magnetic separation device for isolation of infected erythrocytes from the whole blood without a need for shear ﬂow or blood dilution. In this design, the blood can be taken directly from the patients and afterward returned upon puriﬁcation. This system is reported to be about 380 times more eﬃcient comparing microﬂuidic systems. To complete this passage, we refer to a recent review of Kasetsirikul et al. covering malaria diagnostic methods along with techniques for malaria detection and infected RBCs separation including DEP and magnetophoretic approaches [268]. Despite the current popularity of microﬂuidic approaches for magnetic cell separation, these methods have a considerable drawback that is a small volume of processing blood samples and thus the separation of rare circulating objects (e.g., CTCs). Although microﬂuidic isolation can almost completely remove the target objects (with eﬃciency about 90%–95%), a typical sample volume for microﬂuidic investigation is about 5–10 mL, and this can only contain a tiny amount of rare blood biomarkers that is insuﬃcient for adequate analysis and reliable diagnosing [269]. Therefore, alternative approaches for fast processing of large blood volumes including in vivo analysis, are being developed. Vermesh et al. described an immunomagnetic cell enrichment method for the isolation of CTCs in vivo [270]. This implies the immunomagnetic CTCs labeling with antibody-conjugated magnetic beads injected directly to the bloodstream and capturing of the labeled cells with a ﬂexible magnetic wire that was inserted and removed through a standard intravenous catheter. Although the capturing eﬃciency of the system in a model experiment in vivo was shown to be only 8%, it was able to capture from 2500 to 10,000 cells per minute and these are 10 to 80 times more eﬃcient comparing to microﬂuidic platforms. Int. J. Mol. Sci. Magnetic Cell Separation 2020, 21, 2323 yed for the cell separ ples of the setups fo 29 of 60 magnet ti Finally, we refer to some recent reviews covering various issues related to magnetic cell sorting. Plouﬀe et al. have reviewed the application of magnetic particles for cell isolation [157]. This includes an overview of biomedical applications utilizing magnetic separation and various cell separation approaches; a theoretical background of magnetic phenomena and nanoparticle properties that can be employed for the cell separation, including synthesis methods of appropriate magnetic particles; and examples of the setups for manipulation with the cells conjugated with magnetic nanoparticles. Huang et al. have reviewed progress in magnetic manipulation with particles and cells in microﬂuidic chips including their separation, concentration, capture, arrangement, and assembly [271]. The review also includes the theoretical background of particle and cell manipulation in a microﬂuidic environment. Munaz et al. have reviewed advances in magnetophoresis with respect to microﬂuidic systems, including fundamental and theory of magnetophoresis and its applications for mixing, separation, and trapping of particles and cells [272]. Pezzi et al. have evaluated ﬁve diﬀerent types of commercially available magnetic beads with respect to various aspects of cell labeling and isolation [273]. They have tested antibody binding, and surface density, target cell capture eﬃciency and purity, the release of conjugated antibodies and captured cells, an impact of magnetic beads on further cell imaging and analysis, and the possibility of integrating magnetic beads cell isolation with standard nucleic extraction methods. This work may be used as a comprehensive guide to choosing the most suitable magnetic beads for cell manipulations depending on the requirements and possibilities of a particular investigation. al. have reviewed progress in magnetic manipulation with particles an dic chips including their separation, concentration, capture, arrangement, and review also includes the theoretical background of particle and cell manipu dic environment. Munaz et al. have reviewed advances in magnetophoresis with dic systems, including fundamental and theory of magnetophoresis and its appl paration, and trapping of particles and cells [272]. Pezzi et al. have evaluated fiv ommercially available magnetic beads with respect to various aspects of cell la 273]. They have tested antibody binding, and surface density, target cell captur y, the release of conjugated antibodies and captured cells, an impact of magnet l imaging and analysis, and the possibility of integrating magnetic beads cell iso nucleic extraction methods. Magnetic Cell Separation This work may be used as a comprehensive guide t suitable magnetic beads for cell manipulations depending on the require es of a particular investigation. Separation of Cells Acoustic Separation of Cells stophoresis is an applic Acoustophoresis is an application of the acoustic ﬁeld for cell sorting (see Figure 9c). Acoustic waves in the ultrasound range are applied in a number of microﬂuidic devices [165] allowing for separation of cells according to their size, density, and compressibility (Figure 14) [274]. Acoustophoresis operates with an acoustic power and an ultrasound frequency similar to those of ultrasound imaging, and, therefore, does not inﬂuence signiﬁcantly the cell viability [275,276] that opens prospects to use this technique for cell sorting. the ultrasound range are applied in a number of microfluidic devices [165] a n of cells according to their size, density, and compressibility (Figure horesis operates with an acoustic power and an ultrasound frequency similar d imaging, and, therefore, does not influence significantly the cell viability [27 spects to use this technique for cell sorting. e 14. Acoustofluidic manipulations with particles and cells with a positive (a) and neg tic response in a bulk acoustic standing wave. (c) The schematic of an surface standing Figure 14. Acoustoﬂuidic manipulations with particles and cells with a positive (A) and negative (B) acoustic response in a bulk acoustic standing wave. (C) The schematic of an surface standing acoustic waves (SSAW) device with interdigital transducers (IDTs) focusing the cells along well-deﬁned streamlines. (D) The cross-section of an SSAW device with four pressure nodes. Reprinted with permission from [165]. Copyright 2015, Royal Society of Chemistry. Ultrasound waves used for acoustophoresis can be divided into standing and traveling waves [277] In turn, standing waves can be classiﬁed on bulk acoustic waves (BAW) [278] and surface acoustic Acoustofluidic manipulations with particles and cells with a positive (a) and n esponse in a bulk acoustic standing wave. (c) The schematic of an surface standi Figure 14. Acoustoﬂuidic manipulations with particles and cells with a positive (A) and negative (B) acoustic response in a bulk acoustic standing wave. (C) The schematic of an surface standing acoustic waves (SSAW) device with interdigital transducers (IDTs) focusing the cells along well-deﬁned streamlines. (D) The cross-section of an SSAW device with four pressure nodes. Reprinted with permission from [165]. Copyright 2015, Royal Society of Chemistry. Ultrasound waves used for acoustophoresis can be divided into standing and traveling waves [277]. In turn, standing waves can be classiﬁed on bulk acoustic waves (BAW) [278] and surface acoustic Int. J. Mol. Sci. 2020, 21, 2323 30 of 60 30 of 60 waves (SAW) [279]. Acoustic Separation of Cells stophoresis is an applic The raveling acoustic waves occur when ultrasound propagates in the medium with more or less uniform acoustic impedance. The pulses of traveling surface acoustic waves may be employed for isolation of single cells from the main sample stream and divert them in another outlet of the microﬂuidic chip. This was shown to eﬀective for the isolation of ﬁbroblasts, keratinocytes, and melanoma cell lines [280,281]. The standing acoustic waves occur under resonance conditions. To perform acoustophoretic sorting with standing BAW, ultrasound is typically applied transversely to the cell ﬂow direction. The standing BAWs will occur in a microﬂuidic channel if the ultrasound wavelength matches the channel width [165]. This kind of wave is used to redirect cells from the main streamline toward the pressure node to the special outlet. Currently, microﬂuidic chip technology combined with bulk acoustic waves is used for separation of diﬀerent mammalian cell subpopulations, such as red blood cells, white blood cells, platelets [282,283], and cancer cells like neuroblastoma [283]; breast cancer [284,285], prostate cancer [278], pancreas cancer [286]. Moreover, this approach allows for separating of other types of blood emboli like a lipid embolus [287] and bacterial cell [288]. In turn, standing SAWs are formed in the microﬂuidic channel when ultrasound propagates through the longitudinal direction [289]. Standing SAWs may be applied for isolation of either normal cell lines like white blood cells [290], platelets [291], and muscle cells [292] or cancer cells like breast cancer [279,292], melanoma [280,281], and alveolar basal epithelial adenocarcinoma [292]. Furthermore, the tilted-angle standing surface acoustic waves are used for white blood cell sample washing from cell debris formed during red blood cell lysis procedure [293]. At present day, there are microﬂuidic devices employing both standing and traveling surface acoustic waves for the selection of cancer cells from the blood. This multi-stage sorter used standing waves for cell focusing in the central part of the channel and traveling waves for displacing of target cells to the desired outlet [294]. 4.3.2. Passive Cell Separation Methods The sorting approaches described above include active particle or cell separation techniques. There are also passive methods developed for the separation, isolation, or enrichment of particles and cell populations based on typical diﬀerences in their size, shape, or morphological parameters. Inertial forces, hydrodynamic propagation, and deterministic lateral displacement are applied to sort diﬀerent objects [295,296]. Inertial Focusing in Microﬂuidic Channels One of the commonly used approaches for label-free passive cell separation is inertial focusing in microﬂuidic channels. The theoretical background and detailed description of the separation process were summarized by Di Carlo [297]. Brieﬂy, the inertial focusing of the cells or particles can be achieved when the liquid ﬂow in the microchannel is still laminar, but inertia becomes valuable. This ﬂow mode depends on the geometry of the channel and liquid velocity and viscosity and usually observed from very low Reynolds number Re (Stokes ﬂow, inertia is negligible) to ~2000 (lower range limit of turbulence ﬂow). The cells moving in the ﬂow are aﬀected by drag and lift forces and therefore aligned in a deﬁned equilibrium position along the main ﬂow direction (Figure 15a). The equilibrium position is deﬁned by the ratio of the drag and lift forces and varies with the third power exponent of the particle/cell diameter [298]. In this way, kinetic and equilibrium separation can be realized. The kinetic separation is based on the diﬀerent equilibration times for the cells of various size whereas the equilibrium separation implies diﬀerent equilibrium positions for diﬀerent cells. The curved microchannels may be employed to produce inertial forces. Considering the laminar inertial ﬂow, the liquid ﬂowing through the curved channel results in the re-circulating secondary ﬂows that are formed due to a mismatch of the liquid velocity in the channel center and near-wall regions (Figure 15b). These re-circulating ﬂows create a pressure gradient in the radial direction of the channel and form two symmetric vortices inducing secondary ﬂow. The dimensionless number Int. J. Mol. Sci. 2020, 21, 2323 31 of 60 31 of 60 describing the secondary ﬂow was ﬁrst established by Dean and, therefore, called the Dean number, while the secondary ﬂow itself is called the Dean ﬂow. Thus, the cells in curved channels are aﬀected both by inertial migration and Dean ﬂow. This results in the movement of equilibrium line from the channel center that allows improving cell collection; to reduce the channel length required for focusing and separation; to enable an additional equilibrium separation modality for the cells of various sizes (Figure 15c,d). Sci. 2019, 20, x FOR PEER REVIEW 15. Inertial Focusing in Microﬂuidic Channels (a) Formation of the hydrodynamic equilibrium position of a cell moving in the liq ected by the drag and lift forces; (b) formation of re-circulating secondary flow (Dean ved microchannel; (c) separation of the cells depending on their size employing se curved microchannel; (d) the shift of the equilibrium position due to the superpo lift and Dean flow in a curved microchannel Adapted with permission from [297] C Figure 15. (a) Formation of the hydrodynamic equilibrium position of a cell moving in the liquid ﬂow and aﬀected by the drag and lift forces; (b) formation of re-circulating secondary ﬂow (Dean ﬂow) in the curved microchannel; (c) separation of the cells depending on their size employing secondary ﬂow in curved microchannel; (d) the shift of the equilibrium position due to the superposition of inertial lift and Dean ﬂow in a curved microchannel. Adapted with permission from [297] Copyright 2001, the Royal Society of Chemistry. 5. (a) Formation of the hydrodynamic equilibrium position of a cell moving in the liq cted by the drag and lift forces; (b) formation of re-circulating secondary flow (Dean ed microchannel; (c) separation of the cells depending on their size employing se curved microchannel; (d) the shift of the equilibrium position due to the superpo ift and Dean flow in a cur ed microchannel Adapted with permission from [297] C Figure 15. (a) Formation of the hydrodynamic equilibrium position of a cell moving in the liquid ﬂow and aﬀected by the drag and lift forces; (b) formation of re-circulating secondary ﬂow (Dean ﬂow) in the curved microchannel; (c) separation of the cells depending on their size employing secondary ﬂow in curved microchannel; (d) the shift of the equilibrium position due to the superposition of inertial lift and Dean ﬂow in a curved microchannel. Adapted with permission from [297] Copyright 2001, the Royal Society of Chemistry. , the Royal Society of Chemistry. curved microchannels may be employed to produce inertial forces. Considering th low, the liquid flowing through the curved channel results in the re-circulating s at are formed due to a mismatch of the liquid velocity in the channel center and Figure 15b). These re-circulating flows create a pressure gradient in the radial di nel and form two symmetric vortices inducing secondary flow. Inertial Focusing in Microﬂuidic Channels The dimensionles ng the secondary flow was first established by Dean and, therefore, called the Dean e secondary flow itself is called the Dean flow. Thus, the cells in curved channels ar nertial migration and Dean flow. This results in the movement of equilibrium line center that allows improving cell collection; to reduce the channel length req and separation; to enable an additional equilibrium separation modality for th izes (Figure 15c,d). ecent practical examples, Nathamgari et al. used curved microchannel to isolate sin m cell clusters after dissociation from the initial tissue [299]. Son et al. employe on in the curved microchannel to purify sperm cells from RBCs and leukocytes t l fi t d t t d th i fl idi ti f th i f l l ll As recent practical examples, Nathamgari et al. used curved microchannel to isolate single stem cells from cell clusters after dissociation from the initial tissue [299]. Son et al. employed inertial separation in the curved microchannel to purify sperm cells from RBCs and leukocytes [300,301]. Schaap et al. ﬁrst demonstrated the microﬂuidic separation of three specimens of algal cells of various sizes and shapes, including non-spherical ones [302]. Lee et al. used inertial separation in the curved channel for mesenchymal stem cell enrichment from a tissue-digested mouse bone marrow cell mixture [303]. The further development of inertial focusing is related to the preparation of more sophisticated microchannels and the integration of curved channels with other separation techniques. For instance, Shen et al. demonstrated spiral microchannel with series of micro-obstacles inside allowing for the linear acceleration of the secondary ﬂow (Figure 16a) [304]. The acceleration can be tuned employing various geometry of the obstacles and degree of conﬁnement. This results in increased eﬃciency and throughput of separation. Nivedita et al. combined the spiral channel with a lateral cavity acoustic transducer [305]. The microchannel provided a passive separation of non-target cells while the acoustic transducer is used for further active enrichment of the puriﬁed cell sample. Ramachandraiah et al. developed the method for eﬀective isolation of leucocyte subpopulations from the unprocessed whole blood samples based on the combination of inertial microﬂuidics and cell lysis. The reported device allows for complete removal of the RBCs by osmotic lysis along with the separation of the nucleated cells depending on their size in the curved microﬂuidic channel. Zhou et al. Inertial Focusing in Microﬂuidic Channels The device was shown to be eﬀective for the isolation of myogenic cells from the enzymatically processed muscle tissue cell mixture and for the puriﬁcation of them from cell aggregates and debris. Wang et al. developed a microﬂuidic separation device consisting of a wavy microchannel structure followed with a sheet ﬂow-focusing section [308]. The introduction of the sheet ﬂow-focusing area allows for more precise control over particle/cell position and even control the spacing between the separated objects. Furthermore, the concept of multisectioned devices can be extended for preparation of automated integrated microﬂuidic devices containing several focusing and separation areas. Zhang et al. described the example of the device containing ﬂow regulatory and two focusing areas in consequently connected microﬂuidic chips (Figure 16c) [309]. The focusing was performed in spirally shaped microchannels. The device was also equipped with a set of operational and control units to carry out fully automated cell manipulations and was shown to be capable of eﬃcient separation of cancer cells from human blood. ey figured out that size-dependent particle separation could be effectively controll the channel wall and the speed of the carrying fluid. Another issue that may be a ight microchannels is the isolation of small pathogens and blood components like s. However, the conventional inertial focusing of such small bioparticles req nt length of the microchannel and a significant amount of time for processing the concept of oscillatory inertial focusing in the practically infinite channel was p ular, the direction of liquid flow in a symmetrical microchannel is switched to the a high frequency. Due to the symmetry of the velocity field, the particles prese direction, which allows for continuous separation like in infinite channels. For al. demonstrated the successful isolation of submicron particles and Staphylococc exploiting this approach [311]. 16. (a) Principal scheme of the device employing Dean-like secondary flow acce d by micro-bars in the spiral microchannel. Reprinted with permission from [304]. C The Royal Society of Chemistry. (b) Asymmetric reverse wavy microchannel i cally reversible Dean flow. Reprinted with permission from [306]. Copyright 2018, Figure 16. (a) Principal scheme of the device employing Dean-like secondary ﬂow acceleration induced by micro-bars in the spiral microchannel. Reprinted with permission from [304]. Copyright 2017, The Royal Society of Chemistry. (b) Asymmetric reverse wavy microchannel inducing periodically reversible Dean ﬂow. Reprinted with permission from [306]. Copyright 2018, Springer Nature. Inertial Focusing in Microﬂuidic Channels designed an asymmetric reverse wavy microchannel and successfully employed it for the isolation of cancer cells from a blood sample (Figure 16b) [306]. This channel geometry induces periodically reversible Dean 32 of 60 n. For ht micr Int. J. Mol. Sci. 2020, 21, 2323 is the employment et al i e ti ated the ﬂow leading to eﬀective size-depended particle and cell separation. Syverud et al. demonstrated the microﬂuidic device with «Labyrinth» patterned curved channels [307]. The device was shown to be eﬀective for the isolation of myogenic cells from the enzymatically processed muscle tissue cell mixture and for the puriﬁcation of them from cell aggregates and debris. Wang et al. developed a microﬂuidic separation device consisting of a wavy microchannel structure followed with a sheet ﬂow-focusing section [308]. The introduction of the sheet ﬂow-focusing area allows for more precise control over particle/cell position and even control the spacing between the separated objects. Furthermore, the concept of multisectioned devices can be extended for preparation of automated integrated microﬂuidic devices containing several focusing and separation areas. Zhang et al. described the example of the device containing ﬂow regulatory and two focusing areas in consequently connected microﬂuidic chips (Figure 16c) [309]. The focusing was performed in spirally shaped microchannels. The device was also equipped with a set of operational and control units to carry out fully automated cell manipulations and was shown to be capable of eﬃcient separation of cancer cells from human blood. ey figured out that size-dependent particle separation could be effectively controll the channel wall and the speed of the carrying fluid. Another issue that may be a ight microchannels is the isolation of small pathogens and blood components like s. However, the conventional inertial focusing of such small bioparticles req nt length of the microchannel and a significant amount of time for processing the concept of oscillatory inertial focusing in the practically infinite channel was p ular, the direction of liquid flow in a symmetrical microchannel is switched to the a high frequency. Due to the symmetry of the velocity field, the particles prese direction, which allows for continuous separation like in infinite channels. For al. demonstrated the successful isolation of submicron particles and Staphylococc exploiting this approach [311]. ﬂow leading to eﬀective size-depended particle and cell separation. Syverud et al. demonstrated the microﬂuidic device with «Labyrinth» patterned curved channels [307]. Deterministic Lateral Displacement One more separation approach that can be employed to separate particles or cells in continuous ﬂow mode is deterministic lateral displacement (DLD). DLD is a method that utilizes an array of posts placed in the microchannel in a deﬁned manner that is every following raw is shifted latterly toward the previous one at a set distance [312]. This leads to the formation of separate laminar streamlines that follow through the structure in a certain path. If the particle size (Reﬀ) is more than a critical radius (Rc), the particle moving around the post will be deﬂected to the neighbor streamline [313]. This action repeats in the vicinity of every post that results in the displacement of the particle from the initial trajectory with a displacement angle determined by the row shift (“bumping” mode). Otherwise, the particles smaller than RC remain in the same streamline and keep following a “zigzag” but eventually straight path (see Figure 17a). The critical particle radius is deﬁned by the post diameter, the distance between the posts, and the raw shift [313]. First, DLD was introduced as a separation technique by Huang et al. [314] They demonstrated eﬀective isolation of two types of ﬂuorescent beads passing through the post array depending on single bead critical size based on the ﬁxed gap between the posts. Additionally, they have shown the possibility to resolve the spectrum of particles from 0.8 to 1 µm by passing through the separation device containing several separate sections with various gaps. Later, Inglis et al. provided an extended theory of DLD separation supported by experimental measurements for a range of particles from 2.3 to 22 µm [313]. Further, the same group employed DLD for blood cell separation [315]. The authors described two types of DLD separation devices, allowing for separation of blood cells depending on their size or preparation of blood plasma i.e., to remove the cells from the whole blood completely. DLD was shown to be eﬀective for isolation of lymphocyte subpopulations [316], nucleated RBCs from the peripheral blood of pregnant women [317], T. cyclops parasites from RBCs in whole blood [318], and cardiomyocytes form heterogeneous cell mixture [319]. Further development of DLD was related to the improvement of throughput of DLD devices to extend the volume of the processed sample per time to unite and enable the rapid enrichment of rare circulating objects like CTCs. For instance, Liu et al. Inertial Focusing in Microﬂuidic Channels (c) Design of the separation device employing Dean ﬂow with multiple separation areas. Reprinted with permission from [309]. Copyright 2001, The Royal Society of Chemistry. 16. (a) Principal scheme of the device employing Dean-like secondary flow acc by micro-bars in the spiral microchannel. Reprinted with permission from [304]. C he Royal Society of Chemistry. (b) Asymmetric reverse wavy microchannel i ally reversible Dean flow Reprinted with permission from [306] Copyright 2018 Figure 16. (a) Principal scheme of the device employing Dean-like secondary ﬂow acceleration induced by micro-bars in the spiral microchannel. Reprinted with permission from [304]. Copyright 2017, The Royal Society of Chemistry. (b) Asymmetric reverse wavy microchannel inducing periodically reversible Dean ﬂow. Reprinted with permission from [306]. Copyright 2018, Springer Nature. (c) Design of the separation device employing Dean ﬂow with multiple separation areas. Reprinted with permission from [309]. Copyright 2001, The Royal Society of Chemistry. ure. (c) Design of the separation device employing Dean flow with multiple separation a rinted with permission from [309]. Copyright 2001, The Royal Society of Chemistry. However, the preparation of curved and wave-shaped microchannels might be challenging, and, for some applications, the straight microchannels are preferable due to the ease of preparation and parallelization. Thus, one of the possible approaches to improve inertial separation with straight channels is the employment of the channels with an asymmetrical cross-section. For instance, Moloudi et al. investigated the possibility of particle separation in a trapezoidal straight microchannel [310]. They ﬁgured out that size-dependent particle separation could be eﬀectively controlled by the slope of the channel wall and the speed of the carrying ﬂuid. Another issue that may be addressed with straight microchannels is the isolation of small pathogens and blood components like fungi or exosomes. However, the conventional inertial focusing of such small bioparticles requires the signiﬁcant length of the microchannel and a signiﬁcant amount of time for processing. For this purpose, the concept of oscillatory inertial focusing in the practically inﬁnite channel was proposed. In particular, the direction Int. J. Mol. Sci. 2020, 21, 2323 33 of 60 33 of 60 of liquid ﬂow in a symmetrical microchannel is switched to the opposite one with a high frequency. Due to the symmetry of the velocity ﬁeld, the particles preserve their motion direction, which allows for continuous separation like in inﬁnite channels. For instance, Mutlu et al. Inertial Focusing in Microﬂuidic Channels demonstrated the successful isolation of submicron particles and Staphylococcus aureus bacteria exploiting this approach [311]. Deterministic Lateral Displacement proposed the DLD microﬂuidic device to isolate CTCs from the peripheral blood with a throughput of up to 2 mL/min [320]. They ﬁgured out that the CTCs isolation eﬃciency depends on the shape of the post. The triangle posts induce less shear stress and, therefore less eﬀect on the cell hydrodynamic radius compared with the round ones. Thus, the cells keep moving through the streamlines as deﬁned by the DLD pattern that improves the separation eﬃciency. Moreover, the same group demonstrated that the purity of the isolated cells could be further improved if DLD is combined with aﬃnity-based separation techniques [321]. Previously, Loutherback et al. demonstrated the DLD device composed of triangle posts that can be run with a throughput of 10 mL/min and isolate CTCs from the whole blood with an eﬃciency of 85% [322]. However, the increase of the ﬂow rate may lead to undesired side eﬀects in the DLD array. One of the most signiﬁcant is the clot formation when processing the blood samples. Although Loutherback et al. reached the signiﬁcant ﬂow rate in their device, the volume of the processed blood was limited by 200 µL per DLD array due to the clogging. D’Silva et al. carried out the study of clot formation in DLD arrays under various conditions and pointed outed how the clots can aﬀect the DLD performance [323]. The clots increase the ﬂuid resistance, capture the cells passing through the post array, and alter the paths of the ﬂuid streamlines. According to D’Silva et al., clogging in DLD array may be induced by two factors: platelet the activation and coagulation that can be suppressed by inhibiting the action of thrombin and chelating of calcium ions respectively. Additionally, the authors demonstrated that shear-induced platelet activation is an important factor of clogging in microﬂuidic systems. This issue can be addressed by optimization of Int. J. Mol. Sci. 2020, 21, 2323 34 of 60 34 of 60 DLD array conﬁguration, in particular in the injecting area. For instance, Mehendale et al. proposed the design of DLD device with a radial arrangement of round posts (RAPID) of various size combined in 3 separation zones [324]. This design was shown to be eﬀective for continuous separation of spectrum of particles with respect to their size with throughput of 3 mL/min. Deterministic Lateral Displacement The clogging was avoided due to preferential isolation of the largest particles while the rest of the particles can follow multiple parallel paths through the device. Further, they adapted the device for processing of whole blood [325]. Without any pre-processing, the device was able to operate with the whole blood for 6 h and rich almost 60-fold enrichment of the platelets as model target cells. Another issue is that DLD, as other microﬂuidic passive separation approaches, implies a laminar ﬂuid ﬂow with Re less than 1. This is the pre-requisite for the formation of the deﬁned laminar streamlines and a predictable movement of the particles through the post array. This results in a relatively low ﬂuid ﬂow rate and restricts the separation performance. On the other hand, the increase of the ﬂuid velocity at high ﬂow rates leads to the domination of inertial forces that alter the streamlines, which resulted in the formation of the vortices in the wake of the posts. The vortices increase the hydrodynamic radius of the posts reducing the critical radius of the particles. Recently, Dincau et al. reported on the numerical simulation and experimental evaluation of the performance of the DLD device operating in the high-Re mode (10 < Re < 60) [326]. They ﬁgured out that the vortices appear around Re = 25 and are fully developed under Re = 50, reaching the half-size of the post. Thus, the same particle can move through the DLD array in a “zigzag” mode under Re = 0.1 and in a “bumping” mode under Re = 25, which leads to lateral displacement. This may be employed for the development of DLD devices with an adjustable gap between the posts with the separation threshold tuned by the ﬂuid ﬂow rate. Further studies revealed that the main mechanism responsible for switching the displacement mode is associated with the compressing of streamlines under high-Re [327]. This was shown by the separation of vortex formation and streamline compression using the posts of a symmetric airfoil shape that does not produce the vortices even under Re = 100. Additionally, DLD may be employed for separation of particles and cells by their shape involving two types of phenomena: diﬀerences in length scale and diﬀerences in transport properties [328]. For instance, Beech et al. combined size-based sorting with the isolation of cells regarding their susceptibility to shear stress (Figure 17) [329]. Deterministic Lateral Displacement 2019, 20, x FOR PEER REVIEW 34 of 6 Figure 17. Mechanisms of separation by DLD with respect to cell size, shape, and deformability as in the case of RBCs. (a) The principle of DLD separation: particles with Reff < Rc follow the flow direction and those with Reff > Rc are displaced at an angle to the flow direction. For hard spheres, Reff is equa to the radius. (b) Red blood cells are normally disc–shaped but they can adopt other shapes when exposed to different chemicals. (c) Shear forces deform particles changing Reff, and measuring th change in Reff as a function of applied shear rate is equivalent to measuring the deformability of th particle. (d) Reff depends on the orientation of the particle. Controlling orientation and measuring Re gives information about shape. It is also possible to measure deformability in different directions. (e In a deep device RBCs rotate such that Reff (< Rc) is equal to half the thickness. (f) Confinement in shallow device means that the cell radius defines Reff (> Rc). (g) An echinocyte with Reff > Rc. Reprinted with permission from [329]. Copyright 2012, the Royal Society of Chemistry. Figure 17. Mechanisms of separation by DLD with respect to cell size, shape, and deformability as in the case of RBCs. (A) The principle of DLD separation: particles with Reﬀ< Rc follow the ﬂow direction and those with Reﬀ> Rc are displaced at an angle to the ﬂow direction. For hard spheres, Reﬀis equal to the radius. (B) Red blood cells are normally disc–shaped but they can adopt other shapes when exposed to diﬀerent chemicals. (C) Shear forces deform particles changing Reﬀ, and measuring the change in Reﬀas a function of applied shear rate is equivalent to measuring the deformability of the particle. (D) Reﬀdepends on the orientation of the particle. Controlling orientation and measuring Reﬀgives information about shape. It is also possible to measure deformability in diﬀerent directions. (E) In a deep device RBCs rotate such that Reﬀ(< Rc) is equal to half the thickness. (F) Conﬁnement in a shallow device means that the cell radius deﬁnes Reﬀ(> Rc). (G) An echinocyte with Reﬀ> Rc. Reprinted with permission from [329]. Copyright 2012, the Royal Society of Chemistry. igure 17. Mechanisms of separation by DLD with respect to cell size, shape, and deformability as i he case of RBCs. Deterministic Lateral Displacement Further, Kabacao˘glu and Biros theoretically and experimentally studied the deformability-based sorting of same-size RBCs via DLD [330]. They provided an extended theory of RBC separation based on their deformability in DLD array and ﬁgured out that the deformability of RBCs is deﬁned by their membrane stiﬀness and interior ﬂuid viscosity, which in turn depends on the dimensionless capillary number and viscosity constant. Zeming et al. demonstrated complete isolation of RBCs to a focused stream employing an array of I-shaped pillars inducing continuous rotational movement of the cells that results in a greater eﬀective separating size [331]. Au et al. designed the two-stage DLD device for the separation of CTC clusters with respect to their size and shape asymmetry [332]. The ﬁrst stage of the device isolates the large CTC clusters with a size of 30 µm or higher from the whole blood moving through the conventional DLD array with the round posts. The second stage processes the product coming from the outlet of the ﬁrst stage that includes small CTC clusters and blood cells using the array of asymmetric posts and height restrictions. The resulted isolation product contained about 99% of the large clusters and about 65% of small clusters recovered from the whole blood. Although the design of DLD devices still is under consideration for every particular application, they were shown to be eﬀective for cell isolation for cancer diagnostics. The examples include but not are limited to the works on CTC isolation mentioned above. Additionally, Wunsch et al. demonstrated the approach for the preparation of a nano-scaled DLD array that is suitable for the separation of exosomes [333]. More recently, Song et al. designed the DLD device with the posts modiﬁed by EpCAM aptamer covered Au NPs [334]. The aptamer-induced aﬃnity capturing signiﬁcantly increases the size-based enrichment of targeted CTCs in the DLD array. The captured cells can be released for further analysis by simple chemical reactions. Int. J. Mol. Sci. 2020, 21, 2323 35 of 60 35 of 60 To conclude this subsection, we refer to the review of McGrath et al. devoted to the DLD separation [312]. This review provides a deep insight into the theoretical background of DLD principles, as well as the number of examples of various DLD devices, and can be used as a guide to assemble the DLD device for the desired application. nt. J. Mol. Sci. p [ ] Pinched Flow Fractionation inched Flow Fractionation Pinched flow fractionation (PFF) is an approach for the separation of particles according to the izes in laminar flow (Figure 18). It was firstly proposed by Yamada et al. [335] and included tw lements. The first is hydrodynamic focusing of particles in a fluid stream similar to the flow ytometry, but in this case, particles or cells additionally aligned against the microfluidic channe wall. Through this process, a flowing stream with particles or cells is pinched into a narrow channe ross-section-pinched segment. Once the channel broadens, the particles are forced to flow i ifferent trajectories due to their size determines how far from the wall, their center of mass is placed he second element is the flow field fractionation. Different outlet channels conjoined to the mai hannel with particles pinched to the wall. Due to adjusted fluidic resistances, the particles o ifferent sizes are sorted by radially spreading streamlines and follow into different outlet channel article separation efficiency is defined by the ratio of flow rates, the width of the pinched segmen h t t l fl t d th t f th t ll ti th ti l Th t li t Pinched ﬂow fractionation (PFF) is an approach for the separation of particles according to their sizes in laminar ﬂow (Figure 18). It was ﬁrstly proposed by Yamada et al. [335] and included two elements. The ﬁrst is hydrodynamic focusing of particles in a ﬂuid stream similar to the ﬂow cytometry, but in this case, particles or cells additionally aligned against the microﬂuidic channel wall. Through this process, a ﬂowing stream with particles or cells is pinched into a narrow channel cross-section-pinched segment. Once the channel broadens, the particles are forced to ﬂow in diﬀerent trajectories due to their size determines how far from the wall, their center of mass is placed. The second element is the ﬂow ﬁeld fractionation. Diﬀerent outlet channels conjoined to the main channel with particles pinched to the wall. Due to adjusted ﬂuidic resistances, the particles of diﬀerent sizes are sorted by radially spreading streamlines and follow into diﬀerent outlet channels. Particle separation eﬃciency is deﬁned by the ratio of ﬂow rates, the width of the pinched segment, the total ﬂow rate, and the geometry of the segment collecting the particles. The parameters listed above should be adjusted in order to ﬁnd the optimal design. Deterministic Lateral Displacement (a) The principle of DLD separation: particles with Reff < Rc follow the flow directio nd those with Reff > Rc are displaced at an angle to the flow direction. For hard spheres, Reff is equa o the radius. (b) Red blood cells are normally disc–shaped but they can adopt other shapes when xposed to different chemicals. (c) Shear forces deform particles changing Reff, and measuring th hange in Reff as a function of applied shear rate is equivalent to measuring the deformability of th article. (d) Reff depends on the orientation of the particle. Controlling orientation and measuring Re ives information about shape. It is also possible to measure deformability in different directions. (e n a deep device RBCs rotate such that Reff (< Rc) is equal to half the thickness. (f) Confinement in hallow device means that the cell radius defines Reff (> Rc). (g) An echinocyte with Reff > Rc. Reprinte with permission from [329] Copyright 2012 the Royal Society of Chemistry Figure 17. Mechanisms of separation by DLD with respect to cell size, shape, and deformability as in the case of RBCs. (A) The principle of DLD separation: particles with Reﬀ< Rc follow the ﬂow direction and those with Reﬀ> Rc are displaced at an angle to the ﬂow direction. For hard spheres, Reﬀis equal to the radius. (B) Red blood cells are normally disc–shaped but they can adopt other shapes when exposed to diﬀerent chemicals. (C) Shear forces deform particles changing Reﬀ, and measuring the change in Reﬀas a function of applied shear rate is equivalent to measuring the deformability of the particle. (D) Reﬀdepends on the orientation of the particle. Controlling orientation and measuring Reﬀgives information about shape. It is also possible to measure deformability in diﬀerent directions. (E) In a deep device RBCs rotate such that Reﬀ(< Rc) is equal to half the thickness. (F) Conﬁnement in a shallow device means that the cell radius deﬁnes Reﬀ(> Rc). (G) An echinocyte with Reﬀ> Rc. Reprinted with permission from [329]. Copyright 2012, the Royal Society of Chemistry. p [ ] Pinched Flow Fractionation he total flow rate, and the geometry of the segment collecting the particles. The parameters liste bove should be adjusted in order to find the optimal design. Yamada M et al separated polystyrene microspheres with a diameter of 15 µm from 30 µm one Yamada M. et al. separated polystyrene microspheres with a diameter of 15 µm from 30 µm ones [335]. The authors suggested a linear relationship between the particle radius and position in the Int. J. Mol. Sci. 2020, 21, 2323 36 of 60 36 of 60 pinched segment, assuming that all channels were much larger in width than in height. The pinching is taking place in the pinched segment with width 2d that should be similar to the diameter 2R max of the largest particle to be sorted. Therefore, the particles with a size signiﬁcantly smaller than the channel width cannot be eﬀectively separated. In order to overcome this problem and increase the particle displacement after passing the pinched segment, the geometry of one or more of the outlet channels was made asymmetrical to modify ﬂow resistance for better hydrodynamic control. This realization was proposed by Takagi et al. as asymmetric pinched ﬂow fractionation increasing the separation resolution [336]. This modiﬁcation is capable of separating 1 µm and 2.1 µm particles with an eﬃciency of 80%. Additionally, the broadening of the pinched segment allows adding downstream collecting channels in the PFF design [337]. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 35 of 60 an efficiency of 80%. Additionally, the broadening of the pinched segment allows adding downstream collecting channels in the PFF design [337]. Figure 18. Principle of pinched flow fractionation. (a) In the pinched segment, particles are aligned to one sidewall regardless of their sizes by controlling the flow rates from two inlets; (b) particles are separated according to their sizes by the spreading flow profile at the boundary of the pinched and the broadened segments. The liquid containing particles is dark-colored. Reproduced with permission from [335]. Copyright 2004, American Chemical Society. Figure 18. Principle of pinched ﬂow fractionation. (a) In the pinched segment, particles are aligned to one sidewall regardless of their sizes by controlling the ﬂow rates from two inlets; (b) particles are separated according to their sizes by the spreading ﬂow proﬁle at the boundary of the pinched and the broadened segments. The liquid containing particles is dark-colored. p [ ] Pinched Flow Fractionation Reproduced with permission from [335]. Copyright 2004, American Chemical Society. Figure 18. Principle of pinched flow fractionation. (a) In the pinched segment, particles are aligned to one sidewall regardless of their sizes by controlling the flow rates from two inlets; (b) particles are separated according to their sizes by the spreading flow profile at the boundary of the pinched and the broadened segments. The liquid containing particles is dark-colored. Reproduced with permission from [335]. Copyright 2004, American Chemical Society. Figure 18. Principle of pinched ﬂow fractionation. (a) In the pinched segment, particles are aligned to one sidewall regardless of their sizes by controlling the ﬂow rates from two inlets; (b) particles are separated according to their sizes by the spreading ﬂow proﬁle at the boundary of the pinched and the broadened segments. The liquid containing particles is dark-colored. Reproduced with permission from [335]. Copyright 2004, American Chemical Society. Alternatively, the separation resolution was enhanced by a simple geometric modification of the primary PFF design like adding a snakelike part to channel [338]. This results in a more effective separation of particles due to an increase of the lateral distance between the particles after the pinched segment. The enhanced PFF was shown to be capable of separating 7 different sizes of particles with an efficiency of 70%. In addition, PFF has been expanded by the spatial reorientation of the microfluidic device for gravitationally enhanced separation of particles with different sizes and mass [339]. The theoretical and experimental studies on dynamic characteristics of laminar flows and hydrodynamic enhancement of particle separation were carried out. Polystyrene microbeads with different sizes were used as model particles to demonstrate rapid (<1 min) and high-purity (>99.9%) effective separation. As a result, a simple sorting system was developed to perform size profiling and Alternatively, the separation resolution was enhanced by a simple geometric modiﬁcation of the primary PFF design like adding a snakelike part to channel [338]. This results in a more eﬀective separation of particles due to an increase of the lateral distance between the particles after the pinched segment. The enhanced PFF was shown to be capable of separating 7 diﬀerent sizes of particles with an eﬃciency of 70%. In addition, PFF has been expanded by the spatial reorientation of the microﬂuidic device for gravitationally enhanced separation of particles with diﬀerent sizes and mass [339]. p [ ] Pinched Flow Fractionation The theoretical and experimental studies on dynamic characteristics of laminar ﬂows and hydrodynamic enhancement of particle separation were carried out. Polystyrene microbeads with diﬀerent sizes were used as model particles to demonstrate rapid (<1 min) and high-purity (>99.9%) eﬀective separation. As a result, a simple sorting system was developed to perform size proﬁling and following Int. J. Mol. Sci. 2020, 21, 2323 37 of 60 mass-dependent separation of particles using a combination of gravity and hydrodynamic ﬂows and applied to separate polydisperse perﬂuorocarbon droplets emulsions. The ﬁrst step towards nano-scale separation was realized by Y. Sai et al. by separation of 0.5 µm from 0.86 µm polystyrene microspheres [340]. They suggested incorporating microvalves to change the ﬂow resistance in the collecting outlets of the PFF device to enhance separation eﬃciency. Expectedly, the pressure in the outlet varies depending on the microvalve position that leads to changing the ﬂow rate inside the channel and the path of the ﬂow through the particles. Thus, by adjusting the valves, one can force particles of diﬀerent sizes to move to diﬀerent outlets. Additionally, it was established that the separation resolution depends on the microchannel wall roughness that is the particles with a diameter comparable with the wall roughness could not be separated by the PFF technique [341]. In concentrated samples, interactions between particles increased, which leads to decreasing the selectivity of the PFF system. Thus, the inﬂuence of concentration needs to be investigated. The quantitative inﬂuence of the pinching intensity in the balance between the requirements of selectivity and minimal dilution was discussed [342]. As a result, PFF was proved as an eﬃcient technique to separate a semi-concentrated polydisperse suspension of microparticles into sub-populations with tiny overlapping. Eventually, the PFF technology was extended to a passive hydrodynamic cell sorting for leukocyte enrichment [343]. In addition, PFF was used to separate cancer cells (LS174T colorectal adenocarcinoma) from white blood cells (WBC), with that the cells were separated at eﬃciencies above 90% for both cell types [344]. The size overlapping between cancer cells and WBC prevents high-eﬃciency separation, however, it can be improved using a diﬀerence in cell deformability. Further PFF improvements can be developed by using other separation techniques in combination with PFF, for instance, sedimentation [345], centrifugal [346], optical [347], and dielectrophoretic forces [348]. Furthermore, inertial enhanced pinched ﬂow fractionation by taking advantage of inertial forces was devised [349]. 5. Automatic Processing of Cytometry Data The features that provide separation of object subpopulations are selected and plotted as 1D or 2D plots. Then the data points in a range (for 1D) or a region (for 2D) of values are selected manually on the plot by visually monitoring the points clusters and the data for objects subpopulation is extracted, so the next separation can be done on this subset. beginning of flow cytometry development, the analysis was carried out manually [350] and, therefore, consumed a lot of time, while the quality of analysis depended on the operator qualification. Currently, a cytometry device usually provides a user with a toolset for automatic data processing. The data processing pipeline in flow cytometry usually consists of some signal preprocessing, generating a set of features for each object in the flow and then provide separation of object classes based on the combination of features (Figure 19). Signal preprocessing is highly specific for each device design and, thus, it is outside the scope of this review. Figure 19. The deep neural network recognizes cell types by their corresponding waveforms. Different types of cells are categorized and charged with different polarity charges so that they can Figure 19. The deep neural network recognizes cell types by their corresponding waveforms. Diﬀerent types of cells are categorized and charged with diﬀerent polarity charges so that they can be separated into diﬀerent collection tubes. Reprinted with permission from [351]. Copyright 2019, Nature Springer. Figure 19. The deep neural network recognizes cell types by their corresponding waveforms. Different types of cells are categorized and charged with different polarity charges so that they can Figure 19. The deep neural network recognizes cell types by their corresponding waveforms. Diﬀerent types of cells are categorized and charged with diﬀerent polarity charges so that they can be separated into diﬀerent collection tubes. Reprinted with permission from [351]. Copyright 2019, Nature Springer. To perform data analysis after receiving the signals, speciﬁc features should be determined. The combination of these features should characterize the cells of interest in the most accurate way. Until recently, classical computer vision (CV) and machine learning (ML) algorithms, such as thresholding (for feature extraction) or gradient busting and random forest (to solve classiﬁcation problems) were mainly employed in the analysis of ﬂow cytometry data [352]. 5. Automatic Processing of Cytometry Data After the ﬂow cytometry data is collected, a detailed analysis should be performed. At the beginning of ﬂow cytometry development, the analysis was carried out manually [350] and, therefore, consumed a lot of time, while the quality of analysis depended on the operator qualiﬁcation. Currently, a cytometry device usually provides a user with a toolset for automatic data processing. The data processing pipeline in ﬂow cytometry usually consists of some signal preprocessing, generating a set of features for each object in the ﬂow and then provide separation of object classes based on the combination of features (Figure 19). Signal preprocessing is highly speciﬁc for each device design and, thus, it is outside the scope of this review. Conventional ﬂow cytometry methods provide an intrinsic set of features describing single objects in the ﬂow. It usually includes forward scatter and side scatter channels along with signals from PMTs detecting ﬂuorescence in a number of wavelength bands. Imaging ﬂow cytometry, however, requires conversion from raw images of single objects into a set of features that allow discriminating properly diﬀerent object classes. Feature construction and selection of separation criteria for a long time was a complex procedure that requires high qualiﬁcation and a lot of expertise from the device operator. Progress in ﬂow cytometry increased both the number of cells in the experiment and the amount of information obtained from single cells, so the processing methods became more complex. On the other hand, modern progress in machine learning and deep learning methods start to make formerly complex procedures completely automatic and less erroneous in general. In the conventional analysis of ﬂow cytometry data, expert works with a set of features—the values that describe each object in the ﬂow. The most basic is the intensity of ﬂuorescence in diﬀerent spectral channels along with front scatter and side scatter light intensities. The multidimensional space of features is usually orthogonalized by a compensation matrix that describes how to minimize the crosstalk between channels. Afterward, the feature space can be distorted by some transfer function Int. J. Mol. Sci. 2020, 21, 2323 5. Automatic Processing Aft th fl t 38 of 60 At th 38 of 60 At the (e.g., logarithm, biexponential, logistic, tangential) that provides cut-oﬀfor the outliers or compression of the rare high-intensity events into the space near the major data points. The essential part of the data processing is based on gating. 5. Automatic Processing of Cytometry Data This algorithm can use a complete dataset without downsampling and runs visualization procedure much faster than the previous method giving, in general, a similar result. The third automatic approach for the detection of subpopulations from a ﬂow cytometry dataset is based on the t-distributed Stochastic Neighbour Embedding (t-SNE) [369] method. The method builds a projection from the dataset with high dimensionality containing a large number of independent features into 2D space in a way that neighbor points are still nearby and separate clusters become well visible in the projection. In the ﬂow cytometry, this method is usually called viSNE [370]. Information about automatic methods not using dating summarized in a number of comprehensive reviews, that include Aghaeepour et al. [371] and Mair et al. [372] The next major step in the automation of cytometry data processing is based on transfer from manual feature construction to automatic. This process in computer science is heavily based on the transfer from classical machine learning algorithms, which separate the data by manually selected and thoroughly checked set of features, to learning approaches, which construct features by learning. Most of the deep learning approaches were based initially on learning convolutions in the separate layers of neural networks (NNs) architecture. However, nowadays there are various and versatile sets of network architectures providing required functionality and the progress in inventing new ones is very fast. First successful approach to construct deep learning models for computer vision was mostly based on a deep network with a lot of layers in the structure forming a pairs from convolution layer that learns transfer methods to construct a features at each level of abstraction and fully connected layers keeping information as a set of weights of selected features [373]. The more modern neural network architectures can be based on only convolutions [374] or some kind of recurrence (RNN) providing the memory of previously processed data inside the network. Any machine learning model still requires a dataset to be trained on. During the training, deep learning algorithms independently selects geometric features from the available data (a certain combination of curved lines, a combination of pixels of a certain brightness), followed by the derivation of abstract features from, for example, presence of a vacuole in the image or damage of cell nucleus [375]. As a result, the capabilities of ﬂow cytometry are increased. 5. Automatic Processing of Cytometry Data However, there are a few examples of the original algorithms [353,354] to detect rarely seen labeled cells in the sample and to segment the nucleus, respectively. The introduction of these algorithms allowed us to automate the analysis of cytometry data with fairly good accuracy. To date, these approaches can be considered traditional and most widely used. Traditional ML and CV are currently implemented in the majority of cytometry data processing software, for example, IDEAS by AMNIS or FCS ﬂow cytometry by De Novo software, which is often used with the MATLAB package for additional calculations. There are also free open-source software packages, like ImageJ [355,356] and CellProﬁler [357–359]. The pipeline tends to be the following: single images from a cytometer are loaded to the software (for example, CellProﬁler) where they are processed (for instance, brightness is adjusted and borders are highlighted) and placed into separate ﬁles. Then, the ﬁles are transferred to the input of some machine learning system built on the basis of Int. J. Mol. Sci. 2020, 21, 2323 39 of 60 some computational frameworks programmed in MATLAB, Python, or some other general-purpose computer language. A lot of eﬀort was made to deﬁne the automatic gating procedure for ﬂow cytometry data [360–364] or clustering of similar phenotype on multidimensional data [365,366]. A number of machine learning approaches for automatic separation of subpopulations in the feature space was developed. The most well-known are SPADE, FlowSOM, and CITRUS methods along with the viSNE alone and combined with the previous three ones for prior dimensionality reduction. Spanning-tree Progression Analysis of Density-normalized events (SPADE) [367] organizes the cells in a hierarchy of related phenotypes. It allows for high throughput processing of data volumes and draws a tree of diﬀerent phenotypes/subpopulations of objects that can be easily understood, and extract subpopulations of cells with similar properties. The inputs for the algorithm are selected markers/features set, outlier density, target density and desired number of the clusters. The ﬁrst and the last ones are the most important, the second and the third regulate how many cells are excluded and how many will survive the downsampling process. The last parameter governs when the algorithm will stop. FlowSOM [368] is another approach to build Self-Organizing Maps on the dataset and organize it into a minimal spanning tree. 5. Automatic Processing of Cytometry Data For example, a median ﬁlter was applied to decrease image noise, whereas the Canny edge detection allows deﬁning the contours of cells, particles or their groups. The extracted information about the geometry of the object allows building the binary mask. Further, the binary mask was applied to incoming images in order to receive information about their morphological characteristics (area and shape) and scattering and ﬂuorescence intensity to highlight the region of interest. Afterward, the data was transferred to the input of a six- or eight-layer convolution neural network. This operation removes unnecessary information and makes it easier to extract intricate features. To put in other words, in the developed method the traditional ML and DL compensate the disadvantages of each other and combine high speed and accuracy. Testing the eﬀectiveness of the method was done by sorting particles of diﬀerent diameters in the sample (3-mm and 6-mm). This paper also demonstrated a well-developed approach to building the entire computing architecture. Often this issue is neglected in research, giving preference directly to data analysis. However, an emerging trend towards the application of ﬂow cytometers in diagnostics, engineering issues will have to be paid more attention. Properly built and optimized computing architecture can signiﬁcantly increase eﬀectiveness without the need for expensive equipment. In turn, this will make promising ﬂow cytometer-based devices more aﬀordable. Li et al. addressed the problem of imaging cells without ﬂuorescent agents to avoid their impact on cell behavior [351]. The authors developed an unusual approach to the problem of computational complexity. In particular, they discard most complicated operations, like image construction and extraction of features, and work directly with one-dimensional time series that display the shape of the detected wave. In order for the neural network could process with data, it was transformed into a two-dimensional array where one measurement corresponds to laser pulses at a point, the other corresponds to the number of points per pulse. Each signal was further divided into 100 smaller waveforms, with an overlap factor of 50% to create redundancy. This will further reduce noise and increase the sustainability of learning. After all the preparatory operations, the data were transferred to the input of a neural network with architecture based on VGG neural network architecture [374]. It consists of 16 convolutional layers, three layers for max-pooling, and three fully connected layers. 5. Automatic Processing of Cytometry Data Large arrays of images can be processed without distraction by a preliminary search and registration of signiﬁcant features, making the process fully automated. Moreover, with the help of NNs, one can detect those signiﬁcant features, which were not assumed by a researcher [376]. Deep learning approach allows us to solve some complex computer vision problems like semantic segmentation (separation of closely packed objects) or object detection with the accuracy of well-trained human experts and sometimes even better, however, it is achieved by the large size of required datasets. The usual balanced set of objects to train a deep neural network for computer vision problems should Int. J. Mol. Sci. 2020, 21, 2323 40 of 60 40 of 60 consist of tens of thousands of objects from each class. Sometimes it can be achieved by artiﬁcial augmentation of smaller datasets containing only thousands or even hundreds of objects, but it is mostly a required minimum to train a good working model. The initial marking of the dataset should be done by human experts and it is among the most tedious and complex tasks in a deep learning approach. Additionally, a serious obstacle using deep learning methods in diagnostics may be their interpretability [377]. One or another step of the algorithm is diﬃcult to explain in a large part of deep learning methods. In medical diagnostics, this may cause fatal consequences. The high system requirements for computing performance to run the deep learning algorithms is also can be considered as a disadvantage, however, it is now mostly eliminated by the computational power provided by the general-purpose graphics processing unit (GPGPU) or tensor unit acceleration with parallel processing. g p p g p p g p p g Recently, there were a number of publications describing the complex system providing cell sorting by a combination of all the above-mentioned approaches. Nitta et al. demonstrated an integrated approach to the problem of ﬁnding the right cells and sorting them in a real-time combing traditional and deep machine learning [378]. Since neural networks usually have high accuracy, but relatively low speed, ﬁrst for each event in the image a binary mask is created for the bright-ﬁeld image, followed by extraction of the main characteristics from bright-ﬁeld and ﬂuorescent images. In this regard, algorithms of traditional computer vision were applied. 6. Advantages and Limitations of Modern Flow Cytometry Flow cytometry technology has undergone many changes since its invention in the 50s of the last century. The development of new hardware and software for ﬂow cytometry systems as well as ﬂuorophores development expanded the number of science domains in which this technology could be applied. At the dawn of ﬂow cytometry technology, it was used just to count and estimate the size of particles [382]. Currently, ﬂuorescent ﬂow cytometry systems are applied to solve a broad spectrum of issues in the diagnostics [383–385], biotechnology [386], material science [387,388], and other scientiﬁc ﬁelds. The current state-of-the-art in ﬂuorescent ﬂow cytometry allows to estimate up to 20 independent parameters for each cell and possesses a high throughput possibility—thousands of cells per second [389]. However, the simultaneous usage of the number of ﬂuorophores to label diﬀerent cell structures can lead to the overlapping of their emission spectra and requires compensation procedure performance [390]. This step is crucial in the case of studying rare objects, such as CTCs, because their small amount does not allow an operator to create a compensation ﬁle. There are two approaches to solve this problem. The ﬁrst one is to apply antibody capture beads to simulate positive, dim, and negative staining for the creation of a compensation ﬁle [391]. The second one is to use advanced software for automatic correction of the spectral overlapping [389]. Additionally, conventional ﬂuorescent ﬂow cytometry systems do not provide bright ﬁeld and ﬂuorescent images of events that constrain the ability of real-time veriﬁcation. Imaging ﬂow cytometry systems successfully solved this problem by combining the statistical power of conventional ﬂow cytometry with the advantages of ﬂuorescence microscopy [392]. The combination of both these technologies permits to acquire up to 12 ﬂuorescent and bright-ﬁeld images of each object without signiﬁcant loss in throughput ability. However, this technology imposes some limitations on the object size, which is directly determined by the detection limit, dynamic range, and intensity resolution of the system [393]. Conventional ﬂow cytometry also has the size threshold for object detection >500 nm [394,395]. One more disadvantage of ﬂow cytometry is the disability to detect events in the whole blood due to its cell concentration [396] far more than the detection threshold (no more than 108 cell/mL for Amnis, 3 × 107 cell/mL for Bio-Rad, 7.5 × 106 cell/mL for Beckman). 6. Advantages and Limitations of Modern Flow Cytometry However, a more detailed analysis of certain cell populations selected during ﬂow cytometry measurement, for instance, genetic analysis requires some post factum procedure. With this regard, the demand for separation of the sample or its return for further analysis arises. Some devices such as Sony SH800, Bio-Rad S3e cell sorters, or Beckman MoFlo XDP High-Speed Cell Sorter have this ability, but this is the exception rather than the rule. 5. Automatic Processing of Cytometry Data As a result of the work, the network gives the probability of a cell belonging to one of three classes. The result was a score of 95.71% and accuracy 95.75% with high learning stability and recognition time within a few milliseconds. Tanhaemami et al. predicted the lipid content in microalgae cells after nitrogen starvation by application of linear regression and a genetic algorithm (without the use of ﬂuorescent labels) [379]. Eulenberg et al. [380] and Doan et al. [381] studied the cell cycle of Jurkat cells in diabetic retinopathy Int. J. Mol. Sci. 2020, 21, 2323 41 of 60 41 of 60 and morphological changes in red blood cells. Both papers mostly modify the existing model of machine learning and interpret conclusions in terms of biology, without reference to a speciﬁc device. The works employ training by standard images received from the cytometer without adjustment of the element base or the type of signals on the sensors. These examples demonstrate the ﬂexibility of neural networks, as they require fewer preliminary data manipulation to work than traditional ML. Certainty modern machine learning methods will gradually supersede traditional methods in the processing and analysis of ﬂow cytometry data in the long run. ML and DL proved to be powerful and well-suited tools in cytometry. Probably, quite a few tasks can be solved by purely engineering approaches by adapting existing algorithms. Nevertheless, there is still the problem of further increasing the rate and biological interpretation of the results. 7. Conclusions and Outlook The progress in the design of modern ﬂow cytometry devices is strongly related to advances in adjacent areas of research and development. Considering the hardware part, great progress was made in the design of advanced optical systems like structured light illumination schemes, which allow for capturing better quality images on higher speeds of ﬂow with less laser-induced damage Int. J. Mol. Sci. 2020, 21, 2323 42 of 60 42 of 60 of ﬂuorophores. Additionally, the novel optical systems require fewer eﬀorts in precise focusing of detected objects in ﬂow and lens objective on the common spot. Great progress was also made in nonconventional detection methods including ﬂow cytometry approaches for detection in vivo based on photoacoustic and photothermal phenomena. However, these substantial areas are out of the scope of this review. Further, a semiconductor industry constantly provides better detectors that require less light to detect a signal. The modern techniques of image processing are able to take composite digital images by merging processed low-quality signals with properly removed noise from many sensors simultaneously. Semiconductor laser diodes and detectors for the less common part of the optical spectrum starts to be more available in all means and easier to integrate into new device designs. Enormous progress in ﬂow cytometry data processing software is achieved with the development of modern machine learning and deep learning techniques for all stages of data processing. The denoising and sensor fusion, solving computer vision and object detection tasks, grouping objects by the similarity of features that are automatically selected and generated are used everywhere now. All device and processing software vendors are starting now to provide this kind of solution to end-users. With all this combination of novel techniques, the ﬂow cytometry area of research is developing rapidly providing better devices that have better performance and much easier to operate. It pushes ﬂow cytometry towards widely adopted endpoint-of-care devices from the research lab. Author Contributions: Conceptualization, D.V.V. and D.N.B.; writing — original draft preparation, D.V.V. (Section 3.1.1, Section 3.2, Section 4.3, Section 4.3.1, Electrokinetic Separation, Magnetic Separation, Section 4.3.2, Inertial Focusing in Microﬂuidic Channels, Deterministic Lateral Displacement), A.A.K. (Section 1, Section 2.1, Section 2.2, Section 3.1.2), R.A.V. (Section 3.1.3, Section 3.3, Section 4.1, Section 4.1.1, Section 4.1.2, Section 4.2, Section 4.2.1, Section 4.2.2, Acoustic Separation, Section 6), A.V.E. (Section 2.3, ﬁgure’s preparation), M.A.M. (Section 5), O.A.I. (Section 1, Section 4.1.3, Pinched Flow Fractionation), D.N.B. 7. Conclusions and Outlook (Section 1, Section 7); writing ’ reviewing and editing, D.V.V. and D.N.B.; supervision, D.N.B.; project administration D.N.B.; funding acquisition, D.N.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the RUSSIAN SCIENCE FOUNDATION, grant number 18-19-00354. Funding: This research was funded by the RUSSIAN SCIENCE FOUNDATION, grant number 18-19-00354. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Galanzha, E.I.; Menyaev, Y.A.; Yadem, A.C.; Sarimollaoglu, M.; Juratli, M.A.; Nedosekin, D.A.; Foster, S.R.; Jamshidi-Parsian, A.; Siegel, E.R.; Makhoul, I.; et al. In vivo liquid biopsy using Cytophone platform for photoacoustic detection of circulating tumor cells in patients with melanoma. Sci. Transl. Med. 2019, 11, eaat5857. [CrossRef] 2. Nolan, J.; Nedosekin, D.A.; Galanzha, E.I.; Zharov, V.P. 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https://openalex.org/W4282565318	https://www.researchsquare.com/article/rs-1728216/latest.pdf	English	null	Real-time near-infrared spectroscopy monitoring of the paraspinal collateral network: comparison of two clinically available systems	Research Square (Research Square)	2,022	cc-by	7,532	Real-time near-infrared spectroscopy monitoring of the paraspinal collateral network: comparison of two clinically available systems Zara Dietze (  zara.dietze@helios-gesundheit.de ) Leipzig Heart Center Konstantin von Aspern Leipzig Heart Center Josephina Haunschild Leipzig Heart Center Susann Ossmann Leipzig Heart Center Michael A. Borger Leipzig Heart Center Christian D. Etz Leipzig Heart Center Research Article ed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/16 Methods Two systems (FORE-SIGHT®, CAS Medical Systems, USA and INVOS™ 5100C; Medtronic, Ireland) used at 4 paravertebral levels (mid and lower thoracic, upper and lower lumbar) were compared in an acute large animal model during three different experimental sequences mimicking relevant clinical scenarios: segmental artery (SA) occlusion by (1) open-access clip-occlusion (n = 7), (2) endovascular coil-embolization (n = 9) and distal circulatory arrest via (3) aortic cross-clamping (n = 9). Conclusion Both devices provide similar cnNIRS monitoring applicability at mid-thoracic, upper and lower lumbar levels. INVOS seems to have a more rapid and pronounced response to open SA sequential occlusion and aortic cross-clamping at the lower thoracic level. Purpose Efficacy of near-infrared spectroscopy monitoring of paraspinal collateral network (cnNIRS) has been shown to provide additional safety in extensive aortic repair. Better understanding of available cnNIRS monitoring systems regarding differences in performance for this specific purpose is required. Results Significant differences in oxygenation measurements between devices were observed only at the lower thoracic level during SA clipping and aortic cross-clamping (p < 0.001). During SA clipping, FORE-SIGHT and INVOS demonstrated reduction to 85% and 72% of baseline values after occlusion of the mid-thoracic region, with mean differences between devices varying from 12.9 to 20.6% (p > 0.05) throughout the experiment. In the aortic cross-clamp sequence, reduction of values was observed in both devices, with FORE-SIGHT having less pronounces decrease during cross-clamping compared to INVOS (mean differences 1.0-14.7%, p > 0.05), and slower response to reperfusion after declamping (72 to 84% within 1 minute in FORE-SIGHT and 57 to 99% in INVOS). Introduction Thoracoabdominal aortic (TAA) repair continues to be one of the most technically demanding areas of cardiovascular surgery, being associated with high mortality and morbidity despite numerous currently available protective adjuncts. Among possible complications of open and endovascular treatment, the most devastating complication remains paraplegia – a consequence of several pathological pathways leading to spinal cord injury [1]. A promising non-invasive method for indirect, real-time spinal cord tissue monitoring is based on the evaluation of paraspinal collateral network tissue oxygenation (StO2) using near-infrared spectroscopy (cnNIRS). By cnNIRS oxygen tissue saturation of the paraspinal vasculature is assessed using optical signal emission at wavelengths between 760–2500 nm (near-infrared) and their specific adsorption spectra [2]. Previous experimental studies have shown that oxygenation and perfusion of the paraspinal collateral network correlates with the spinal cord tissue perfusion [2, 3]. The underlying theory is based on the modern collateral network concept, which has been developed and experimentally proven by Griepp and Etz [4, 5]. Through this modern concept it is demonstrated that spinal cord perfusion is provided not only by direct blood flow from segmental arteries (SA)—originating from the thoracoabdominal aorta, or from internal thoracic arteries (from subclavian arteries) or caudally originating hypogastric arteries—but also by vast para- and intraspinal arterial collaterals [4–7]. This network is Thoracoabdominal aortic (TAA) repair continues to be one of the most technically demanding areas of cardiovascular surgery, being associated with high mortality and morbidity despite numerous currently available protective adjuncts. Among possible complications of open and endovascular treatment, the most devastating complication remains paraplegia Page 2/16 Page 2/16 capable of readjusting blood supply to the spinal cord tissue in response to acute or chronic segmental artery occlusion. The cnNIRS technique has been experimentally proven and clinically implemented as an additional monitoring modality prior to, during and after open and endovascular TAA repair [2, 8]. For clinical practice cnNIRS has the advantage that it can be safely and non-invasively placed on the back of the patient [8]. capable of readjusting blood supply to the spinal cord tissue in response to acute or chronic segmental artery occlusion. The cnNIRS technique has been experimentally proven and clinically implemented as an additional monitoring modality prior to, during and after open and endovascular TAA repair [2, 8]. For clinical practice cnNIRS has the advantage that it can be safely and non-invasively placed on the back of the patient [8]. Ethics statement All presented acute animal experiments were approved by the Institutional Animal Care and Use Committee in accordance with the local Veterinary Office and the principals stated by the National Society for Medical Research and the guidelines for the care and use of laboratory animals [10]. Introduction Initial clinical application of cnNIRS in the setting of extensive aortic repair is promising and its utilization is increasing [8, 9]. However, before cnNIRS can become a standard tool for intra- and postoperative monitoring, more clinical experience and reference data is needed. Furthermore, differences in handling and measurements between commercially available cnNIRS monitoring equipment have not been investigated. The aim of this study was therefore to experimentally compare two currently available monitoring systems in a well-established aortic large animal model of ischemic spinal cord injury. Experimental setup Twenty-five juvenile female pigs (German landrace, 31–55 kg) were operated on. Data sets of two animals were invalid and could not be analyzed due to technical problems, resulting in a total of 25 animals used for analysis. The study design included three different experimental settings: 1) open, sequential occlusion of segmental arteries (SA, Clipping) simulating open surgical TAA aortic replacement without SA reimplantation (n = 7); 2) minimally invasive coil- and plug embolization (MIS2ACE) of SAs, simulating TEVAR or modern priming modality [11] (n = 9) and 3) cross-clamping of the descending thoracic aorta, simulating acute distal aortic ischemia during open surgery (n = 9). Sample sizes were calculated a priori at a power of 80%, an alpha error probability of 5% with an effect size (d) of 1.5. All animals were sedated (15 mg/kg ketamine and 5mg/kg diazepam i.m.) prior to being transferred to the operating room. After placement of a peripheral catheter into a large ear vein, the animals were intubated endotracheally. Ventilation was set to an oxygen fraction of 0.5. Anesthesia was maintained by intravenous propofol (1–2 mg/kg) and 0.03-0.05mg/kg fentanyl (Pfizer, New York, NY, USA). The animal was turned on its right side. For evaluation of collateral network regional oxygen saturation cnNIRS optodes of two different monitoring systems (FORE-SIGHT Elite®, CAS Medical Systems, Inc., Branford, Connecticut, USA and INVOS™ Oximeter 5100C; Medtronic, Dublin, Ireland) were placed paravertebrally at four levels (mid and lower thoracic; upper and lower lumbar levels) in an alternating sequence (Fig. 1). The cnNIRS measurements were recorded manually as well as digitally and the stored data exported afterwards from both monitoring devices. The optode sequence (placement sides) was switched for each experiment in order to avoid potential measurement bias associated with surgical access and ride sided positioning. Haemodynamic and clinical monitoring was performed, including continuous invasive blood pressure monitoring through arterial catheters (right carotid artery and a femoral artery), pulse oximetry, 5-lead electrocardiogram and rectal temperature monitoring. A 7F, 20-cm multilumen central venous catheter was placed via the left jugular vein for additional infusions and blood sampling. Aortic Cross-Clamp Group (n = 9) After left lateral thoracotomy at the 5th intercostal space, the descending thoracic aorta was mobilized proximal to SA T4 (Fig. 2). In order to ensure correct positioning of the aortic clamp, a loop was placed around the aorta. The cross-clamp was then applied for a period of 10 minutes (clamp on) followed by 10 minutes of reperfusion (clamp off). The cnNIRS measurements were recorded manually every 10 minutes before aortic cross-clamping and every minute during the “clamp on” and” clamp off" periods as well as continuously via the internal system storage at a five seconds interval. Additionally, several static and dynamic parameters of cnNIRS were evaluated in this group, since aortic occlusion (as expected for any vascular occlusion test) was associated with the most rapid decrease and increase of cnNIRS values. Changes were expressed as percent changes from baseline: aseline to 2 min occlusion” Desaturation – difference between baseline and 2 minutes af “Baseline to 2 min occlusion” Desaturation – difference between baseline and 2 m “Baseline to 2 min occlusion” Desaturation – difference between baseline and 2 minutes after aortic cross-clamping “Baseline to 2 min occlusion” Desaturation – difference between baseline and 2 minutes after aortic cross-clamping “Baseline to 10 min occlusion” Desaturation – difference between baseline and 10 minutes after aortic cross-clamping (at the end of the occlusion period) “Baseline to 10 min occlusion” Desaturation – difference between baseline and 10 minutes after aortic cross-clamping (at the end of the occlusion period) “Baseline to 10 min occlusion” Desaturation – difference between baseline and 10 mi (at the end of the occlusion period) Desaturation slope, 1st min – desaturation rate from baseline to the first minute after cross clamping lope, 1st min – desaturation rate from baseline to the first minute after cross clamping Resaturation slope, 1st min – resaturation rate from the 10th minute of occlusion to the first minute of reperfusion Minimum – the lowest cnNIRS value during the occlusion period Resaturation slope, 1st min – resaturation rate from the 10th minute of occlusion to t Minimum – the lowest cnNIRS value during the occlusion period Maximum – the highest cnNIRS value during the reperfusion period After the procedure, the animals were euthanatized. In order to validate correct and complete S harvested and examined. Surgical approach and experimental sequence Open occlusion Group (Clipping; n = 7) Page 3/16 In the open SA occlusion group three surgical incisions were performed: 1) upper left lateral thoracotomy at the 5th intercostal space, where the heart, descending thoracic aorta and SAs T4-T6 were visualized; 2) lower left lateral thoracotomy at the 8th intercostal space and 3) left lateral retroperitoneal access for exposure of the thoracic and abdominal aorta as well as SAs T7-L5. All SAs were mobilized (starting from T4) and sequentially occluded by clip placement (WECK® Horizon™ Metal Ligation System, Teleflex, Morrisville, NC, USA) (Fig. 2). The cnNIRS measurements were manually recorded every 10 minutes before and after SA clipping and every minute during the clipping process as we as continuously stored at 5 seconds intervals in the cnNIRS monitor. Coil-Embolization Group (Coiling; n = 9) Left lateral thoracotomy at the 5th intercostal space was performed. A 7F sheet was introduced via femoral artery using the Seldinger technique. After heparinization (5000 IU), a 5.2F IM catheter was placed in order to identify the thoracoabdominal SAs (from T4 to L5, 15 pairs) by selective angiography. The visualized SAs underwent coil insertion using 60 mm and 70 mm platinum endovascular coils (Trufill Pushable Coils, Cordis, Waterloo, Belgium). Success of SA embolization was verified by contrast injection after each coil implantation and at the end of the procedure. Two to three coils were usually required for complete embolization of a proximal SA and 3–4 coils for larger, distal SA portions (Fig. 2). The cnNIRS measurements were recorded prior to and after coil-embolization analogous to the open SA occlusion group. Within-system cnNIRS changes during the procedures Both devices demonstrated significant changes in oxygenation consequential to open SA clipping, coil-embolization and aortic cross-clamping for measurements at upper and lower lumbar levels (within-system ANOVA p < 0.05 in all of the cases), and most of the measurements at lower thoracic level (Fig. 3). At this level, no statistically significant changes within each of the systems were observed in FORE-SIGHT performance during SA coiling (baseline to end-ischemia mean difference 6.4%). In the clipping group, baseline to end-ischemia mean differences varied from 36.6 to 38.3 in INVOS, and from 16.0 to 39.43 in FORE-SIGHT. In the coiling experiments, these values were 17.9–34.3 for INVOS, and 6.4–30.3 for FORE-SIGHT. Aortic cross-clamping was associated with a significant decrease in values in both systems (baseline to end-ischemia mean differences ranging from 38.7 to 43.3 in INVOS, and from 28.1 to 41.7 in FORE-SIGHT). Segmental artery sequential occlusion (Clipping and Coiling Groups) Comparison of cnNIRS values during sequential open clipping of SAs demonstrated a statistically significant difference only at the lower thoracic level (F (7, 84) = 4.565, p < 0.001) (Fig. 3). INVOS and FORE-SIGHT showed a decrease in cnNIRS values to 72% and 85% from baseline after mid-thoracic SA occlusion, respectively (mean difference 27.7, p = 0.010 in INVOS and 14.9, p = 0.018 in FORE-SIGHT). Multiple mean comparisons between the devices, however, did not reach statistical significance. FORE-SIGHT oxygenation values never crossed the 70% from baseline threshold (84% for FORE- SIGHT vs. 63% for INVOS 60 minutes after completed SA occlusion) (Fig. 3). In the SA coil-embolization group no statistically significant difference between the two monitoring devices was observed at any measurement level (Fig. 3). Since SA occlusion using coil-embolization technique is more time-consuming compared to SA clipping (Supplementary Table 4), an additional comparison of these two occlusion strategies within each device was performed. A significant difference was found in measurements of INVOS at lower thoracic level (Clipping vs. Coiling, p = 0.002). After T4 – T7 SA occlusion, cnNIRS values decreased to 72% from baseline in the Clipping, and only to 97% in the Coiling groups (mean difference 24.3, p = 0.020). This discrepancy was observed also during following time-points (60 minutes after total SA occlusion: 63% in the Clipping and 82% in the Coiling groups). The patterns of cnNIRS values' changes were quite similar at the lower, and both lumbar levels in both devices: clipping values were lower than those during SA coiling (Supplementary Fig. 1). Statistical analysis Measurements were analyzed using GraphPad Prism Version 9.00 for Macintosh (GraphPad Software, La Jolla California, USA). Distribution of continuous variables was evaluated using Q-Q plots and the Kolmogorov-Smirnov test. Continuous variables are expressed as mean ± standard deviation. Normally distributed continuous variables with unequal variance were compared using the Welch’s t-test, while not normally distributed data was analyzed by means of the Mann-Whitney U- test. Paired dependent continuous variables were compared using the paired t-test or Wilcoxon matched-groups signed-rank test, depending on the distribution of differences. Within- and between-group changes of cnNIRS values were analyzed using mixed-design split-plot ANOVA. The Greenhouse–Geisser method was used to correct the violation of the sphericity assumption. When required, correction for multiple comparison was performed using the Bonferroni and Šidák methods. Page 4/16 Page 4/16 Bland-Altman plots were used for device agreement evaluation. Statistical significance was assumed at a p-value ≤ 0.05 for 2-tailed testing, unless otherwise specified. Device comparison The comparison of baseline measurements (absolute values) between both devices (n = 25) was significantly different at mid thoracic (p = 0.004), upper lumbar (p < 0.001) and lower lumbar levels (p = 0.012). An overview of absolute values in all the three experimental groups is presented in Supplementary Tables 1–3. the three experimental groups is presented in Supplementary Tables 1–3. Aortic Cross-Clamp Group In the Aortic Cross-Clamp experiments, a statistically significant difference in between the devices was seen, again, at lower thoracic level (F (12, 192) = 3.253, p < 0.001), however without any statistically significant differences between the devices in multiple mean comparisons observed (Fig. 4). At this level, INVOS seemed to have a stronger response to ischemia (with decrease of oxygenation values after 2 minutes to 64% compared to 72 % by FORE-SIGHT). INVOS also had a faster response to reperfusion with return to baseline immediately after clamp off (mean difference − 41.4, p < 0.001), compared to the increase to 84% during the same time period in FORE-SIGHT (mean difference − 11.7, p = 0.22). No difference between Page 5/16 Page 5/16 devices was detected at other levels. The comparison of dynamic changes of cnNIRS (Table 1) did not reveal any statistically significant differences between the devices. Bland-Altman plots showed agreement between the device-systems in Desaturation slopes at both lumbar levels, however with wide Levels of Agreement (mean bias at upper lumbar level 2.6 with Levels of Agreement from − 14.3 to 19.6; mean bias at lower level − 0.54 with Levels of Agreement from − 29.3 to 28.2). Similar results were observed at lumbar levels for minimal cnNIRS values (mean bias at upper lumbar level 1.5 with Levels of Agreement from − 30.5 to 33.5; mean bias at lower lumbar level 2.4 with Levels of Agreement from − 31.5 to 36.3). Bland- Altman plots for agreement of the two devices in Desaturation and Resaturation slopes, Minimum, Maximum cnNIRS values are presented in Supplementary Figs. 2 and 3. Table 1 Performance parameters of two devices during aortic Cross-Clamp experiments. Aortic Cross-Clamp Group Mid thoracic level Lower thoracic level Upper lumbar level Lower lumbar level FORE- SIGHT INVOS FORE- SIGHT INVOS FORE- SIGHT INVOS FORE- SIGHT INVOS Baseline (absolute values) 56.6 ± 6.0 50.6 ± 6.4 57.9 ± 5.6 55.8 ± 7.1 64.4 ± 7.9 60.9 ± 6.5 62.8 ± 10.8 60.9 ± 8.4 p = 0.0595 p = 0.3281 p = 0.0634 p = 0.6353 “Baseline to 2 min occlusion” Desaturation (%) 10.3 ± 18.7 18.7 ± 15.6 28.4 ± 6.9 36.1 ± 10.1 39.3 ± 12.2 38.4 ± 8.1 40.7 ± 8.7 37.3 ± 12.9 p = 0.0279 p = 0.0992 p = 0.8631 p = 0.5433 “Baseline to 10 min occlusion” Desaturation (%) 7.1 ± 16.0 18.9 ± 16.1 28.2 ± 6.4 42.8 ± 12.4 41.5 ± 13.2 43.3 ± 11.3 39.9 ± 12.2 38.6 ± 12.5 p = 0.0354 p = 0.0058 * p = 0.6707 p = 0.8205 Desaturation slope, 1st min (%/min) 7.5 ± 13.8 15.7 ± 15.1 25.2 ± 5.4 32.9 ± 9.5 32.7 ± 7.9 35.3 ± 8.3 36.9 ± 9.5 36.3 ± 14.8 p = 0.0428 p = 0.0248 p = 0.3888 p = 0.9147 Minimum (%) 84.8 ± 17.9 76.2 ± 17.3 67.7 ± 7.7 56.4 ± 12.5 53.9 ± 13.2 55.4 ± 10.3 54.1 ± 12.1 56.5 ± 11.6 p = 0.0341 p = 0.0577 p = 0.7883 p = 0.6523 Resaturation slope, 1st min (%/min) 3.7 ± 14.1 14.1 ± 25.6 10.9 ± 19.9 40.5 ± 21.7 32.3 ± 17.0 39.3 ± 19.5 26.2 ± 13.9 37.8 ± 22.0 p = 0.0901 p = 0.0023 * p = 0.1085 p = 0.0742 Maximum (%) 107.1 ± 11.9 110.1 ± 1 9.1 100.0 ± 14.0 106.0 ± 17.8 98.8 ± 14.2 105.0 ± 9.9 94.6 ± 12.9 105.5 ± 19.9 p = 0.5979 p = 0.1260 p = 0.0174 p = 0.0742 Values are expressed as mean ± standard deviation (in absolute values or % of baseline). Paired t-test or Wilcoxon matched-pairs signed-rank test were used in order to compare the devices. – statistically significant difference after Bonferroni correction for multiple comparisons (statistical significance set at p-value < Table 1 Performance parameters of two devices during aortic Cross-Clam Table 1 (%) Paired t-test or Wilcoxon matched-pairs signed-rank test were used in order to compare the devices. Discussion Before cnNIRS can emerge as a widely used monitoring method for procedures involving the thoracoabdominal aorta, it is crucial to recognize differences and similarities between these devices in measuring regional oxygenation during aortic procedures. The aim of this series was to compare performance of two currently available NIRS systems in three different experimental settings mimicking three clinical situations. The group of open SA clipping aimed to simulate an extensive, permanent loss of direct blood flow to the spinal cord. Using cnNIRS in this clinical setting could provide guidance in decision to sacrifice or reimplant certain SAs. The second group reproducing endovascular coil-embolization of the SAs, was mimicking the clinical setting of TEVAR (decision on extent of coverage) or MIS2ACE priming technique (estimating the number of SAs to be coiled). The aortic cross-clamping experiments recreating an acute insult during surgery, would provide clinicians with guidance regarding safe ischemia duration and, if required, adjustment of temperature and/or distal perfusion settings The aim of this series was to compare performance of two currently available NIRS systems in three different experimental settings mimicking three clinical situations. The group of open SA clipping aimed to simulate an extensive, permanent loss of direct blood flow to the spinal cord. Using cnNIRS in this clinical setting could provide guidance in decision to sacrifice or reimplant certain SAs. The second group reproducing endovascular coil-embolization of the SAs, was mimicking the clinical setting of TEVAR (decision on extent of coverage) or MIS2ACE priming technique (estimating the number of SAs to be coiled). The aortic cross-clamping experiments recreating an acute insult during surgery, would provide clinicians with guidance regarding safe ischemia duration and, if required, adjustment of temperature and/or distal perfusion settings during open surgical repair. Both of the devices, like most of the NIRS systems available on the market, use wavelengths between 670 and 880 nm. This ensures, that the absorption spectra of deoxygenated and oxygenated hemoglobin can be analyzed correctly and separately from the water absorption spectrum (970 nm) [15]. The INVOS 5100C device generates light signals at two wavelengths (730 and 810 nm), while FORE-SIGHT Elite features five wavelengths (680, 730, 770, 805, and 870 nm) [16]. Another important variation is the distance between light source and light detectors (one – located near the LED source and the other one – located far from it) [17]. Discussion Nervous tissue is known to be extremely susceptible to ischemic injury and therefore requires a scrutinous continuous monitoring in a variety of clinical settings. This is especially important for high-risk situations such as the surgical treatment of aortic pathologies in general, but in particular during and after extensive thoracoabdominal aortic repair. Regardless of advanced techniques and rapidly improving perioperative standards, the rate of spinal cord injury remains unacceptably high. Numerous intra- and perioperative adjuncts and novel strategies have been suggested in order to decrease the risk of paraplegia, occurring both after open and endovascular interventions. However, even the best aortic centers have not completely eradicated this dramatic complication. Thus, an accurate and reliable intra- and postoperative monitoring method is needed in order to improve postoperative outcomes. Nervous tissue is known to be extremely susceptible to ischemic injury and ther monitoring in a variety of clinical settings. This is especially important for high- treatment of aortic pathologies in general, but in particular during and after exte Regardless of advanced techniques and rapidly improving perioperative standa unacceptably high. Numerous intra- and perioperative adjuncts and novel strate decrease the risk of paraplegia, occurring both after open and endovascular inte centers have not completely eradicated this dramatic complication. Thus, an ac monitoring method is needed in order to improve postoperative outcomes. Near-infrared spectroscopy is a promising non-invasive monitoring modality, that has been successfully implemented in a number of clinical situations, including evaluation of cerebral perfusion during aortic arch surgery [12–14]. Based on the collateral network concept developed in the last decade, cnNIRS has been introduced and increasingly recognized as a non- invasive quick-response method for evaluation of paraspinal tissue oxygen saturation. Despite of currently lacking data regarding cost-effectiveness of cnNIRS use in thoracoabdominal repair, both the reduction in quality of life due to paraplegia and costs of treatment and rehabilitation after this complication are extreme. Also, in comparison to other clinically available monitoring methods, such as somatosensory evoked or motor evoked potentials, cnNIRS does not require a neurophysiologist for its analysis. Moreover, the preparation of the cnNIRS setup lasts no longer than several minutes, the height of placement of the optodes can be standardized and, most importantly, the monitoring can be performed also in postoperative period. Neither does cnNIRS require any specific anesthetic management in order to avoid interactions with neuroblocking agents. To date few different NIRS devices are available for clinical use. Aortic Cross-Clamp Group – statistically significant difference after Bonferroni correction for multiple comparisons (statistical significance set at p-value < 0.0071). Page 6/16 Discussion However, most of the previous publications, except Hyttel-Sorensen et al., who compared INVOS with the first generation of FORE-SIGHT, did not find any difference between INVOS and FORE-SIGHT baseline measurements of peripheral tissue oxygenation [20, 25]. Nevertheless, none of the previous publications compared the performance of these or any other NIRS devices on the back muscles. Considering the different depth of penetration and variations in bias, one could assume that the two devices may be analyzing different portions (layers) of the paraspinous collateral network. During the Clipping and Coiling experiments, the devices reflected the collateral network oxygenation changes almost equally: INVOS had a more pronounced response only to SA clipping at the lower thoracic level. Interestingly, since SA occlusion using the coil-embolization technique was more time-consuming compared to the open SA occlusion method, it was also associated with a less pronounced decrease of cnNIRS values in both devices (yet statistically significant only in INVOS at the lower thoracic level), meaning that both devices can be interchangeably used when the spinal cord perfusion is decreasing slowly and gradually (for example, during SA coil-embolization). In the Aortic Cross-Clamp group the devices differed significantly in cnNIRS changes at the lower thoracic level, moreover INVOS had lower oxygenation rates during desaturation. This trend has been previously observed by our colleagues: it has been shown that INVOS has a faster and more pronounced reaction to blood flow changes: it has higher desaturation and resaturation rates and lower minimal values compared to FORE-SIGHT [20]. This observation has also been confirmed in cerebral oximetry measurements [23, 24], furthermore INVOS was associated with a more rapid reaction to StO2 changes compared to other NIRS devices (other than FORE-SIGHT) [26, 27]. In experiments with stable isocapnic hypoxia in healthy volunteers, Bickler et al. compared performance of several devices (including INVOS 5100C and FORE-SIGHT) with a reference regional tissue oxygenation calculated based on jugular bulb and arterial blood: FORE-SIGHT demonstrated a better accuracy compared to INVOS (accuracy root mean square 4.26% versus 9.69% respectively) [22]. In a prospective study on patients undergoing cardiac surgery, Kobayashi et al. compared preoperative regional cerebral oxygenation values between FORE-SIGHT Elite and INVOS 5100C and found out that inter-patient variations, caused by differences in hemoglobin concentration, left ventricular ejection fraction or sculp-cortex distance, were greater in INVOS compared to FORE-SIGHT [28]. Discussion This distance determines the depth of photon penetration: the greater it is, the less is the bias caused by superficial tissues [16]. In INVOS 5100C the two photodiode detectors of adult sensors are located in 30 mm and 40 mm distance from the emitter and in FORE-SIGHT Elite – in 15 mm and 50 mm [18–19]. Theoretically, both increased number of wavelengths used and greater difference in emitter-detector distances should provide FORE-SIGHT with better control for bias and more precise performance. This could explain the fact that FORE-SIGHT showed in the most of the experimental measurements higher values with overall less variation. The only exception was the lower lumbar level Page 7/16 Page 7/16 during aortic cross-clamping – the level of definitive spinal cord ischemia. Here the oxygenation values depicted by FORE- SIGHT were lower than those of INVOS. One should also consider different shapes of light emitters and, theoretically, sensitivity of the light detectors. The last, but not the least factor seriously affecting the measurements, are the algorithms used to derive the oxygen saturation values. These are based on various assumptions used for calculation, different calibration techniques and corrections for anatomical regions where measurements are performed [20–22]. Unfortunately, these algorithms are company classified, leaving the question about interchangeability of devices unanswered. Numerous attempts have been made during the last decades to evaluate the factors influencing NIRS measurements and the variability between devices. However, most of the currently available publications are focused on cerebral oxygenation monitoring, a highly specific anatomical area where great variations in ratio of arterial to venous blood and the well-known extracranial interference problem exist [22–24]. Fewer authors evaluated differences between monitoring systems on peripheral tissue during vascular occlusion tests [17, 18, 20]. Nevertheless, comparison of results of each new study with those provided in the literature is limited due to dissimilarity of experimental settings (animal experiments, tests on patients, adult or pediatric, healthy or during surgical treatment), devices (and subsequent device generations) used and anatomical areas of measurement (brain, upper extremity, lower extremity, back muscles). In the present study, we aimed to compare the performance of two currently available NIRS devices in three experimental settings, simulating possible clinical situations in patients undergoing treatment for thoracic or thoracoabdominal aortic pathologies. According to our observations, the baseline measurements in all animals demonstrated that FORE-SIGHT tends to have higher cnNIRS values compared to INVOS. Conflict of interest None declared. The authors thank Urszula D. Simoniuk for the help provided during the experimental studies. The authors thank Urszula D. Simoniuk for the help provided during the experimental studies. Limitations Since the results are based on an experimental large animal model, one should consider the anatomical (vascular anatomy, skin thickness, etc.) and physiological (hemodynamics) differences between the two species (swine versus human) [2, 3]. Further experiments are required to evaluate the correlation of cnNIRS (and each of cnNIRS devices) with other currently available monitoring techniques (e.g., somatosensory and motor evoked potentials). Discussion In our series, the Aortic Cross-Clamping group was also evaluated using slightly modified principles of Page 8/16 Page 8/16 vascular occlusion tests [17]. The desaturation and resaturation slopes were analyzed only for the first minute since only then the cnNIRS changes were rapid and linear. The inter-device variations in these parameters seemed to be more prominent at thoracic levels (reaching statistical significance in Resaturation slope and “Baseline to 10 min” Desaturation), whereas at both lumbar levels the parameters were similar. Additionally, the agreement between devices in dynamic parameters was evaluated using Bland-Altman plots. The Bland-Altman plots comparing desaturation parameters also showed a better agreement in devices at lumbar levels compared to thoracic measurements, however with quite wide Levels of Agreement, suggesting that although both devices are effective in reflecting decrease of perfusion in the collateral network, the interpretation of their performance (for example, critical cnNIRS ischemia levels) in clinical practice should be carried out separately. It is important to mention, that all of the observed statistically significant differences in performance of devices (cnNIRS changes during the experiments and dynamic parameters during aortic cross-clamping) were observed at lower thoracic level. The possible explanation for this phenomenon could be the greater inter-subject variations in perfusion of collateral network in this area (due to variations in arterial pressure, different collateralization etc.), compared to mid-thoracic (satisfactory perfusion due to upper body collaterals) and both lumbar (notably decreased perfusion) levels. This perfusion variations, together with the differences in technical characteristics (depth of penetration) and algorithms, could lead to significant discrepancies in cnNIRS measurements at this level. Conclusions Both INVOS 5100C and FORE-SIGHT Elite provide adequate cnNIRS monitoring with similar performance at mid-thoracic, upper and lower lumbar levels. The sequential SA occlusion methods (clipping or coil-embolization) and aortic cross- clamping may be well reflected by both NIRS devices. INVOS, however, seems to have a more rapid and stronger response at the lower thoracic level. Moreover, during aortic cross-clamping, acceptable clinical agreement during ischemia in performance between devices was observed only at lumbar levels. These findings yield important inter-device variations that should be acknowledged in clinical practice, for example setting the ischemic threshold (critical reduction point) for each of the devices separately. Author contributions Conceptualization: K. von Aspern, J. Haunschild, Z. Dietze, C. D. Etz; Methodology: Z. Dietze, K. von Aspern; Formal analysis and investigation: Z. Dietze, K. von Aspern, J. Haunschild; Writing - original draft preparation: Z. Dietze; Writing - review and Page 9/16 editing: K. von Aspern, J. Haunschild, C. D. Etz; Funding acquisition: K. von Aspern, J. Haunschild, C. D. Etz; Resources: C. D. Etz , S. Ossmann, M. A. Borger; Supervision: C. D. Etz. editing: K. von Aspern, J. Haunschild, C. D. Etz; Funding acquisition: K. von Aspern, J. Haunschild, C. D. Etz; Resources: C. D. Etz , S. Ossmann, M. A. Borger; Supervision: C. D. Etz. editing: K. von Aspern, J. Haunschild, C. D. Etz; Funding acquisition: K. von Aspern, J. Haunschild, C. D. Etz; Resources: C. D. Etz , S. Ossmann, M. A. Borger; Supervision: C. D. Etz. References 1. Etz CD, Weigang E, Hartert M, Lonn L, Mestres CA, Di Bartolomeo R, et al. Contemporary spinal cord protection during thoracic and thoracoabdominal aortic surgery and endovascular aortic repair: a position paper of the vascular domain of the European Association for Cardio-Thoracic Surgery. Eur J Cardiothorac Surg. 2015;47:943–57. 2. von Aspern K, Haunschild J, Hoyer A, Luehr M, Bakhtiary F, Misfeld, et al. Non-invasive spinal cord oxygenation monitoring: validating collateral network near-infrared spectroscopy for thoracoabdominal aortic aneurysm repair. Eur J Cardiothorac Surg. 2016;50:675–83. 2. von Aspern K, Haunschild J, Hoyer A, Luehr M, Bakhtiary F, Misfeld, et al. Non-invasive spinal cord oxygenation monitoring: validating collateral network near-infrared spectroscopy for thoracoabdominal aortic aneurysm repair. Eur J Cardiothorac Surg. 2016;50:675–83. 3. von Aspern K, Haunschild J, Ziemann M, Misfeld M, Mohr FW, Borger MA, Etz CD. Evaluation of collateral network near- infrared spectroscopy during and after segmental artery occlusion in a chronic large animal model. J Thorac Cardiovasc Surg. 2019 Jul;158(1):155–64.e5. 3. von Aspern K, Haunschild J, Ziemann M, Misfeld M, Mohr FW, Borger MA, Etz CD. Evaluation of collateral network near- infrared spectroscopy during and after segmental artery occlusion in a chronic large animal model. J Thorac Cardiovasc Surg. 2019 Jul;158(1):155–64.e5. 4. Etz CD, Kari FA, Mueller CS, Silovitz D, Brenner RM, Lin HM, et al. The collateral network concept: a reassessment of the anatomy of spinal cord perfusion. J Thorac Cardiovasc Surg. 2011;141(4):1020–8. 4. 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Washington (DC): National Academies Press (US); 2011 11. Petroff D, Czerny M, Kölbel T, et al. Paraplegia prevention in aortic aneurysm repair by thoracoabdominal staging with ‘minimally invasive staged segmental artery coil embolisation’ (MIS²ACE): trial protocol for a randomised controlled multicentre trial. BMJ Open. 2019;9(3):e025488. 12. De Paulis R, Czerny M, Weltert L, Bavaria J, Borger M, Carrel TP, et al. Current trends in cannulation and neuroprotection during surgery of the aortic arch in Europe. Eur J Cardiothorac Surg. May 2015;47(5):917–23. 13. Urbanski PP, Luehr M, Di Bartolomeo R, Diegeler A, De Paulis R, Esposito G, et al. Multicentre analysis of current strategies and outcomes in open aortic arch surgery: heterogeneity is still an issue. Eur J Cardiothorac Surg. 2016 Aug;50(2):249–55. 14. Czerny M, Schmidli J, Adler S, van den Berg JC, Bertoglio L, Carrel T, et al. Current options and recommendations for the treatment of thoracic aortic pathologies involving the aortic arch: an expert consensus document of the European Association for Cardio-Thoracic surgery (EACTS) and the European Society for Vascular Surgery (ESVS), European Journal of Cardio-Thoracic Surgery, Volume 55, Issue 1, January 2019, Pages 133–162. 15. Wilson RH, Nadeau KP, Jaworski FB, Tromberg BJ, Durkin AJ. Review of short-wave infrared spectroscopy and imaging methods for biological tissue characterization. J Biomed Opt. 2015;20(3):030901. 15. Wilson RH, Nadeau KP, Jaworski FB, Tromberg BJ, Durkin AJ. Review of short-wave infrared spectroscopy and imaging methods for biological tissue characterization. J Biomed Opt. 2015;20(3):030901. Page 10/16 Page 10/16 16. References Impact of extracranial contamination on regional cerebral oxygen saturation: a comparison of three cerebral oximetry technologies. Anesthesiology. 2012 Apr;116(4):834–40. 23. Davie SN, Grocott HP. Impact of extracranial contamination on regional cerebral oxygen saturation: a comparison of three cerebral oximetry technologies. Anesthesiology. 2012 Apr;116(4):834–40. 24. Naguib AN, Winch PD, Sebastian R, Gomez D, Guzman L, Rice J, et al. The Correlation of Two Cerebral Saturation Monitors With Jugular Bulb Oxygen Saturation in Children Undergoing Cardiopulmonary Bypass for Congenital Heart Surgery. J Intensive Care Med. 2017;32(10):603–8. 24. Naguib AN, Winch PD, Sebastian R, Gomez D, Guzman L, Rice J, et al. The Correlation of Two Cerebral Saturation Monitors With Jugular Bulb Oxygen Saturation in Children Undergoing Cardiopulmonary Bypass for Congenital Heart Surgery. J Intensive Care Med. 2017;32(10):603–8. 25. Hyttel-Sorensen S, Hessel TW, Greisen G. Peripheral tissue oximetry: comparing three commercial near-infrared spectroscopy oximeters on the forearm. J Clin Monit Comput. 2014;28(2):149–55. 25. Hyttel-Sorensen S, Hessel TW, Greisen G. Peripheral tissue oximetry: comparing three commercial near-infrared spectroscopy oximeters on the forearm. J Clin Monit Comput. 2014;28(2):149–55. 26. Fellahi JL, Butin G, Fischer MO, Zamparini G, Ge´rard JL, Hanouz JL. Dynamic evaluation of near-infrared peripheral oximetry in healthy volunteers: a comparison between INVOS and EQUANOX. J Crit Care. 2013;28:881.e1–6. 26. Fellahi JL, Butin G, Fischer MO, Zamparini G, Ge´rard JL, Hanouz JL. Dynamic evaluation of near-infrared peripheral oximetry in healthy volunteers: a comparison between INVOS and EQUANOX. J Crit Care. 2013;28:881.e1–6. 27. Lee JH, Park YH, Kim HS, Kim JT. Comparison of two devices using near-infrared spectroscopy for the measurement of tissue oxygenation during a vascular occlusion test in healthy volunteers (INVOS_ vs. InSpectraTM). J Clin Monit Comput. 2015;29:271–8. 27. Lee JH, Park YH, Kim HS, Kim JT. Comparison of two devices using near-infrared spectroscopy for the measurement of tissue oxygenation during a vascular occlusion test in healthy volunteers (INVOS_ vs. InSpectraTM). J Clin Monit Comput. 2015;29:271–8. 28. Kobayashi K, Kitamura T, Kohira S, Torii S, Mishima T, Ohkubo H, et al. Cerebral oximetry for cardiac surgery: a preoperative comparison of device characteristics and pitfalls in interpretation. J Artif Organs. 2018 Dec;21(4)412 – 18 28. Kobayashi K, Kitamura T, Kohira S, Torii S, Mishima T, Ohkubo H, et al. Cerebral oximetry for cardiac surgery: a preoperative comparison of device characteristics and pitfalls in interpretation. J Artif Organs. 2018 Dec;21(4)412 – 18. Figures Page 11/16 Figure 1 Experimental setting. References A – lateral view of the animal with surgical incision performed groups, lower left thoracotomy and left retroperitoneal access in the SA Clipping gro location of NIRS optodes placed at four levels. INVS - INVOS; FST – FORE-SIGHT. Figure 1 Experimental setting. A – lateral view of the animal with surgical incision performed in groups: upper left thoracotom groups, lower left thoracotomy and left retroperitoneal access in the SA Clipping group. B – dorsal view of the anima location of NIRS optodes placed at four levels. INVS - INVOS; FST – FORE-SIGHT. Figure 1 Figure 1 Experimental setting. A – lateral view of the animal with surgical incision performed in groups: upper left thoracotomy in all groups, lower left thoracotomy and left retroperitoneal access in the SA Clipping group. B – dorsal view of the animal with location of NIRS optodes placed at four levels. INVS - INVOS; FST – FORE-SIGHT. Experimental setting. A – lateral view of the animal with surgical incision performed in groups: upper left thoracotomy in all groups, lower left thoracotomy and left retroperitoneal access in the SA Clipping group. B – dorsal view of the animal with location of NIRS optodes placed at four levels. INVS - INVOS; FST – FORE-SIGHT. Page 12/16 Page 12/16 Figure 2 Figure 4 Comparison of paraspinal muscle oxygenation measurements (percent of baseline) performed by two devices in the Aortic Cross-Clamp groups. CN – collateral network. X-clamp – cnNIRS immediately after aortic cross-clamping. Brackets show statistically significant within-device changes during the experiment (blue in FORE-SIGHT and green in INVOS). Figure 2 Illustration of experimental concept in SA Clipping group, SA Coiling group and Aortic Cross-Clamp group. SA – segmental arteries. Page 13/16 Page 13/16 Page 13/16 Page 13/16 Figure 3 Comparison of paraspinal muscle oxygenation measurements (percent of b Clipping and the SA Coiling. CN – collateral network. Brackets show statistic the experiment (blue in FORE-SIGHT and green in INVOS). Fi 3 Figure 3 Comparison of paraspinal muscle oxygenation measurements (percent of baseline) performed by two devices in the SA Clipping and the SA Coiling. CN – collateral network. Brackets show statistically significant within-device changes during the experiment (blue in FORE-SIGHT and green in INVOS). Figure 3 Comparison of paraspinal muscle oxygenation measurements (percent of baseline) performed by two devices in the SA Clipping and the SA Coiling. CN – collateral network. Brackets show statistically significant within-device changes during the experiment (blue in FORE-SIGHT and green in INVOS). Comparison of paraspinal muscle oxygenation measurements (percent of baseline) performed by two devices in the SA Clipping and the SA Coiling. CN – collateral network. Brackets show statistically significant within-device changes during the experiment (blue in FORE-SIGHT and green in INVOS). Page 14/16 Figure 4 Comparison of paraspinal muscle oxygenation measurements (percent of baseline) performed by two devices in the Aort Cross-Clamp groups. CN – collateral network. X-clamp – cnNIRS immediately after aortic cross-clamping. Brackets show statistically significant within-device changes during the experiment (blue in FORE-SIGHT and green in INVOS). Supplementary Files Figure 4 Comparison of paraspinal muscle oxygenation measurements (percent of baseline) performed by two devices in the Aortic Cross-Clamp groups. CN – collateral network. X-clamp – cnNIRS immediately after aortic cross-clamping. Brackets show statistically significant within-device changes during the experiment (blue in FORE-SIGHT and green in INVOS). SupplTable1.docx SupplTable2.docx SupplTable3.docx SupplTable4.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SuppFig1.tiff SuppFig2.tiff SuppFig3.tiff Page 15/16 Page 15/16 SupplTable1.docx SupplTable2.docx SupplTable3.docx SupplTable4.docx Page 16/16
https://openalex.org/W4385890947	https://zenodo.org/record/8253359/files/INC_081623.pdf	English	null	Goldbach's Conjecture — Towards the Inconsistency of Arithmetic	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,229	Goldbach's Conjecture — Towards the Inconsistency of Arithmetic Ralf Wüsthofen Abstract. This paper proves an inconsistency in Peano arithmetic (PA). We express a strengthened form of the strong Goldbach conjecture using a specific set. This set varies according to whether the conjecture or its negation is assumed. We show that, on the other hand, the set remains unchanged under these two assumptions. This causes a contradiction. Notations. Let denote the natural numbers starting from 1, let n denote the natural numbers starting from n > 1 and let 3 denote the prime numbers starting from 3. Strengthened strong Goldbach conjecture (SSGB): Every even number greater than 6 is the sum of two different odd primes. Theorem. PA is contradictory, i.e. the statement FALSE can be derived. Theorem. PA is contradictory, i.e. the statement FALSE can be derived. Proof. We define the set Sg := { (pk, mk, qk) \| k, m ; p, q 3, p < q; m = (p + q) / 2 }. SSGB is equivalent to saying that every integer n ≥ 4 is the arithmetic mean of two different odd primes and so it is equivalent to saying that all integers n ≥ 4 appear as m in a middle component mk of Sg. So, by the definition of Sg we have SSGB <=> n 4 Ǝ (pk, mk, qk) Sg n = m SSGB <=> Ǝ n 4 (pk, mk, qk) Sg n ≠ m. SSGB <=> n 4 Ǝ (pk, mk, qk) Sg n = m SSGB <=> Ǝ n 4 (pk, mk, qk) Sg n ≠ m. The set Sg has the following two properties. The set Sg has the following two properties. First, the whole range of 3 can be expressed by the triple components of Sg (”covering”), because every integer x ≥ 3 can be written as some pk with k = 1 when x is prime, as some pk with k ≠ 1 when x is composite and not a power of 2, or as (3 + 5)k / 2 when x is a power of 2; p 3, k . So we have (C) x 3 Ǝ (pk, mk, qk) Sg x = pk x = mk. (C) x 3 Ǝ (pk, mk, qk) Sg x = pk x = mk. 1 A few examples of the covering: A few examples of the covering: x = 19: (19∙1, 21∙1, 23∙1), (19∙1, 60∙1, 101∙1) x = 27: (3∙9, 7∙9, 11∙9) x = 42: (3∙14, 5∙14, 7∙14), (7∙6, 9∙6, 11∙6) x = 4096: (3∙1024, 4∙1024, 5∙1024) x = 10000: (5∙2000, 6∙2000, 7∙2000). Second, all pairs (p, q) of distinct odd primes are used in the definition of the set Sg (“maximality”). So we have M) p, q 3, p < q k (pk, mk, qk) Sg, where m = (p + q) / 2. (C) would immediately imply SSGB since an n ≥ 4 that is different from all Sg triple components pk and mk is in particular different from all m in Sg. So the property (C) excludes this possibility. Theorem. PA is contradictory, i.e. the statement FALSE can be derived. The property (M) excludes the possibility that if there is an n ≥ 4 different from all m in Sg, then n is the arithmetic mean of a pair of distinct odd primes not used in Sg. So (M) rules out the possibility that the question of whether SSGB holds or not depends on whether (M) holds or not. (The proof would no longer be possible if we left out any pair of distinct odd primes in the formulation of SSGB and Sg.) Therefore, in both cases SSGB and SSGB, neither (C) nor (M) applies. Therefore, in both cases SSGB and SSGB, neither (C) nor (M) applies. The basic idea is now the following. The basic idea is now the following. There are two possibilities for Sg, exactly one of which must occur: Either there is an n 4 in addition to all the numbers m defined in Sg or there is not. The latter is equivalent to SSGB and the former is equivalent to SSGB. Since, due to (M), an n 4 different from all m cannot be the arithmetic mean of a pair of primes not used in Sg and since, due to (C), this n equals a component of some Sg triple that exists by definition, the covering of 3 by the Sg triples in the case n exists ( SSGB) is equal to that in the case n does not exist (SSGB). This causes a contradiction because in the case SSGB the numbers m defined in Sg take all integer values x ≥ 4 whereas in the case SSGB they don’t. 2 The following steps are independent of the choice of n if, in the case of SSGB, there is more than one that is different from all m. For example, the minimal such n works. We split Sg into two complementary subsets in the following way. For any y Sg = Sg+(y) ∪ Sg-(y), with Sg+(y) := { (pk, mk, qk) Sg \| Ǝ k' pk = yk' mk = yk' qk = yk' } Sg-(y) := { (pk, mk, qk) Sg \| k' pk ≠ yk' mk ≠ yk' qk ≠ yk' }. Sg+(y) := { (pk, mk, qk) Sg \| Ǝ k' pk = yk' mk = yk' qk = yk' } Sg-(y) := { (pk, mk, qk) Sg \| k' pk ≠ yk' mk ≠ yk' qk ≠ yk' }. We define We define S1 := { (pk, mk, qk) Sg \| SSGB } S2 := { (pk, mk, qk) Sg \| SSGB }. Under the assumption SSGB there is an n 4 as described above and under the assumption SSGB there is no such n. Then, ( ( y 3 SSGB => S1 = Sg+(y) ∪ Sg-(y) ) ( SSGB => S2 = Sg+(n) ∪ Sg-(n) ) ) (1) (1) (1) ( (C) (M) ). Since (C) and (M) are both ruled out and since Sg+(n) ∪ Sg-(n) is independent of n, we get Since (C) and (M) are both ruled out and since Sg+(n) ∪ Sg-(n) is independent of n, we get (1.1) y 3 SSGB => S1 = Sg+(y) ∪ Sg-(y) (1.2) y 3 SSGB => S2 = Sg+(y) ∪ Sg-(y). (1.1) y 3 SSGB => S1 = Sg+(y) ∪ Sg-(y) (1.2) y 3 SSGB => S2 = Sg+(y) ∪ Sg-(y). (1.1) y 3 SSGB => S1 = Sg+(y) ∪ Sg-(y) (1.2) y 3 SSGB => S2 = Sg+(y) ∪ Sg-(y). 3 3 Now, we will make use of the following principle. If two sets of (possibly infinitely many) x-tuples are equal, then the sets of their corresponding i-th components are equal; 1 ≤ i ≤ x. To this end, for each k we define M1(k) := { mk \| (pk, mk, qk) S1 } M2(k) := { mk \| (pk, mk, qk) S2 }. To this end, for each k we define M1(k) := { mk \| (pk, mk, qk) S1 } M2(k) := { mk \| (pk, mk, qk) S2 }. To this end, for each k we define M1(k) := { mk \| (pk, mk, qk) S1 } M2(k) := { mk \| (pk, mk, qk) S2 }. Then, applying the principle above to the middle component of the triples (pk, mk, qk), ( (1.1) (1.2) ) implies (2.1) k y 3 SSGB => M1(k) = { mk \| (pk, mk, qk) Sg+(y) ∪ Sg-(y) } (2.2) k y 3 SSGB => M2(k) = { mk \| (pk, mk, qk) Sg+(y) ∪ Sg-(y) }. (2.1) k y 3 SSGB => M1(k) = { mk \| (pk, mk, qk) Sg+(y) ∪ Sg-(y) } (2.2) k y 3 SSGB => M2(k) = { mk \| (pk, mk, qk) Sg+(y) ∪ Sg-(y) }. We define k y 3 SSGB => M1(k) = { mk \| (pk, mk, qk) Sg+(y) ∪ Sg-(y) } Setting M1 := M1(1) and M2 := M2(1), we get Setting M1 := M1(1) and M2 := M2(1), we get (2.1') y 3 SSGB => M1 = { m \| (p, m, q) Sg+(y) ∪ Sg-(y) } (2.2') y 3 SSGB => M2 = { m \| (p, m, q) Sg+(y) ∪ Sg-(y) }. (2.2') y 3 SSGB => M2 = { m \| (p, m, q) Sg+(y) ∪ Sg-(y) }. Since for every y 3 Sg+(y) ∪ Sg-(y) equals Sg by definition, for every y 3 { m \| (p, m, q) Sg+(y) ∪ Sg-(y) } equals the set M := { m \| (p, m, q) Sg }. So, from ( (2.1') (2.2') ) we obtain (3) ( SSGB => M1 = M ) ( SSGB => M2 = M ). (3) ( SSGB => M1 = M ) ( SSGB => M2 = M ). (3) 4 4 M is a bound variable that is equal to either 4 if SSGB is true or some non-empty proper subset Y of 4 if SSGB is false. Now, we make use of the following rule. Now, we make use of the following rule. Let P = P(A) be a proposition that depends on a set A. Then, for any set B, ( we have a proof of P(A) we have a proof of A = B ) => we have a proof of P(B). In the special case that A is a variable that is replaced by the value B, this reduces to we have a proof of P(A) => we have a proof of P(B). Since we have a proof of (3) and since M is a variable in (3), we can apply the above rule with P = (3). If M = 4 we use the rule with A = M and B = 4, and if M = Y we use it with A = M and B = Y. Then, since either M = 4 or M = Y, from (3) we obtain (3.1) we have a proof of ( SSGB => M1 = 4 SSGB => M2 = 4 ) (3.2) we have a proof of ( SSGB => M1 = Y ≠ 4 SSGB => M2 = Y ≠ 4 ). Now, we will establish a contradiction to ( (3.1') (3.2') ). Now, we will establish a contradiction to ( (3.1') (3.2') ). Under the assumption SSGB the set M = { m \| (p, m, q) Sg } is equal to 4 and under SSGB it is equal to Y ≠ 4. Therefore, (4.1) we have a proof of ( SSGB => M1 = 4 ) (4.2) we have a proof of ( SSGB => M2 = Y ≠ 4 ). Then, ( (3.1') (3.2') ) together with ( (4.1) (4.2) ) implies (5.1) we have a proof of ( SSGB => M2 = 4 ) Because of ( (4.1) (4.2) ) and because We define we have a proof of ( SSGB => M1 = 4 SSGB => M2 = 4 ) (3.1) we have a proof of ( SSGB => M1 = 4 SSGB => M2 = 4 ) (3.1) we have a proof of ( SSGB => M1 = 4 SSGB => M2 = 4 ) we have a proof of ( SSGB => M1 = Y ≠ 4 SSGB => M2 = Y ≠ 4 (3.2) we have a proof of ( SSGB => M1 = Y ≠ 4 SSGB => M2 = Y ≠ 4 ). (3.2) we have a proof of ( SSGB => M1 = Y ≠ 4 SSGB => M2 = Y ≠ 4 ). (3.2) we have a proof of ( SSGB => M1 = Y ≠ 4 SSGB => M2 = Y ≠ 4 ). This implies (3.1') ( we have a proof of ( SSGB => M1 = 4 ) we have a proof of ( SSGB => M2 = 4 ) ) (3.1') ( we have a proof of ( SSGB => M1 = 4 ) we have a proof of ( SSGB => M2 = 4 ) ) (3.2') ( we have a proof of ( SSGB => M1 = Y ≠ 4 ) we have a proof of ( SSGB => M2 = Y ≠ 4 ) ). (3.2') ( we have a proof of ( SSGB => M1 = Y ≠ 4 ) we have a proof of ( SSGB => M2 = Y ≠ 4 ) ). 5 5 Because of ( (4.1) (4.2) ) and because SSGB => M2 = { } ≠ 4 SSGB => M1 = { } ≠ Y, we have a proof that (M2 = 4) is false and we have a proof that (M1 = Y ≠ 4) is false. we have a proof that (M2 = 4) is false and we have a proof that (M1 = Y ≠ 4) is false. So, ( (5.1) (5.2) ) yields 6 6 (6.1) we have a proof of SSGB (6.2) we have a proof of SSGB. Since we have neither a proof of SSGB nor of SSGB, both (6.1) and (6.2) are false. Therefore, we obtain (FALSE FALSE) and thus FALSE. □ Remark. The term Sg isn't a standard part of Peano arithmetic, but it can easily be defined within Peano arithmetic. This also applies to all other sets used in the proof. 7 7
https://openalex.org/W3015743795	https://europepmc.org/articles/pmc7149857?pdf=render	English	null	Transfer of motor skill between virtual reality viewed using a head-mounted display and conventional screen environments	Journal of neuroengineering and rehabilitation	2,020	cc-by	10,793	Juliano and Liew Journal of NeuroEngineering and Rehabilitation (2020) 17:48 https://doi.org/10.1186/s12984-020-00678-2 Juliano and Liew Journal of NeuroEngineering and Rehabilitation (2020) 17:48 https://doi.org/10.1186/s12984-020-00678-2 Juliano and Liew Journal of NeuroEngineering and Rehabilitation https://doi.org/10.1186/s12984-020-00678-2 Abstract Background: Virtual reality viewed using a head-mounted display (HMD-VR) has the potential to be a useful tool for motor learning and rehabilitation. However, when developing tools for these purposes, it is important to design applications that will effectively transfer to the real world. Therefore, it is essential to understand whether motor skills transfer between HMD-VR and conventional screen-based environments and what factors predict transfer. Methods: We randomized 70 healthy participants into two groups. Both groups trained on a well-established measure of motor skill acquisition, the Sequential Visual Isometric Pinch Task (SVIPT), either in HMD-VR or in a conventional environment (i.e., computer screen). We then tested whether the motor skills transferred from HMD- VR to the computer screen, and vice versa. After the completion of the experiment, participants responded to questions relating to their presence in their respective training environment, age, gender, video game use, and previous HMD-VR experience. Using multivariate and univariate linear regression, we then examined whether any personal factors from the questionnaires predicted individual differences in motor skill transfer between environments Results: Our results suggest that motor skill acquisition of this task occurs at the same rate in both HMD-VR and conventional screen environments. However, the motor skills acquired in HMD-VR did not transfer to the screen environment. While this decrease in motor skill performance when moving to the screen environment was not significantly predicted by self-reported factors, there were trends for correlations with presence and previous HMD- VR experience. Conversely, motor skills acquired in a conventional screen environment not only transferred but improved in HMD-VR, and this increase in motor skill performance could be predicted by self-reported factors of presence, gender, age and video game use. (Continued on next page) * Correspondence: sliew@usc.edu 2Neural Plasticity and Neurorehabilitation Laboratory, Division of Occupational Science and Occupational Therapy, University of Southern California, Los Angeles, CA, USA 3USC Stevens Neuroimaging and Informatics Institute, Department of Neurology, University of Southern California, Los Angeles, CA, USA Full list of author information is available at the end of the article Transfer of motor skill between virtual reality viewed using a head-mounted display and conventional screen environments Julia M. Juliano1 and Sook-Lei Liew2,3* © 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. 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Juliano and Liew Journal of NeuroEngineering and Rehabilitation (2020) 17:48 Page 2 of 13 (2020) 17:48 Juliano and Liew Journal of NeuroEngineering and Rehabilitation p p g Conclusions: These findings suggest that personal factors may predict who is likely to have better transfer of motor skill to and from HMD-VR. Future work should examine whether these and other predictors (i.e., additional personal factors such as immersive tendencies and task-specific factors such as fidelity or feedback) also apply to motor skill transfer from HMD-VR to more dynamic physical environments. Keywords: Virtual reality, Head-mounted display, Motor skill acquisition, Transfer, Presence, There is also large interindividual variability within the results, and this variability suggests there may be particular tasks or particular individuals that will be more successful in transferring HMD-VR motor skills to the real world. Un- derstanding the task-related or personal factors that medi- ate learning and transfer from HMD-VR environments should be examined in order to understand what makes HMD-VR interventions effective. One advantage of HMD- VR over conventional screen environments is the ability to realistically simulate the real world which allows for greater task specificity [27]. Background The use of virtual reality (VR) in rehabilitation has been growing exponentially over recent years [1, 2]. Clinical applications of VR have been shown to be engaging and motivating [3, 4] with promising results suggesting VR interventions are comparable [5] or in some cases super- ior [6, 7] to conventional rehabilitation. However, while a number of studies have reported benefits of using VR for cognitive and motor rehabilitation, there are also re- ports on the limitations of using these devices for clin- ical applications [8, 9]. In particular, some studies have shown that VR interventions are not effective at improv- ing motor performance in the real world due to a lack of motor skill transfer (i.e., the application of a motor skill in a novel task or environment [10]) [11, 12]. Concerns about motor skill transfer from virtual to real environments are even greater when specifically considering the use of VR viewed using a head-mounted display (HMD-VR). HMD-VR provides a more immer- sive experience compared to conventional environments (e.g., computer screens) and results in increased levels of presence (i.e., the illusion of actually being present in the virtual environment) and embodiment (i.e., the percep- tual ownership of a virtual body in a virtual space) [13, 14] that modulate behavior [15] and impact performance on motor learning and rehabilitation applications (e.g., gait, balance, neurofeedback tasks) [16–18]. Additionally, motor learning in HMD-VR (e.g., upper extremity visuo- motor adaptation) has been shown to rely on different learning processes compared to a conventional screen en- vironment [19]. Given the differences in immersive experi- ences and learning processes between HMD-VR and conventional environments, it can be assumed that indi- viduals may experience these environments as separate contexts. Studies have found the context of the training environment to affect the transfer of motor skills [20], where motor performance may decrease when testing oc- curs in an environment different from training [21]. How- ever, only a small number of studies have specifically explored motor skill transfer of from an HMD-VR train- ing environment to a more conventional environment (e.g., computer screen or real world) [22–26]. Among these studies, there are again conflicting results, with some studies finding successful motor skill transfer from HMD- VR to the real world [22, 23], and others not [24–26]. Task-related factors such as fidelity (i.e., imitation of the real environment) and dimensionality (i.e., matching dimensions between virtual and real environ- ments) between HMD-VR and the real world have been shown to influence lower extremity motor performance [28] and have been suggested to have an influence on trans- fer in both lower and upper extremity motor transfer [29, 30]. Individual differences in personal factors such as gen- der, age, video game experience, prior technical computer literacy, and computer efficacy seemed to influence transfer from HMD-VR to the real world in studies examining the transfer of spatial knowledge acquired in an HMD-VR en- vironment [26, 31]. However, the individual differences on both task-related and personal factors have not been exten- sively examined in HMD-VR motor skill transfer. We begin to address this gap by examining whether individual per- sonal factors facilitate better transfer from upper extremity motor skill acquisition in HMD-VR to a conventional screen environment. Background In the current study, we examined: (1) whether transfer of upper extremity motor skills occurs between HMD-VR and conventional screen environments, and (2) what per- sonal factors predict transfer between environments. Given the variability of motor skill learning and transfer in previous studies [22–26, 29], we hypothesized that indi- vidual motor performance would vary after transfer to a novel environment, and that this variability could be pre- dicted by individual differences in variables such as pres- ence in the training environment, prior experience with HMD-VR, or non-VR video games. Participants A two-second time interval was given be- tween each trial. Participants completed 4 training blocks (Training Blocks 1–4) consisting of 30 SVIPT trials either in an HMD-VR (Fig. 2a; Train-HMD-VR) environment or on a computer screen (Fig. 2b; Train-Screen). Block 1 was considered the Baseline training block for each group. After completion of the training blocks, all participants completed 2 counter-balanced testing blocks consisting of 20 SVIPT trials in an HMD-VR environment and on a computer screen. We defined the testing block that matched the training condition as the “Acquired Skill” Participants Seventy-four healthy adults were recruited. Participants were randomized into two groups (Train-HMD-VR, Juliano and Liew Journal of NeuroEngineering and Rehabilitation Page 3 of 13 Page 3 of 13 (2020) 17:48 Train-Screen). Three participants in the Train-Screen group were excluded from the analysis as a result of per- forming all trials in the Baseline training block incor- rectly (see Analyses) and one participant in the Train- HMD-VR group was excluded from the analysis as a re- sult of being an outlier, which was defined as being be- yond three standard deviations from the group mean motor skill in at least one of the blocks. This resulted in a total of seventy participants (53 females/16 males/1 other, aged: M = 25.81, SD = 4.71) with thirty-five partici- pants in each group included in the analysis. A statistical power analysis was performed for sample size estimation based on data from a pilot study of this work (N = 12) [32]. The effect size in this study was d = 0.38. With an alpha = 0.05 and power = 0.60, the projected sample size need with this effect size was approximately N = 35. Eli- gibility criteria included healthy, self-reported right- handed individuals and no previous experience with the motor skill task (see Experimental design). Written in- formed consent was obtained from all subjects. The ex- perimental protocol was approved by the University of Southern California Institutional Review Board and per- formed in accordance with the 1964 Declaration of Helsinki. blocks in which participants completed a modified ver- sion of the Sequential Visual Isometric Pinch Task (SVIPT) [33]. In this task, participants were instructed to apply varying degrees of isometric force between their thumb and index finger to a small pinch force sensor (Futek Pinch Sensor FSH01465; Futek IPM FSH03633; Fig. 1b) to move a cursor between numbered colored gates as quickly and accurately as possible (Fig. 1c). A small circle at the bottom of the screen changed from red to green to indicate the start of each trial. For each trial, no time limit was given and trial completion time was recorded. At the end of each trial, the small circle at the bottom of the screen changed from green to red and participants received auditory feedback (a pleasant “ding” if the cursor correctly entered all the gates or an unpleasant “buzz” if the cursor missed one or more of the gates). Experimental design Figure 1a provides an overview of the experimental de- sign. The experiment consisted of training and testing Fig. 1 Experimental paradigm. a Experimental design. b Pinch force between the thumb and index finger was applied to a small force transducer to move the cursor in the SVIPT. c Sequential Visual Isometric Pinch Task (SVIPT) display. Participants were asked to apply force to the force transducer, which translated into the movement of a small black cursor (shown at the home position in the white bar) moving horizontally to the right in the environment. The cursor moved left by reducing force. Instructions were to move the cursor between the gates, in order from 1 to 5, as quickly and accurately as possible, without over- or under-shooting any of the gates Fig. 1 Experimental paradigm. a Experimental design. b Pinch force between the thumb and index finger was applied to a small force transducer to move the cursor in the SVIPT. c Sequential Visual Isometric Pinch Task (SVIPT) display. Participants were asked to apply force to the force transducer, which translated into the movement of a small black cursor (shown at the home position in the white bar) moving horizontally to the right in the environment. The cursor moved left by reducing force. Instructions were to move the cursor between the gates, in order from 1 to 5, as quickly and accurately as possible, without over- or under-shooting any of the gates Juliano and Liew Journal of NeuroEngineering and Rehabilitation (2020) 17:48 Page 4 of 13 Juliano and Liew Journal of NeuroEngineering and Rehabilitation Page 4 of 13 (2020) 17:48 Fig. 2 Training and testing environments. Participants were trained on a motor skill task in either the HMD-VR or Screen environment and were then tested on the same task in both environments. a HMD-VR environment; the stimulus shown in the HMD-VR display is also shown on the computer screen. b Screen environment Fig 2 Training and testing environments Participants were trained on a m Fig. 2 Training and testing environments. Participants were trained on a motor skill task in either the HMD-VR or Screen environment and were then tested on the same task in both environments. a HMD-VR environment; the stimulus shown in the HMD-VR display is also shown on the computer screen. b Screen environment testing block (e.g., train in HMD-VR, test in HMD-VR). Training and testing environments The environments for all blocks were designed using the game engine development tool, Unity 3D (Version 5.6.6). In the blocks where participants were in the HMD-VR environment, participants performed the task in a head- mounted display (Oculus Rift DK2). In the blocks where participants were in the Screen environment, partici- pants performed the task on a 17.3 in., 1920 × 1080 pixel resolution computer laptop (ASUS ROG G751JY-DH71). The HMD-VR environment was created based on a fixed coordinate system that did not depend on the partici- pant’s head position. All participants were physically seated in the same location for all blocks and used the same force transducer for the task. The only difference between HMD-VR and Screen blocks is that participants put on the HMD-VR headset for HMD-VR blocks. Experimental design There was no difference in either group between the last block of training (Block 4) and the Acquired Skill testing block; thus, the Acquired Skill testing block was used as a proxy for total amount of motor skill within the assigned training environment. We defined the testing block that was different from the training condition as the “Transfer” testing block (e.g., train in HMD-VR, test in Screen). Importantly, there was no difference in the order of testing block completion for either group. Lastly, after completion of both training and testing blocks, participants were asked to complete three ques- tionnaires (see Questionnaires). Questionnaires Participants were asked to complete two Likert-scale questionnaires regarding their reaction to the training environment; the first questionnaire related to partici- pants’ simulator sickness and the second questionnaire related to participants’ level of presence. The first ques- tionnaire was the simulator sickness questionnaire, adapted from Kennedy, Lane, Berbaum, & Lilienthal (1993) [34], and consisted of a series of questions to gauge participant sickness level and was given both be- fore and after the task. Questions were collapsed along four main themes: nausea, oculomotor reactions, dis- orientation, and overall simulator sickness. The second questionnaire was the presence questionnaire, which was adapted from Witmer & Singer (1998) [35] and revised by the UQO Cyberpsychology Lab (2004). It consisted of a series of questions to gauge the participant’s sense of presence in the training environment. Questions were measured along five main themes: realism, possibility to act, quality of interface, possibility to examine, and self- evaluation of performance. Participants were also asked questions regarding their gender, age, whether or not they played video games, and whether or not they had previous experience using HMD-VR. Both these ques- tions and the presence questionnaire were administered at the end of the experiment. Simulator sickness, presence, and subjective measures Simulator sickness, presence, and subjective measures To examine any differences between groups in the vari- ables calculated based on simulator sickness questionnaire responses (i.e., nausea, oculomotor reactions, disorienta- tion, and overall simulator sickness), we use a Mann- Whitney U test as either skewness or kurtosis values were greater than 1.96 for each variable. To examine any differ- ences between groups in the variables calculated based on presence and subjective measures questionnaire re- sponses, we used an unpaired t-test for quantitative vari- ables (i.e., realism, possibility to act, quality of interface, possibility to examine, self-evaluation of performance, age) and a chi-squared test for qualitative variables (i.e., gender, video game use, and previous HMD-VR experi- ence). Significance was defined as p < 0.0125 for the four simulator sickness variables and defined as p < 0.0055 for the nine presence and subjective variables. Finally, to examine which individual factors predicted motor skill transfer, we considered the nine presence and subjective variables in a multivariate linear regres- sion model (i.e., realism, possibility to act, quality of interface, possibility to examine, self-evaluation of per- formance, age, gender, video game use, and previous HMD-VR experience) for each group. To identify vari- ables that strongly predicted motor skill transfer, we used the regularization technique lasso [37] with 10-fold cross-validation, which shrinks some coefficients and sets others to zero. Shrinking coefficient estimates through lasso can reduce the variance at the cost of a small increase in bias [38] and has been suggested for datasets with a similar sample size to predictor ratio [39, 40]. Specifically, we trained a lasso model with cross- validation on 75% of the dataset using the glmnet R function [41]. Then, using the tuning parameter lambda that produced the minimum mean square error (MSE), we calculated the prediction error on the remaining 25% of the dataset and refit the lasso model using the full dataset. This resulted in a sparse linear model that is more interpretable and only includes a subset of the var- iables included in the initial linear model. Variance infla- tion factor (VIF) was calculated for each predictor to check for multicollinearity; we considered a VIF value less than 3.3 as meeting the assumption of collinearity [42]. For exploratory purposes, we then individually ex- amined the variables in a univariate linear regression model to determine whether any variables on their own could explain motor skill transfer. Analyses All analyses were complete in R (Version 3.5.3) using R Studio (Version 1.1.423). We assessed the normality of Page 5 of 13 Juliano and Liew Journal of NeuroEngineering and Rehabilitation Page 5 of 13 (2020) 17:48 Transfer testing block for each group. A paired t-test was used for these within-group comparisons with significance defined as p < 0.025 since comparisons were made for each group. Lastly, to quantify individual motor skill trans- fer, we took the difference in motor skill between the Transfer testing block and the Acquired Skill testing block across individuals and compared overall motor skill trans- fer between the two groups; an unpaired t-test was used for this between group comparison. each variable using skewness and kurtosis of the distri- bution. Given our sample size, we considered a variable with an absolute skewness or kurtosis value of less than 1.96 as normally distributed [36]. Only simulator sickness variables were considered non-normally distributed (see Simulator sickness, presence, and subjective measures). Sig- nificance was defined as p < 0.05 unless corrected with multiple comparisons using a Bonferroni correction; these corrections are specified for each analysis. Motor skill acquisition and motor skill transfer Motor skill was calculated based on a formula first pre- sented in Reis et al. 2009 [33], which measures the ratio of speed to accuracy over the trials in each block. Motor skill for each block is calculated as: Motor Skill ¼ ln 1−error rate error rateðln ðdurationÞbÞ ! ; Motor Skill ¼ ln 1−error rate error rateðln ðdurationÞbÞ ! ; Motor Skill ¼ ln 1−error rate error rateðln ðdurationÞbÞ ! ; where error rate and duration are averaged over trials in each block and b is a free parameter that equals 5.424 [33]. If error rate = 0 for a given block (i.e., all trials were incorrect), the resulting motor skill calculation would be undefined. For each group, we assessed motor skill ac- quisition and motor skill transfer; motor skill acquisition is the increased performance in the trained environment and motor skill transfer is the maintained performance in the untrained environment after training. To calculate motor skill acquisition, we compared motor skill from the Baseline training block to the Acquired Skill testing block for each group. Additionally, we compared the motor skill on the Baseline training block to the Trans- fer testing block to assess whether training in one enviorment had an effect on motor skill performance in the other environment. To calculate motor skill transfer we compared the Acquired Skill testing block to the where error rate and duration are averaged over trials in each block and b is a free parameter that equals 5.424 [33]. If error rate = 0 for a given block (i.e., all trials were incorrect), the resulting motor skill calculation would be undefined. For each group, we assessed motor skill ac- quisition and motor skill transfer; motor skill acquisition is the increased performance in the trained environment and motor skill transfer is the maintained performance in the untrained environment after training. To calculate motor skill acquisition, we compared motor skill from the Baseline training block to the Acquired Skill testing block for each group. Additionally, we compared the motor skill on the Baseline training block to the Trans- fer testing block to assess whether training in one enviorment had an effect on motor skill performance in the other environment. To calculate motor skill transfer we compared the Acquired Skill testing block to the Simulator sickness, presence, and subjective measures Qualitative predictors (i.e., gender, video game use, and previous HMD-VR ex- perience) in both multivariate and univariate linear re- gression models were encoded as dummy variables. No differences in motor skill acquisition between training environments No differences in motor skill acquisition between training environments No differences in motor skill acquisition between training environments To assess initial and end of training performance be- tween training environments, we compared motor skill between groups at the Baseline training block (Block 1) and the Last training block (Block 4). There were no differences between groups in Base- line training blocks (t (68.0) = 0.44, p = 0.6626; Train- HMD-VR: M = −4.83, SD = 1.12; Train-Screen: M = − 4.95, SD = 1.13) and in the Last training blocks (t (66.3) = 0.26, p = 0.7941; Train-HMD-VR: M = −2.93, SD = 1.06; Train-Screen: M = −3.00, SD = 0.90). To compute individual participant acquisition rates, we applied a linear-log linear regression to motor skill across the four training blocks [43]. We found simi- lar acquisition rates (i.e., slopes from regression model) between the two groups (t (58.8) = −0.41, p = 0.6839; Train-HMD-VR: M = 1.36, SD = 0.84; Train- Screen: M = 1.43, SD = 0.56), suggesting that motor skill acquisition occurred at a similar rate across HMD-VR and Screen groups. (2020) 17:48 to the Transfer testing block for the Train-HMD-VR group. We found a significant difference in performance between the HMD-VR Baseline training block (M = − 4.83, SD = 1.12) and the computer screen Transfer test- ing block (M = −3.20, SD = 0.96; t (34) = −9.12; p < 0.0001; Fig. 3a), suggesting that motor skill training in HMD-VR increased the motor skill performance on a computer screen, compared to if no HMD-VR training occurred. To assess whether training on a computer screen had an effect on motor skill performance in HMD-VR, we compared the motor skills on the Baseline training block to the Transfer testing block for the Train-Screen group. We found a significant difference in performance between the computer screen Baseline training block (M = −4.95, SD = 1.13) and the HMD-VR Transfer testing block (M = −2.73, SD = 1.01; t(34) = − 12.52; p < 0.0001; Fig. 3b), suggesting that motor skill training on a computer screen increased the motor skill performance in HMD-VR, compared to if no training on a computer screen occurred. Table 1), suggesting that this type of motor skill training does not produce any additional simulator sickness side effects in HMD-VR compared to what is experienced when training on a computer screen. Additionally, we compared each of the nine prediction variables (realism, possibility to act, quality of interface, possibility to exam- ine, self-evaluation of performance, age, gender, video game use, and previous HMD-VR experience) and found no significant differences between the two groups (Sup- plementary Table 2). Table 1), suggesting that this type of motor skill training does not produce any additional simulator sickness side effects in HMD-VR compared to what is experienced when training on a computer screen. Additionally, we compared each of the nine prediction variables (realism, possibility to act, quality of interface, possibility to exam- ine, self-evaluation of performance, age, gender, video game use, and previous HMD-VR experience) and found no significant differences between the two groups (Sup- plementary Table 2). Motor skill transfer to computer screen: performance decreases To assess motor skill transfer to a computer screen after training in HMD-VR, we compared the Acquired Skill testing block (HMD-VR) to the Transfer testing block (Screen) and found a significant difference (t(34) = 2.83, p = 0.0078; Fig. 3a), where motor skill was lower in the Transfer testing block (M = −3.20, SD = 0.96) compared to the Acquired Skill testing block (M = −2.88, SD = 1.14). This suggests that performance decreased after transfer to the untrained computer screen environment. As a re- minder, there was no significant difference in motor skill based on the order of the Acquired Skill and Transfer blocks, which were counterbalanced across individuals. Motor skill acquisition and motor skill transfer Motor skill acquisition occurs in both environments To ensure that motor skill acquisition occurred in both environments, we compared the motor skill be- tween the Baseline training block and the Acquired Skill testing block for the Train-HMD-VR group and for the Train-Screen group separately. On average, we found motor skill acquisition occurred after training in HMD-VR (Train-HMD-VR: t (34) = −11.42, p < 0.0001; Baseline (Block 1): M = −4.83, SD = 1.12, Ac- quired Skill (HMD-VR): M = −2.88, SD = 1.14; Fig. 3a) and after training on a computer screen (Train-Screen: t (34) = −9.68, p < 0.0001; Baseline (Block 1): M = − 4.95, SD = 1.13, Acquired Skill (Screen): M = −3.14, SD = 0.92; Fig. 3b). This suggests that motor skill ac- quisition on an isometric pinch force task can occur both in HMD-VR as well as on a more conventional screen environment. Motor skill transfer to HMD-VR: performance increases Motor skill transfer to HMD-VR: performance increases We then assessed motor skill transfer to HMD-VR after training on a computer screen. To examine this, we compared the Acquired Skill testing block (Screen) to the Transfer testing block (HMD-VR) and found a sig- nificant difference (t(34) = −2.59, p = 0.0142; Fig. 3b), where motor skill was higher in the Transfer testing block (M = −2.73, SD = 1.01) compared to the Acquired Skill testing block (M = −3.14, SD = 0.92). This suggests that performance increased after transfer to the un- trained HMD-VR environment. In this group, there was also no significant difference in motor skill based on the order of the Acquired Skill and Transfer blocks, which were counterbalanced across individuals. No differences in simulator sickness, presence, and subjective measures between environments To assess differences in simulator sickness level between training environments, we compared scores of nausea, oculomotor reactions, disorientation, and overall simula- tor sickness between groups and found no significant difference for each of the measures (Supplementary Juliano and Liew Journal of NeuroEngineering and Rehabilitation Page 6 of 13 Individual motor skill transfer Participants increased their motor skill after training on a computer screen (t(34) = −9.68, p < 0.0001). Transfer to HMD-VR occurred as a result of computer screen training (t(34) = −12.52; p < 0.0001); however, the motor skills transferred to HMD-VR was greater than the motor skill on a computer screen (t(34) = −2.59, p = 0.0142). Indicators of significance: p < 0.05, p < 0.01, p < 0.0001*** Fig. 3 Motor skill shown for the Train-HMD-VR group in (a) and the Train-Screen group in (b). Light yellow blocks are HMD-VR training blocks, dark yellow blocks are HMD-VR testing blocks. Light blue blocks are Screen training blocks, dark blue blocks are Screen testing blocks. a Motor skill across training blocks in Train-HMD-VR group and both corresponding testing blocks. Participants increased their motor skill after training in HMD-VR (t(34) = −11.42, p < 0.0001). Transfer to a computer screen occurred as a result of HMD-VR training (t(34) = −9.12; p < 0.0001); however, the motor skills transferred to a computer screen was less than the motor skills in HMD-VR (t(34) = 2.83, p = 0.0078). b Motor skill across training blocks in Train-Screen group and both corresponding testing blocks. Participants increased their motor skill after training on a computer screen (t(34) = −9.68, p < 0.0001). Transfer to HMD-VR occurred as a result of computer screen training (t(34) = −12.52; p < 0.0001); however, the motor skills transferred to HMD-VR was greater than the motor skill on a computer screen (t(34) = −2.59, p = 0.0142). Indicators of significance: p < 0.05, p < 0.01, p < 0.0001*** 0.0004; Fig. 4b), where the motor skill transfer to a computer screen in the Train-HMD-VR group (M = −0.31, SD = 0.67) was significantly lower that the motor skill transfer to HMD-VR in the Train-Screen group (M = 0.41, SD = 0.94). This suggests that the type of training environment during motor skill ac- quisition may affect the overall transfer of the motor skills to another environment; specifically, training in an HMD-VR environment may not transfer to a con- ventional environment. However, as seen in Fig. 4a, not all participants had similar transfer, suggesting that individual differences may predict the transfer of motor skill acquisition between environments. testing block compared to the Acquired Skill testing block (Fig. 4a, left). Individual motor skill transfer To assess whether training in HMD-VR had an effect on motor skill performance on a computer screen, we compared the motor skills on the Baseline training block Individual motor skill transfer In the Train-HMD-VR group, a greater proportion of participants performed worse on the Transfer Juliano and Liew Journal of NeuroEngineering and Rehabilitation (2020) 17:48 Page 7 of 13 Juliano and Liew Journal of NeuroEngineering and Rehabilitation Page 7 of 13 (2020) 17:48 Fig. 3 Motor skill shown for the Train-HMD-VR group in (a) and the Train-Screen group in (b). Light yellow blocks are HMD-VR training blocks, dark yellow blocks are HMD-VR testing blocks. Light blue blocks are Screen training blocks, dark blue blocks are Screen testing blocks. a Motor skill across training blocks in Train-HMD-VR group and both corresponding testing blocks. Participants increased their motor skill after training in HMD-VR (t(34) = −11.42, p < 0.0001). Transfer to a computer screen occurred as a result of HMD-VR training (t(34) = −9.12; p < 0.0001); however, the motor skills transferred to a computer screen was less than the motor skills in HMD-VR (t(34) = 2.83, p = 0.0078). b Motor skill across training blocks in Train-Screen group and both corresponding testing blocks. Participants increased their motor skill after training on a computer screen (t(34) = −9.68, p < 0.0001). Transfer to HMD-VR occurred as a result of computer screen training (t(34) = −12.52; p < 0.0001); however, the motor skills transferred to HMD-VR was greater than the motor skill on a computer screen (t(34) = −2.59, p = 0.0142). Indicators of significance: p < 0.05, p < 0.01, p < 0.0001* Fig. 3 Motor skill shown for the Train-HMD-VR group in (a) and the Train-Screen group in (b). Light yellow blocks are HMD-VR training blocks, dark yellow blocks are HMD-VR testing blocks. Light blue blocks are Screen training blocks, dark blue blocks are Screen testing blocks. a Motor skill across training blocks in Train-HMD-VR group and both corresponding testing blocks. Participants increased their motor skill after training in HMD-VR (t(34) = −11.42, p < 0.0001). Transfer to a computer screen occurred as a result of HMD-VR training (t(34) = −9.12; p < 0.0001); however, the motor skills transferred to a computer screen was less than the motor skills in HMD-VR (t(34) = 2.83, p = 0.0078). b Motor skill across training blocks in Train-Screen group and both corresponding testing blocks. Predicting motor skill transfer performance. Multicollinearity was not an issue as the VIF for each variable was < 3.3. Given the interindividual variability of motor skill trans- fer (Fig. 4a), we were interested in whether any self- reported measurements collected (i.e., realism, possibility to act, quality of interface, possibility to examine, self- evaluation of performance, age, gender, video game use, and previous HMD-VR experience) could predict the motor skill transfer in each group. Using lasso with cross-validation to select the penalty term lambda, we performed variable selection to examine which of the nine variables most strongly predicted individual motor skill transfer (see Analyses). Additionally, we examined variables individually in each group with a univariate lin- ear regression model for exploratory purposes. We did not find any significant results in examining vari- ables individually in the univariate linear regression models (Supplementary Table 3). However, there was non- significant evidence of a difference in motor skill transfer in reported previous HMD-VR experience (F(1,33) = 2.90, R2 = 0.081, p = 0.0982) where individuals with previous HMD-VR experience had higher motor skill transfer (M = −0.19, SD = 0.69) compared to individuals who had never tried HMD-VR (M = −0.58, SD = 0.57; Fig. 5). Although these results are weak, they provide a preliminary suggestion that individual characteristics in these areas may explain why a reduction in motor skill may occur during HMD-VR transfer to a con- ventional environment. However, further research is needed to confirm these findings in a larger sample and with multiple tasks. Predicting HMD-VR motor skill transfer to a computer screen For the Train-HMD-VR group, the resulting multivariate linear regression model retained four variables (Table 1) and explained 25.5% of the variance but was not statisti- cally significant (F(4,30) = 2.57, R2 = 0.255, p = 0.0580). The model contained two presence variables predicting the motor skill transfer: positively correlated possibility to act and negatively correlated self-evaluation of Predicting computer screen motor skill transfer to HMD-VR For the Train-Screen group, the resulting multivariate lin- ear regression model retained all nine variables (Table 2) and explained 59.7% of the variance (F (10,24) = 3.55, R2 = Table 1 Results from a multivariate regression model for the Train-HMD-VR group Predictor Estimate Std. Individual motor skill transfer Conversely, in the Train-Screen group, a greater proportion of participants performed better on the Transfer testing block compared to the Leaned Skill testing block (Fig. 4a, right). To exam- ine group and individual differences in transfer for each group, we first calculated the amount of motor skill transfer for each individual. To do this, we took the difference in motor skill between the Transfer testing block and the Acquired Skill testing block for each individual. At the group level, we compared the average motor skill transfer between the two groups and found a significant difference (t(61.5) = 3.75, p = Juliano and Liew Journal of NeuroEngineering and Rehabilitation (2020) 17:48 Page 8 of 13 Juliano and Liew Journal of NeuroEngineering and Rehabilitation Page 8 of 13 (2020) 17:48 Fig. 4 a Individual motor skill differences on Acquired Skill and Transfer testing blocks for both the Train-HMD-VR group (left) and the Train- Screen group (right). Purple represents individuals with greater motor skill on the Acquired Skill testing block and green represents individuals with greater motor skill on the Transfer testing block. b The y-axis shows “Motor Skill Transfer”, which is defined as the motor skill on the Transfer block minus the motor skill on the Acquired Skill block for each individual. There was a significant difference in average motor skill transfer between the Train-HMD-VR group (left) and Train-Screen group (right; t(61.5) = 3.75, p = 0.0004). Dots represent individuals, the box represents the first and third quartiles, and the line represents the median. p < 0.001* Fig. 4 a Individual motor skill differences on Acquired Skill and Transfer testing blocks for both the Train-HMD-VR group (left) and the Train- Screen group (right). Purple represents individuals with greater motor skill on the Acquired Skill testing block and green represents individuals with greater motor skill on the Transfer testing block. b The y-axis shows “Motor Skill Transfer”, which is defined as the motor skill on the Transfer block minus the motor skill on the Acquired Skill block for each individual. There was a significant difference in average motor skill transfer between the Train-HMD-VR group (left) and Train-Screen group (right; t(61.5) = 3.75, p = 0.0004). Dots represent individuals, the box represents the first and third quartiles, and the line represents the median. p < 0.001*** Discussion In this study, we examined motor skill transfer from an HMD-VR environment to a conventional environment (i.e., computer screen), and vice-versa. First, we con- firmed that motor skill acquisition occurs in both HMD- VR and conventional screen environments and demon- strated that acquisition occurs at a similar rate in both environments, suggesting that task difficulty was not dif- ferent between the environments. We then demon- strated that while motor skill transfer occurs after training in either environment, there are individual dif- ferences in the amount of motor skill that transferred. Fig. 5 Train-HMD-VR Group: Individuals with previous HMD-VR experience had higher motor skill transfer to the screen compared to individuals who had never tried HMD-VR (F(1,33) = 2.90, R2 = 0.081, p = 0.0982). p < 0.1† 0.597, p = 0.0053) with quality of interface, gender, age, and video game use significantly predicting the motor skill transfer, suggesting that the combination of these vari- ables may be important for predicting computer screen motor skill transfer to HMD-VR. Multicollinearity was not an issue as the VIF for each variable was < 3.3. In examining whether motor skills acquired during training in HMD-VR transferred to a conventional screen environment, we found a significant decrease in motor skill performance as a result of the transfer. To see if this decrease in motor skill transfer could be ex- plained, we examined whether individual differences in five presence themes (realism, possibility to act, quality of interface, possibility to examine, self-evaluation of performance), age, gender, video game use, and previous HMD-VR experience could be used as predictors. We Examining variables individually in the univariate lin- ear regression models, we found significant results for age and video game use. Age was negatively correlated with motor skill transfer (F(1,33) = 4.75, R2 = 0.126, p = 0.0366; Fig. 6a), suggesting that younger age may facili- tate transfer of the acquired motor skill to an HMD-VR Table 2 Results from multivariate regression model for Train-Screen Predictor Estimate Std. Predicting motor skill transfer Error t-value p-value (Intercept) −0.7396 0.7741 −0.9555 0.3470 Possibility to Act 0.0835 0.0382 2.1880 0.0366* Self-Evaluation of Performance −0.1515 0.0716 −2.1170 0.0426* Video Game Play = Yes 0.1138 0.2380 0.4779 0.6362 Previous HMD-VR Experience = Yes 0.2836 0.2284 1.2420 0.2240 Possibility to act and self-evaluation of performance significantly predicted the amount of transfer from HMD-VR to a computer screen. p < 0.05* (2020) 17:48 Juliano and Liew Journal of NeuroEngineering and Rehabilitation Page 9 of 13 Fig. 5 Train-HMD-VR Group: Individuals with previous HMD-VR experience had higher motor skill transfer to the screen compared to individuals who had never tried HMD-VR (F(1,33) = 2.90, R2 = 0.081, p = 0.0982). p < 0.1† environment. Additionally, there was significant evi- dence of a difference in motor skill transfer in reported video game use (F(1,33) = 4.15, R2 = 0.112, p = 0.0498; Fig. 6b) where individuals who did not play video games had overall higher motor skill transfer (M = 0.82, SD = 0.78) compared to individuals who played video games (M = 0.17, SD = 0.96). Furthermore, we found a non- significant positive trend between the quality of interface and motor skill transfer (F(1,33) = 3.61, R2 = 0.099, p = 0.0663; Fig. 6c), suggesting that this presence variable may be important in predicting computer screen motor skill transfer to HMD-VR; however, this should be fur- ther examined. Univariate linear regression results for Train-Screen can be found in Supplementary Table 4. of interface, gender, age, and video game use significantly predicted the amount of transfer from a computer screen to HMD-VR. p < 0 Discussion Overall, these results suggest that while the motor skills acquired in HMD-VR may not transfer to a conventional environment, the factors mentioned could mitigate this decrease. Our work adds to the limited knowledge of personal factors that could potentially drive motor acquisition in HMD-VR and the transfer of motor skill to other envi- ronments. While other studies have identified potential mechanisms for HMD-VR transfer by examining exist- ing literature [7], there is inconclusive evidence for why motor skill acquisition in HMD-VR and transfer to other environments may be more effective for some individ- uals compared to others. The two presence themes iden- tified support previous findings that levels of presence relate to motor performance in an HMD-VR enviorment [46] and extend these findings to the transfer of motor skill acquisition. Additionally, previous experience with the training device, which is HMD-VR in the present case, support findings that the transfer of spatial know- ledge is influenced by previous experience with the en- vironment [31]. Increased exposure to HMD-VR may decrease the novelty, and subsequent attention evoked during the task, which may decrease motor performance. Future studies should examine whether individuals with more HMD-VR experience have greater motor skill transfer to the real world. We also examined whether motor skills acquired dur- ing training on a conventional screen environment transferred to HMD-VR. We found that motor skills learned in a conventional screen environment transfer to HMD-VR; however, not only do the motor skills trans- fer, but performances seem to improve in the novel HMD-VR environment. We found that the combination of the quality of interface, gender, age, and video game use best predicted this motor skill transfer. Additionally, we found evidence that age and video game use inde- pendently may predict the increase in motor skill trans- fer between computer screen and HMD-VR. This supports previous findings that age and video game use affect acquisition and transfer in non-immersive virtual environments [44, 45]. We also found trending evidence that the quality of interface independently may predict the increase in motor skill transfer between a computer screen and HMD-VR, further supporting the involve- ment of presence in the transfer of motor skill. Discussion Error t-value p-value (Intercept) 1.205 1.191 1.012 0.3218 Realism −0.04472 0.02562 −1.745 0.09372 Possibility to Act −0.06161 0.05833 −1.056 0.3014 Quality of Interface 0.1027 0.0493 2.084 0.04798* Possibility to Examine 0.07857 0.04586 1.713 0.09958 Self-evaluation of Performance 0.1428 0.08006 1.784 0.08715 Age −0.0543 0.02388 −2.274 0.03222* Gender = Male 0.9471 0.3572 2.652 0.01396* Gender = Other −0.1832 0.7773 −0.2357 0.8157 Video Game Use = Yes −0.8933 0.2903 −3.077 0.005164** Previous HMD-VR Experience = Yes −0.2622 0.2848 −0.9208 0.3663 Quality of interface, gender, age, and video game use significantly predicted the amount of transfer from a computer screen to HMD-VR. p < 0.05, p < 0.01* Table 2 Results from multivariate regression model for Train-Screen Juliano and Liew Journal of NeuroEngineering and Rehabilitation Page 10 of 13 (2020) 17:48 Fig. 6 Train-Screen Group: a Younger age was significantly related to increased screen-based motor skill transfer to HMD-VR (F(1,33) = 4.75, R2 = 0.126, p = 0.0366). b Individuals who did not play video games had overall higher motor skill transfer to HMD-VR than individuals who played video games (F(1,33) = 4.15, R2 = 0.112, p = 0.0498). c Higher reports on the quality of the interface during training on a computer screen was related to increased computer screen motor skill transfer to HMD-VR; however, this result was non-significantly correlated (F(1,33) = 3.61, R2 = 0.099, p = 0.0663). p < 0.05* Fig. 6 Train-Screen Group: a Younger age was significantly related to increased screen-based motor skill transfer to HMD-VR (F(1,33) = 4.75, R2 = 0.126, p = 0.0366). b Individuals who did not play video games had overall higher motor skill transfer to HMD-VR than individuals who played video games (F(1,33) = 4.15, R2 = 0.112, p = 0.0498). c Higher reports on the quality of the interface during training on a computer screen was related to increased computer screen motor skill transfer to HMD-VR; however, this result was non-significantly correlated (F(1,33) = 3.61, R2 = 0.099, p = 0.0663). p < 0.05* motor skills have already been acquired in the real world. found trending but nonsignificant evidence that a com- bination of two presence themes, positively correlated possibility to act and negatively correlated self- evaluation of performance, best predicted this observed decrease in motor skill. Additionally, we found trending evidence that previous experience using HMD-VR inde- pendently may predict the decrease in the motor skill transfer. Discussion These predictors may be useful to consider in cases when a HMD-VR rehabilitation intervention is introduced after In addition to the personal factors that we have exam- ined in this study, there are undoubtably more mecha- nisms that could either drive or predict HMD-VR motor skill transfer, and this should be further explored. Future studies should also consider other personal factors such as participants’ immersive tendencies, the likelihood that an individual will feel immersed in a new environment [47] as well as avatar embodiment, if applicable [48]. In Page 11 of 13 Page 11 of 13 Juliano and Liew Journal of NeuroEngineering and Rehabilitation (2020) 17:48 (2020) 17:48 addition to personal factors, task-related factors likely contribute to differences in motor skill acquisi- tion and transfer from HMD-VR to conventional en- vironments, and vice versa. Previous findings have suggested that fidelity and dimensionality influences the transfer of motor skills from HMD-VR environ- ments [29]. In the current study, a possible explan- ation for the decrease in performance on a computer screen could be that the visual representation of the HMD-VR environment did not reflect what individ- uals expected and therefore, motor skill performance was not maintained with transfer. Future studies should consider examining the level of fidelity and dimensionally in HMD-VR needed to optimize motor skill transfer to the real world, and vice versa. Motor skill transfer has also been shown to be influenced by other task-related factors such as task variability, engagement, and feedback [49, 50]. In the current study, the increase in performance in HMD-VR could be a result of an increase in attention or en- gagement after transfer from the computer screen. Future studies should also examine how these task- related factors influence HMD-VR motor transfer to the real world, and vice versa. throwing a ball in HMD-VR versus throwing a ball in real life). Future research should also look to see if the identified factors apply to different clinical populations and examine whether mechanisms such as functional in- dependence or cognitive status could predict success of HMD-VR rehabilitation interventions [55, 56]. Another limitation was that our definition of motor skill transfer reflects the transfer of motor skill acquisition rather than motor skill learning. Experimental designs of motor skill learning typically examine transfer after a retention interval and compare transfer performance to baseline performance in the transfer context [22]. Discussion Future studies should examine whether the personal factors identified here are also predictors for this type of experimental de- sign. Lastly, the use of a subjective questionnaire to measure presence is also a limitation; future work should use alternative objective measures, such as physiological responses, in addition [57]. Overall, despite these limita- tions, we believe that the work presented in this study provides an initial examination into the transfer of motor skills between HMD-VR and conventional screen environments as well as insight into the factors that may mediate this transfer. It has also been suggested that HMD-VR may require additional cognitive resources and that additional infor- mation and stimuli must be processed in order to solve tasks in virtual reality [51]. One study found that the motor skills acquired in HMD-VR through the reliance of spatial cognitive capabilities did not transfer to the same task in the real world [25]. Our own previous work has shown that visuomotor adaptation in HMD-VR requires a greater reliance on cognitive strategies than performing the same task on a computer screen [19]. Taken together, this suggests that the decrease in motor skill transfer ob- served when moving to a conventional screen environ- ment could also be due to less engagement of the cognitive processes used when in HMD-VR. The utilization of these cognitive processes during perform- ance in either environment could be influenced by any of the personal or task-related factors described. Future work should examine whether specific cognitive processes have a role in HMD-VR motor skill transfer to the real world. Conclusion Both HMD-VR and conventional screen environments re- sulted in the acquisition of a motor skill at a similar rate, as well as transfer to a different environment. However, motor skill performance decreased when transferring from HMD-VR to a conventional screen environment, while motor skill performance increased when transferring from a conventional screen environment to HMD-VR. Further- more, themes of presence, gender, age, and video game use significantly predicted the motor skill transfer in individuals training on the screen, while themes of pres- ence and previous HMD-VR experience were loosely re- lated to the motor skill transfer in individuals training in HMD-VR. As HMD-VR becomes an increasingly popular medium for motor learning and rehabilitation applica- tions, it is important to understand how to optimize inter- ventions to ensure the complete transfer of motor skills to the target environment. Future studies should examine in- dividual differences in other personal factors and in task- related factors. One limitation of this study was the use of a computer screen as the transfer condition from HMD-VR. Al- though this was purposefully designed to provide the most well-controlled and subtle differences between HMD-VR and the conventional environment, and previ- ous studies have reported significant differences between HMD-VR and computer screen environments [19, 52– 54], future work should examine whether presence, gen- der, age, video game use, or previous HMD-VR experi- ence has an effect on HMD-VR motor skill transfer to more dynamic, real world physical applications (e.g., Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12984-020-00678-2. Additional file 1: Table S1. Differences in simulator sickness level between Train-HMD-VR and Train-Screen. Table S2. Differences in themes of presence (top) and other self-reported measures (bottom) be- tween Train-HMD-VR and Train-Screen. Table S3. Train-HMD-VR results from univariate analysis of predicting HMD-VR motor skill transfer to a Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12984-020-00678-2. Additional file 1: Table S1. Differences in simulator sickness level between Train-HMD-VR and Train-Screen. Table S2. Differences in themes of presence (top) and other self-reported measures (bottom) be- tween Train-HMD-VR and Train-Screen. Table S3. Train-HMD-VR results from univariate analysis of predicting HMD-VR motor skill transfer to a Funding 14. Slater M, Sanchez-Vives MV. Enhancing our lives with immersive virtual reality. Front Robot AI. 2016;3:74. This study was funded by the National Institutes of Health National Center for Medical Rehabilitation Research (Grant #K01HD091283). 15. 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Author details 1 1Neural Plasticity and Neurorehabilitation Laboratory, Neuroscience Graduate Program, University of Southern California, Los Angeles, CA, USA. 2Neural Plasticity and Neurorehabilitation Laboratory, Division of Occupational Science and Occupational Therapy, University of Southern California, Los Angeles, CA, USA. 3USC Stevens Neuroimaging and Informatics Institute, Department of Neurology, University of Southern California, Los Angeles, CA, USA. 1Neural Plasticity and Neurorehabilitation Laboratory, Neuroscience Graduate Program, University of Southern California, Los Angeles, CA, USA. 2Neural Plasticity and Neurorehabilitation Laboratory, Division of Occupational Science and Occupational Therapy, University of Southern California, Los Angeles, CA, USA. 3USC Stevens Neuroimaging and Informatics Institute, 23. Petri K, Emmermacher P, Danneberg M, Masik S, Eckardt F, Weichelt S, et al. Training using virtual reality improves response behavior in karate kumite. 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Consent for publication 19. Anglin JM, Sugiyama T, Liew S-L. Visuomotor adaptation in head-mounted virtual reality versus conventional training. Sci Rep. 2017;7:45469. Written informed consent for the publication of the images used in this study was obtained from the individual depicted. 20. Taylor JA, Ivry RB. Context-dependent generalization. Front Hum Neurosci. 2013;7:171. Availability of data and materials The data used in the current study are available from the corresponding author on reasonable request. The data used in the current study are available from the corresponding author on reasonable request. 16. Iruthayarajah J, McIntyre A, Cotoi A, Macaluso S, Teasell R. The use of virtual reality for balance among individuals with chronic stroke: a systematic review and meta-analysis. Top Stroke Rehabil. 2017;24:68–79. Competing interests 21. Smith SM, Vela E. Environmental context-dependent memory: a review and meta-analysis. Psychon Bull Rev. 2001;8:203–20. 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https://openalex.org/W1558828517	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0132559&type=printable	English	null	Epinephrine Activation of the β2-Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells	PloS one	2,015	cc-by	10,923	RESEARCH ARTICLE Epinephrine Activation of the β2- Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells Nour Al-Sawalha1¤, Indira Pokkunuri1, Ozozoma Omoluabi2, Hosu Kim1, Vaidehi J. Thanawala1, Adrian Hernandez2, Richard A. Bond1,2, Brian J. Knoll1,2* Nour Al-Sawalha1¤, Indira Pokkunuri1, Ozozoma Omoluabi2, Hosu Kim1, Vaidehi J. Thanawala1, Adrian Hernandez2, Richard A. Bond1,2, Brian J. Knoll1,2* a11111 a11111 1 Department of Pharmacological and Pharmaceutical Sciences, University of Houston, 4800 Calhoun Road, Houston, Texas, 77204, United States of America, 2 Department of Biology and Biochemistry, University of Houston, 4800 Calhoun Road, Houston, Texas, 77204, United States of America ¤ Current address: Department of Clinical Pharmacy, College of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan * bknoll@Central.UH.edu ¤ Current address: Department of Clinical Pharmacy, College of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan * bknoll@Central.UH.edu Abstract Citation: Al-Sawalha N, Pokkunuri I, Omoluabi O, Kim H, Thanawala VJ, Hernandez A, et al. (2015) Epinephrine Activation of the β2-Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells. PLoS ONE 10(7): e0132559. doi:10.1371/journal.pone.0132559 Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a TH2 cytokine, IL-13 is sufficient to cause mucus hypersecretion in vivo and in vitro. Asthma therapy often utilizes β2-adrenoceptor (β2AR) agonists, which are effective acutely as bron- chodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether β2AR signaling in normal human airway epithelial (NHBE) cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI- 118,551, a preferential β2AR antagonist, but not by CGP-20712A, a preferential β1AR antagonist. Constitutive β2AR activity was not sufficient for IL-13 induced mucin production and β-agonist-induced signaling is required. A clinically important long-acting β-agonist, for- moterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcrip- tion. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125). Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL- 13. Our findings suggest that β2AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that β2AR-agonists may contribute to asthma exacer- bations by increasing mucin production via activation of β2ARs on epithelial cells. Editor: Shama Ahmad, University of Alabama at Birmingham, UNITED STATES Editor: Shama Ahmad, University of Alabama at Birmingham, UNITED STATES Received: July 29, 2014 Accepted: June 17, 2015 Published: July 10, 2015 Editor: Shama Ahmad, University of Alabama at Birmingham, UNITED STATES Received: July 29, 2014 Accepted: June 17, 2015 Published: July 10, 2015 Copyright: © 2015 Al-Sawalha et al. Abstract This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction agonists." At this point this patent has not been assigned and has no value or license agreement in place or pending issuance. RA Bond is also a minor shareholder in Invion, a company in which he was scientific co-founder. RA Bond sold off almost his entire stock ownership and now owns ~$10,000 worth of stock (as of 12 June 2015). RA Bond has no other role than shareholder with Invion, and is not on any board and has no consulting agreement. This does not alter the authors' adherence to PLOS ONE Editorial policies and criteria. Asthma is a chronic inflammatory disease characterized by airway hyperreactivity, subepithe- lial fibrosis, airway smooth muscle hyperplasia and mucous metaplasia [1]. Mucous metaplasia is an increase in the number of mucus-secreting goblet cells in the epithelium [2] that results in increased mucus synthesis and secretion. Excessive accumulation of airway mucus leads to the formation of mucous plugs that reduce the effective airway diameter and increase airway resis- tance. Patients who die of severe asthma attacks often exhibit goblet cell hyperplasia, mucus accumulation and large mucus plugs of unusual solidity due to high mucin content in their peripheral airways compared to asthmatic patients who did not die of acute attacks [3]. Allergic asthma has properties of a type I hypersensitivity, in which type 2 T-helper lympho- cytes and type 2 innate lymphoid cells contribute to produce a distinctive set of cytokines in the airways, including IL-5, IL-9 and IL-13 [4]. Although the allergic airway also contains diverse hematopoietic and parenchymal cells, and factors secreted by them, airway epithelial overexpression of IL-13 or airway instillation of IL-13 is sufficient to induce mucous metaplasia in mice [5, 6]. Airway epithelium is essential and sufficient for mucous metaplasia induced by IL-13, and this is dependent on the expression of STAT6 (which mediates the action of IL-13) in the epithelium [7]. The epithelium has been suggested by many studies to play an important role in the pathogenesis of asthma [7–10] as well as being a key contributor to the mucus plugs responsible for asthma mortality [11]. Sputum of asthmatic patients show elevated levels of IL- 13 and its presence is negatively associated with therapeutic responsiveness [12]. The MUC5AC gene encodes the major component of mucin in human airways, and induc- tion of MUC5AC transcription by IL-13 is observed in cultured human airway epithelium [10, 13]. Introduction However other factors also are required for MUC5AC transcription in these cells. Signal- ing through the EGF [10] and TGF-β2 [14] receptors is required for IL-13 to induce MUC5AC transcription, and in the promoter region of the gene there are binding sites for numerous diverse transcription factors [15], although none for STAT-6, suggesting the action of multiple intersecting and possibly indirect pathways. Recently, we found evidence for the involvement of β-adrenoceptor (β2AR) signaling in the pathogenesis of asthma. Pharmacologic or genetic ablation of β2AR signaling causes reductions in mucous metaplasia, airway cellularity and air- way hyperreactivity (AHR) in a murine asthma model [16, 17]. Thus, among the other path- ways already mentioned, some that are initiated or influenced by β2ARs may also be involved in the regulation of MUC5AC transcription. Due to the complexity of the signaling pathways that are involved in mediating mucus pro- duction, and the involvement of diverse cell types in whole animal models, we undertook to study human airway epithelial cells cultured in low concentrations of retinoic acid, conditions where mucin expression is normally minimal. We investigated the requirement for β2AR sig- naling in the transcription of the MUC5AC gene, the expression of MUC5AC protein and intracellular mucin accumulation in response to IL-13. In addition, we examined signaling components downstream of β2AR that may be required for this response. β2-Adrenoceptor Signaling Required for Mucin Production Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: BJK and RAB were supported by National Institutes of Health RO1A179236. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Brian J. Knoll is an Academic Editor for PLOS ONE. RA Bond is a co-inventor on a provisional patent application 2011, joint between MD Anderson and the University of Houston - "The steroid-sparing effects of betaadrenergic inverse 1 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 Immunoblotting NHBE cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glyceropho- sphate, 1 mM Na3VO4 and 1 μg/ml leupeptin (Cell Signaling, Danvers, MA) combined with protease inhibitor cocktail tablet (Roche Applied Sciences, Indianapolis, IN). Total protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific Inc, Rock- ford, IL), according to the manufacture’s protocols. Thirty micrograms of protein per lane were subjected to SDS-PAGE using 10% Tris-HCl gels (Bio-Rad Laboratories, Hercules, CA) and transblotted to PVDF membrane (EMD Millipore, Billerica, MA). Membranes were blocked in 3% BSA for 1 hour at room temperature and then incubated with antibodies against phospho-ERK1/2 (Cell Signaling, #4370S), phospho-p38 (Santa Cruz Biotechnology, #sc- 17852-R) or phospho-c-Jun (EMD Millipore, #06–659) overnight at 4°C followed by treatment for 1 hour with HRP conjugated secondary antibody. The protein bands were developed using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) according to manufacturer’s recommendations. A CCD camera (Fluorochem 8800) was used to collect the digital images and AlphaEase software (ProteinSimple, Santa Clara, CA) to quantify band den- sity. The membranes were then stripped and probed with GAPDH antibody (EMD Millipore, #MAB374)). The signal density of the phosphorylated proteins was normalized with that of GAPDH. Materials and Methods Cell Culture Normal human bronchial epithelial (NHBE) cells were obtained from Lonza (Walkersville, MD). The cells were seeded on Transwell-culture inserts (0.45 μm pore size) at 2 x 104 / cm2 and grown in 5% CO2/95% air at 37°C in differentiation medium: 50% bronchial epithelial basal media, 50% DMEM high glucose and supplemented with 30 μg/ml bovine pituitary extract, 0.5 μg/ml BSA, 0.5 μg/ml epinephrine, 50 μg/ml gentamycin, 50 ng/ml amphotericin B, 2 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production 0.5 ng/ml human EGF, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 7 ng/ml triiodothyronine, 10 μg/ml transferrin and 0.1 ng/ml retinoic acid The cells were cultured with epinephrine for ~8 days until they reached confluence, then the apical medium was removed and air-liquid interface (ALI) was established. The cells were then treated with 20 ng/ml of IL-13 combined with different antagonists or inhibitors. In some experiments, the cells were grown in the absence of epinephrine 72 hours before they reached ALI. Compound-related toxicity was assessed through the dryness of the apical surface of the cultured NHBE cells [18]. Cells grown in the absence or presence of epinephrine formed tight junctions that produced equal increases in transepithelial electrical resistance (TEER) suggesting similar epithelial barrier function, consistent with previously published findings [19]. The formation of tight junctions was also confirmed by the presence of ZO-1 on the cell peripheries by immunofluorescence (data not shown). Real-Time PCR Analysis of MUC5AC Expression Total RNA was extracted from cells using Trizol according to manufacturer’s protocol. cDNA was generated from 5 μg of total RNA and MUC5AC and 18S mRNA were quantified using the Taqman Gene Expression Assay (Applied Biosystems, Grand Island, NY) and analyzed by real time quantitative PCR (ABI PRISM 7000 Sequence Detection System, Applied Biosys- tems). The threshold cycles (Ct) for MUC5AC of treated groups was compared to the control groups and normalized to 18S. Relative MUC5AC expression was calculated using Delta-Delta CT method. Periodic Acid Fluorescent Schiff’s (PAFS) Staining The apical surfaces of NHBE cells were washed with PBS, fixed in 4% paraformaldehyde (PFA) and permeabilized with Triton X-100. The inserts were stained with PAFS as described previ- ously [20]. Red fluorescence of mucin was detected when the slides were excited at 380–580 nm and observed at 600–650 nm while the nucleic acid and cytoplasm of the cells fluorescence green when observed at a lower wavelength (380–500 nm and 450–475 nm excitation and 3 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production emission wavelengths respectively) [20]. Images were captured using an Olympus DUS spin- ning disc confocal microscope maintained in the College of Pharmacy Imaging Core. To cor- rect for insert background, empty inserts were stained and used as a negative control. To maintain consistency in subsequent image analysis, we used the same channel-specific thresh- old when capturing all images. The ratio of integrated mucin density to integrated nucleic acid and cytoplasm density was calculated using Image J (NIH). Statistical Analysis Data are presented as means ± SEM. All experiments were done with NHBECs from 3 donors (N = 3). One-way ANOVA followed by Tukey's multicomparison test for multiple group statis- tical analysis was performed using GraphPad Prism 4 software. p<0.05 was considered statisti- cally significant. Immunofluorescence Labeling for Mucin 5AC The apical surfaces of NHBE cells were washed with PBS, fixed in 4% paraformaldehyde (PFA) and permeabilized with Triton X-100. The inserts were incubated for 15 minutes with 10% normal goat serum at room temperature followed by Mucin 5AC Antibody (H-160, Santa Cruz Biotechnology) [21] overnight at 4°C. After washing the inserts with PBS, they were incu- bated with Alexa 594 goat anti-rabbit secondary antibody for 1 hour at room temperature. DAPI, at final concentration of 1 μg/ml, was used to counterstain the nuclei. Slides incubated with primary antibody diluents were used as negative controls. Images were captured by confo- cal microscopy and the same channel-specific threshold was maintained when capturing the images. The ratio of integrated mucin 5AC density of each group to the integrated mucin 5AC density of the corresponding control group was calculated using Image J software (NIH). Results B: Representative images of immunofluorescence with a rabbit antibody against human mucin 5AC (red) (scale bar = 100 μm). The Transwell membranes were incubated with DAPI to counterstain the nuclei (blue). Incubation with antibody diluent showed no red fluorescence (data not shown). The ratio of integrated fluorescence density of each group to the integrated mucin 5AC density of the corresponding control group Fig 1. Epinephrine is required for mucin production in response to IL-13 in NH were grown in the presence or absence of 3 μM epinephrine. At ALI, the cells were t 13 for 14 days, total RNA was harvested and then MUC5AC transcripts were measu are presented as fold change compared to the corresponding treatment control (in t Representative images of immunofluorescence with a rabbit antibody against huma bar = 100 μm). The Transwell membranes were incubated with DAPI to counterstain Incubation with antibody diluent showed no red fluorescence (data not shown). The fluorescence density of each group to the integrated mucin 5AC density of the corre Fig 1. Epinephrine is required for mucin production in resp Fig 1. Epinephrine is required for mucin production in response to IL-13 in NHBE cells. A: NHBE cells were grown in the presence or absence of 3 μM epinephrine. At ALI, the cells were treated with 20 ng/ml IL- 13 for 14 days, total RNA was harvested and then MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to the corresponding treatment control (in the absence of IL13). B: Representative images of immunofluorescence with a rabbit antibody against human mucin 5AC (red) (scale bar = 100 μm). The Transwell membranes were incubated with DAPI to counterstain the nuclei (blue). Incubation with antibody diluent showed no red fluorescence (data not shown). The ratio of integrated fluorescence density of each group to the integrated mucin 5AC density of the corresponding control group Fig 1. Epinephrine is required for mucin production in response to IL-13 in NHBE cells. A: NHBE cells were grown in the presence or absence of 3 μM epinephrine. At ALI, the cells were treated with 20 ng/ml IL- 13 for 14 days, total RNA was harvested and then MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to the corresponding treatment control (in the absence of IL13). Results To determine whether epinephrine increases mucus production in NHBE cells, we cultured them for 72 hours before reaching ALI in a medium that lacks epinephrine and then stimulated with 20 ng/ml of IL-13 for 14 days, also in the absence of epinephrine. Under these conditions, IL-13 did not increase the expression of MUC5AC mRNA, and in the presence of epinephrine alone, MUC5AC mRNA was scarcely detectable above baseline. But with IL-13 treatment in the presence of epinephrine, the MUC5AC expression level was increased by ~15 fold (Fig 1A). Similar results were obtained with cells grown in the presence of 50 nM retinoic acid (S1 Fig). To correlate mRNA expression levels with intracellular mucin 5AC accumulation and mucin glycoprotein, immunofluorescence with anti-mucin 5AC antibody and PAFS staining were used respectively. Epinephrine was required for IL-13-induced increases in the intracellular mucin 5AC and total mucin glycoproteins (Fig 1B and 1C). To determine if a therapeutic β-agonist showed an effect similar to epinephrine, we substi- tuted formoterol. NHBE cells were cultivated with physiological levels of RA to more closely mimic the in vivo state. Under these conditions, IL-13 again did not significantly increase MUC5AC transcription (though levels were slightly higher). However the addition of formo- terol increased MUC5Ac transcripts to a similar degree as epinephrine (S2 Fig). To determine the βAR subtype involved in MUC5AC expression in response to IL-13 in the presence of epinephrine, NHBE cells were incubated with either a preferential β2AR antagonist (1 μM ICI-118,551) or a preferential β1AR antagonist (3 μM CGP-20712A). ICI-118,551 completely abolished (>99%) IL-13 induced MUC5AC expression (0.039 ± 0.038 fold vs 15.99 ± 1.48 fold increase by IL-13. p<0.05). On the other hand, CGP-20712A did not affect the MUC5AC expression level (14.75 ± 0.96 fold vs 15.99 ± 1.48 fold increase by IL-13, 4 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production Fig 1. Epinephrine is required for mucin production in response to IL-13 in NHBE cells. A: NHBE cells were grown in the presence or absence of 3 μM epinephrine. At ALI, the cells were treated with 20 ng/ml IL- 13 for 14 days, total RNA was harvested and then MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to the corresponding treatment control (in the absence of IL13). Results B: Representative images of immunofluorescence with a rabbit antibody against human mucin 5AC (red) (scale bar = 100 μm). The Transwell membranes were incubated with DAPI to counterstain the nuclei (blue). Incubation with antibody diluent showed no red fluorescence (data not shown). The ratio of integrated fluorescence density of each group to the integrated mucin 5AC density of the corresponding control group 5 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production was calculated and expressed as fold change. C: PAFS staining of NHBE cells to quantify total intracellular mucin glycoproteins. Representative images are shown. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to the corresponding control cells (in the absence of IL-13 treatment). Data are presented as means ± SEM from three donors. , † and ¥ indicate p<0.05 significance as compared to + epinephrine,−epinephrine and −epinephrine + IL-13 treated cells respectively. doi:10.1371/journal.pone.0132559.g001 doi:10.1371/journal.pone.0132559.g001 p>0.05) (Fig 2A). CGP-20712A did not affect the intracellular mucin levels induced by IL-13 while ICI-118,551 brought the levels back to baseline (Fig 2B and 2C; for representative images see S3A and S3B Fig). We next asked if the increased MUC5AC expression in response to IL-13 is due to agonist induced or constitutive β2AR signaling. NHBE cells were treated with 10 μM nadolol, a non- selective βAR ligand with inverse agonist activity at β2ARs that blocks both constitutive and agonist-induced receptor activity, or with 10 μM alprenolol, a non-selective βAR antagonist with no inverse agonist activity, for 14 days in combination with IL-13 and in the presence of epinephrine. Treatment with nadolol reduced IL-13 induced MUC5AC expression (3.36 ± 4.10 fold vs 25.37 ± 16.30 fold increase by IL-13, p<0.05), intracellular mucin 5AC protein and mucin content (Fig 3A, 3B and 3C; for representative images see S4A and S4B Fig). Treatment with alprenolol reduced IL-13-induced MUC5AC expression to a similar extent (3.19 ± 3.73 fold vs 25.37 ± 16.30 fold increase by IL-13, p<0.05) and also reduced intracellular mucin 5AC and mucin content (Fig 3A, 3B and 3C, and S4A and S4B Fig for representative images). g g p g To investigate the role of mitogen activated protein kinases (MAPKs), we examined their activation using antibodies specific for phosphorylated (activated) MAPKs. Results In the absence of epinephrine, IL-13 did not affect the phosphorylation of ERK1/2 (Fig 4A), c-Jun (Fig 4B) or p38 (Fig 4C) as compared to their corresponding controls. When epinephrine was included in the medium, IL-13 induced an approximately 3-fold increase in the phosphorylation of ERK1/ 2 and c-Jun when compared to their corresponding controls (Fig 4A and 4B). However, phos- phorylation of p38 was unaffected by IL-13 even in the presence of epinephrine (Fig 4C). Next, we treated NHBE cells with 3 μM FR180204, SP600125 or SB203580 (inhibitors of ERK1/2, JNK and p38 respectively) in combination with IL-13 and epinephrine for 14 days. All three MAPKs inhibitors significantly reduced MUC5AC gene expression (15.18 ± 3.76 fold increase by IL-13 vs 1.82 ± 0.68, 0.77 ± 0.39 and 0.80 ±0.65 fold by FR180204, SP600125 and SB203580 respectively) (Fig 4D). While all MAPK inhibitors reduced the intracellular mucin 5AC protein (see Fig 4E and S5A Fig for representative images), only FR180204 and SP600125 reduced intracellular mucin content when compared to IL-13 treated cells (see Fig 4F and S5B Fig for representative images). representative images). To explore a possible role for PKA in the induction of MUC5AC, we treated NHBE cells with a competitive cAMP analogue, Rp-cAMPS, for 14 days in combination with IL-13 and epi- nephrine. Rp-cAMPS did not significantly reduce the levels of MUC5AC expression at 50 μM (5.97 ± 4.29 fold vs 12.50 ± 5.38 fold increase by IL-13, p>0.05,) while at 100 μM, there was a significant reduction (2.35 ± 1.63 fold vs 12.50 ± 5.38 fold increase by IL-13, p<0.05)(Fig 5A). The intracellular mucin 5AC protein level was significantly reduced when the cells were treated with 100 μM Rp-cAMPS but not at 50 μM, while mucin glycoproteins levels were reduced at both concentrations (see Fig 5B and 5C, and S6A and S6B Fig for representative images). To provide more evidence for a role for cAMP in mucin production in response to IL-13, we treated cells with 10 μM forskolin combined with 100 μM 3-isobutyl-l-methylxan-thine (IBMX), in the absence of epinephrine. This treatment caused a dramatic increase in MUC5AC expres- sion (75.73 ± 66.59 fold vs 0.56 ± 0.40 fold increase by IL-13, p<0.05) (Fig 6A) when the cells were treated with IL-13. Results The same trend was also observed at the level of intracellular mucin To explore a possible role for PKA in the induction of MUC5AC, we treated NHBE cells with a competitive cAMP analogue, Rp-cAMPS, for 14 days in combination with IL-13 and epi- nephrine. Rp-cAMPS did not significantly reduce the levels of MUC5AC expression at 50 μM (5.97 ± 4.29 fold vs 12.50 ± 5.38 fold increase by IL-13, p>0.05,) while at 100 μM, there was a significant reduction (2.35 ± 1.63 fold vs 12.50 ± 5.38 fold increase by IL-13, p<0.05)(Fig 5A). The intracellular mucin 5AC protein level was significantly reduced when the cells were treated with 100 μM Rp-cAMPS but not at 50 μM, while mucin glycoproteins levels were reduced at both concentrations (see Fig 5B and 5C, and S6A and S6B Fig for representative images). To provide more evidence for a role for cAMP in mucin production in response to IL-13, we treated cells with 10 μM forskolin combined with 100 μM 3-isobutyl-l-methylxan-thine (IBMX), in the absence of epinephrine. This treatment caused a dramatic increase in MUC5AC expres- sion (75.73 ± 66.59 fold vs 0.56 ± 0.40 fold increase by IL-13, p<0.05) (Fig 6A) when the cells were treated with IL-13. The same trend was also observed at the level of intracellular mucin PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 6 / 18 β2-Adrenoceptor Signaling Required for Mucin Production Fig 2. β2ARs are required for mucin production in response to IL-13 in NHBE cells. NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ml IL-13 in combination with 3 μM CGP-20712A (a preferential β1AR antagonist) or 1 μM ICI-118,551 (a preferential β2AR antagonist) for 14 days. A: MUC5AC transcripts were quantified from extracted total RNA by qRT-PCR. Data are presented as fold change compared to cells grown in the presence of epinephrine only. B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S3A Fig). C: Quantification of intracellular mucin glycoproteins in response to different ligands. Discussion IL-13 plays an important role in the mucus over-production characteristic of bronchial asthma, and MUC5AC is the major mucin gene that is overexpressed by airway epithelium in asthmatic patients [22, 23]. We present evidence that signaling through β2AR increases IL-13 induced mucin production in human bronchial epithelium cultured in low RA concentrations. Most previous studies using these cells included epinephrine in the medium (e.g., [10, 18, 24, 25]) and to our knowledge, this is the first study showing an important role for β2AR signaling in the IL-13 mediated induction of MUC5AC expression and mucin content in NHBE cells. These results are consistent with our earlier findings that β2AR signaling is required for mucous metaplasia in the airways of mice [16, 17, 26]. The clinical relevance of these findings is shown by the ability of formoterol, a commonly used long-acting β-agonist bronchodilator, to mimic epinephrine in the stimulation of MUC5AC transcripts by IL-13 (S2 Fig). β2AR is the principal subtype present in human airway epithelium [27]. To verify that this subtype mediates the effects of IL-13 in cultured NHBE cells, we used preferential βAR antago- nists. The selectivity of ICI-118,551 toward β2AR, and of CGP 20712A toward β1AR is least 500 fold or greater [28]. Treating NHBE cells with the preferential β2AR antagonist completely abolished MUC5AC expression in response to IL-13 and epinephrine. In contrast, the prefer- ential β1AR antagonist did not alter MUC5AC expression levels under the same conditions. Intracellular mucin 5AC protein levels and mucin glycoprotein were not induced by IL-13 + epinephrine treatment in the presence of ICI-118,551 while CGP 20712A had no effect, par- alleling the MUC5AC mRNA expression results (Fig 2A–2C). Our in vitro results are also con- sistent with our recent animal data where genetic ablation of β2ARs in mice resulted in attenuation of mucous metaplasia in an allergic murine model of asthma [16, 26]. p g β2AR signaling can proceed from agonist-activated receptors and from constitutively active receptors [29]. Cells that were grown in the absence of epinephrine showed no response to IL- 13 treatment in terms of MUC5AC expression, mucin 5AC protein levels and mucin content while cells grown in the presence of epinephrine showed an increase in all three parameters (Fig 1A–1C), suggesting that agonist-induced β2AR activation is necessary. Results The ratio of mucin integrated density and nucleic acid/ cytoplasm integrated density was calculated and the data presented as fold change compared to control Fig 2. β2ARs are required for mucin production in response to IL-13 in NHBE cells. NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ml IL-13 in combination with 3 μM CGP-20712A (a preferential β1AR antagonist) or 1 μM ICI-118,551 (a preferential β2AR antagonist) for 14 days. A: MUC5AC transcripts were quantified from extracted total RNA by qRT-PCR. Data are presented as fold change compared to cells grown in the presence of epinephrine only. B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S3A Fig). C: Quantification of intracellular mucin glycoproteins in response to different ligands. The ratio of mucin integrated density and nucleic acid/ cytoplasm integrated density was calculated and the data presented as fold change compared to control 7 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production cells. See the supplement for the representative images (S3B Fig). Data are presented as means ± SEM from three donors. , # and @ indicate p<0.05 significance as compared to + epinephrine, + epinephrine + IL-13 and + epinephrine + IL-13 + CGP-20721A treated cells respectively. doi:10.1371/journal.pone.0132559.g002 cells. See the supplement for the representative images (S3B Fig). Data are presented as means ± SEM from three donors. , # and @ indicate p<0.05 significance as compared to + epinephrine, + epinephrine + IL-13 and + epinephrine + IL-13 + CGP-20721A treated cells respectively. doi:10.1371/journal.pone.0132559.g002 5AC protein accumulation and mucin content of NHBE cells (Fig 6B and 6C; for representative images see S7A and S7B Fig). PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 Discussion To further distin- guish between constitutive versus agonist induced β2AR signaling, we treated NHBE cells with nadolol or alprenolol in combination with IL-13 in the presence of epinephrine. Nadolol is a non-selective βAR ligand that has full inverse agonist activity at β2ARs, thus it blocks both induced and constitutive β2AR signaling. Alprenolol lacks inverse agonist activity [29] but has weak β2AR agonist activity [30] and behaves as an antagonist in the presence of epinephrine, thus preserving constitutive β2AR signaling. Both nadolol and alprenolol blocked the effect of IL-13 on MUC5AC mRNA expression, protein levels and intracellular mucin content to simi- lar extents (Fig 3A–3C). Thus, we provide evidence that constitutive β2AR receptor activity is not sufficient to drive the increase in mucin in response to IL-13 in human bronchial epithelial cells. Again, our in vitro results are also consistent with our recent mouse data where genetic or pharmacological depletion of epinephrine in mice resulted in attenuation of mucous metapla- sia in an antigen-driven murine model of asthma [16, 17, 26] 8 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production Fig 3. Agonist induced β2AR signaling is required for mucin production in response to IL-13 in NHBE cells. NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ ml IL-13 in combination with 10 μM nadolol (a non-selective inverse agonist of βARs) or 10 μM alprenolol (a non-selective β2AR blocker with no inverse agonist activity) for 14 days. A: MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to cells grown in the presence of epinephrine only. B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated Fig 3. Agonist induced β2AR signaling is required for mucin production in response to IL-13 in NHBE cells. NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ ml IL-13 in combination with 10 μM nadolol (a non-selective inverse agonist of βARs) or 10 μM alprenolol (a non-selective β2AR blocker with no inverse agonist activity) for 14 days. A: MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to cells grown in the presence of epinephrine only. B: Quantification of intracellular mucin 5AC content. Discussion The ratio of mucin 5AC integrated PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 9 / 18 β2-Adrenoceptor Signaling Required for Mucin Production density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S4A Fig). C: Quantification of intracellular mucin glycoproteins in response to different ligands. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S4B Fig). Data are presented as means ± SEM from three donors. and # indicate p<0.05 significance as compared to + epinephrine and + epinephrine + IL-13 treated cells respectively. We have begun determining the basis for the interaction between the β2AR and IL-13 sig- naling pathways. Since β2AR can activate MAPKs through cAMP-dependent and independent pathways, we tested the role of these kinases in the expression of MUC5AC. Since cells that were grown in the absence of epinephrine did not show induction of MUC5AC expression by IL-13, we examined only cells grown in the presence of epinephrine. All three families of MAPKs, JNKs, ERKs and p38, are known to be involved in the production of cytokine and che- mokines by airway epithelium [31]. Phosphorylated p38 and ERK1/2 are detectable in the epi- thelium of asthmatic patients and the level of phosphorylation correlates with asthma severity [32]. In NHBE cells, IL-13 induced the phosphorylation of ERK1/2 and the ERK1/2 selective inhibitor, FR180204, attenuated MUC5AC expression, mucin 5AC protein levels and mucin content in response to IL-13 (Fig 4A, 4D–4F). A selective MEK 1/2 inhibitor, U0126, also Fig 4. MAPK signaling is required for mucin production in response to IL-13 in NHBE cells. A-C. NHBE cells were grown in the presence or absence of epinephrine or IL-13 as indicated for 14 days after ALI before harvesting for total proteins. Immunoblots were performed using antibodies to the indicated phosphorylated MAP-kinases. The signal densities of the phosphorylated proteins were normalized to GAPDH protein density and the relative intensities were reported as the degree of activation of the protein. The data presented as fold change compared to the corresponding control cells. Discussion D-F: NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ml IL-13 in combination with 3 μM FR180204 (ERK1/2 inhibitor), 3 μM SP600125 (JNK inhibitor) or 3 μM SB203580 (p38 inhibitor) for 14 days. D: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the presence of epinephrine only. E: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S5A Fig). F: Quantification of total intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S5B Fig) Data are presented as means ± SEM from three donors. * and # indicate p<0.05 significance as compared to +epinephrine and + epinephrine + IL-13 treated cells respectively. d i 10 1371/j l 0132559 004 Fig 4. MAPK signaling is required for mucin production in response to IL-13 in NHBE cells. A-C. NHBE cells w Fig 4. MAPK signaling is required for mucin production in response to IL-13 in NHBE cells. A-C. NHBE cells were grown in the presence or absence of epinephrine or IL-13 as indicated for 14 days after ALI before harvesting for total proteins. Immunoblots were performed using antibodies to the indicated phosphorylated MAP-kinases. The signal densities of the phosphorylated proteins were normalized to GAPDH protein density and the relative intensities were reported as the degree of activation of the protein. The data presented as fold change compared to the corresponding control cells. D-F: NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ml IL-13 in combination with 3 μM FR180204 (ERK1/2 inhibitor), 3 μM SP600125 (JNK inhibitor) or 3 μM SB203580 (p38 inhibitor) for 14 days. D: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the presence of epinephrine only. E: Quantification of intracellular mucin 5AC content. doi:10.1371/journal.pone.0132559.g004 Discussion doi:10.1371/journal.pone.0132559.g004 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 10 / 18 β2-Adrenoceptor Signaling Required for Mucin Production Fig 5. Inhibiting PKA signaling reduced mucin production in response to IL-13 in NHBE cells. Cells were grown in the presence of 3 μM epinephrine, then after ALI, they were treated with 20 ng/ml IL-13 in combination with 50 μM or 100 μM Rp-cAMP (cAMP-dependent protein kinase inhibitor) for 14 days. A: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of inhibitor. B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S6A Fig). C: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S6B Fig). Data are presented as means ± SEM from three donors. * and # indicate p<0.05 significance as compared to + epinephrine and + epinephrine + IL-13 treated cells respectively. d i 10 1371/j l 0132559 005 Fig 5. Inhibiting PKA signaling reduced mucin production in response to IL-13 in NHBE cells. Cells were grown in the presence of 3 μM epinephrine, then after ALI, they were treated with 20 ng/ml IL-13 in combination with 50 μM or 100 μM Rp-cAMP (cAMP-dependent protein kinase inhibitor) for 14 days. A: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of inhibitor. B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S6A Fig). C: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S6B Fig). Data are presented as means ± SEM from three donors. Discussion The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S5A Fig). F: Quantification of total intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S5B Fig) Data are presented as means ± SEM from three donors. * and # indicate p<0.05 significance as compared to +epinephrine and + epinephrine + IL-13 treated cells respectively. Fig 4. MAPK signaling is required for mucin production in response to IL-13 in NHBE cells. A-C. NHBE cells were grown in the presence or absence of epinephrine or IL-13 as indicated for 14 days after ALI before harvesting for total proteins. Immunoblots were performed using antibodies to the indicated phosphorylated MAP-kinases. The signal densities of the phosphorylated proteins were normalized to GAPDH protein density and the relative intensities were reported as the degree of activation of the protein. The data presented as fold change compared to the corresponding control cells. D-F: NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ml IL-13 in combination with 3 μM FR180204 (ERK1/2 inhibitor), 3 μM SP600125 (JNK inhibitor) or 3 μM SB203580 (p38 inhibitor) for 14 days. D: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the presence of epinephrine only. E: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S5A Fig). F: Quantification of total intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S5B Fig) Data are presented as means ± SEM from three donors. * and # indicate p<0.05 significance as compared to +epinephrine and + epinephrine + IL-13 treated cells respectively. doi:10.1371/journal.pone.0132559.g005 doi:10.1371/journal.pone.0132559.g006 β2-Adrenoceptor Signaling Required for Mucin Production reduced MUC5AC expression in NHBE cells in response to IL-13 (Nguyen et al, in prepara- tion). These results are consistent with other similar studies in NHBE cells [18]. JNK is involved in regulating pro-inflammatory gene expression and remodeling in airway diseases [33]. In NHBE cells, IL-13 induced the phosphorylation of c-Jun, a distinct JNK down- stream target, and a JNK inhibitor, SP600125, inhibited MUC5AC expression and mucin con- tent response to IL-13 (Fig 4B, 4D–4F). Consistent with this finding, attenuation of the asthma phenotype, including mucous metaplasia, by SP600125 has been shown in allergen driven murine model of asthma [34]. The other MAPK that we examined, p38, has four known isoforms: α, β, γ and δ [31]. The transcript levels of the α and β isoforms in the human lung is higher than that of other p38 iso- forms of [35]. Inhaled p38α antisense oligonucleotide attenuates mucus production in IL-13 trangenic mice [36]. We did not observe an increase in p38 phosphorylation in response to IL- 13 (Fig 4C), however a selective inhibitor of both α and β isoforms [37], SB203580, reduced lev- els of MUC5AC expression and mucin 5AC content induced by IL-13 and epinephrine (Fig 4D–4F). These data provide evidence for the involvement of p38 in IL-13 induced mucous metaplasia, consisent with previous studies [18, 38]. Even though p38 phosphorylation was not affected by IL-13, basal levels of p38 activation may still be required for MUC5AC expression and mucin 5AC production, perhaps by integrating signals from the β2- and IL-13 receptors. There was no significant decrease in total mucin content after inhibiting p38, perhaps because mucin 5AC production decreases while the production of other mucin glycoproteins increases. All of these MAPKs (ERK1/2, JNK and p38) can be activated by β2AR via the Gs-adenylyl cyclase pathway, and by G protein-independent, β-arrestin2-dependent pathways [39]. MAPKs then act through downstream components that lead to the activation or translocation to the nucleus of transcriptional factors that act upon the MUC5AC and other genes [40]. p p g [ ] To asses the possible involvement of cAMP mediated mechanisms, we used the Rp-isomer of adenosine-30, 50-cyclic monophosphorothioate (Rp-cAMPS). Rp-cAMPS binds to PKA pre- venting its activation by cAMP [41]. PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 Discussion * and # indicate p<0.05 significance as compared to + epinephrine and + epinephrine + IL-13 treated cells respectively. 11 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production Fig 6. cAMP potentiates mucin production in response to IL-13 in NHBE cells. Cells were grown in the absence of epinephrine, then at ALI, they were incubated with or without 20 ng/ml IL-13 with or without 10 μM forskolin and 100 μM IBMX for 14 days. A: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of IL-13, IBMX and forskolin (control cells) B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S7A Fig). C: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/ cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S7B Fig). Data are presented as means ± SEM from three donors. † and ¥ indicate p<0.05 significance as compared to−epinephrine and −epinephrine + IL-13 treated cells respectively. doi:10.1371/journal.pone.0132559.g006 12 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production phosphorylated upon treatment with IL-13 + epinephrine. It may also be suggested that cAMP is needed for p38 to integrate signals from the β2- and IL-13 receptors. How do β2AR and IL-13 signaling interact? Due to the multiplicity of pathways proceeding from each receptor, it is not possible at present to firmly identify nodes of cross-talk. However, one potential factor could be an isoform of phosphoinositide 3-kinase (PI3K). Mice deficient in PI3Kγ show reductions in most indices of airway inflammation in OVA-sensitized and chal- lenged mice, though there was disagreement over effects on mucin production [45, 46]. Similar results were obtained, including decreases in mucin production, after treatment of mouse air- ways with inhibitors of PI3Kγ or δ [47–50]. Studies in NHBE cells showed that pharmacologic inhibition of PI3Kγ reduces IL-13 induced increases in goblet cell density [18]. Possibly, β2AR could amplify the IL-13 induction of PI3K isoforms by way of Gi [51] or Gβ/γ[52]. An argu- ment also could also be made for p38, which by being constitutive in activity in this system, might therefore be a key transducer in more than one signaling pathway. These possibilities are currently under examination. In the experiments reported here, only IL-13 and β2AR ligands were manipulated, and this reductionist approach does not take into account numerous other cytokines, chemokines and hormones present in the allergic airways. Also, leucocytes and other lung parenchymal cell types are absent from NHBE cultures. Nevertheless, the requirement for β2AR signaling in the induction of MUC5AC by IL-13 shown here is consistent with results we have obtained previ- ously with β2AR knockout mice, mice chronically treated with β blockers, and mice deficient in epinephrine [16, 17, 26]. A further limitation regards possible off target effects from the use of chemical inhibitors and activators. Future studies utilizing more specific genetic approaches will be needed to delineate the precise signaling pathways involved. In particular, a role for β- arrestin2 cannot be excluded from the present data, and information from in vivo studies sug- gests that there is an involvement of β-arrestin2 in the pathogenesis of asthma [53, 54]. In conclusion, our results reveal an important role for β2AR signaling in mediating mucus production in response to IL-13 in NHBE cells. Constitutive β2AR activity alone is not suffi- cient to mediate this effect and it requires agonist activation of the receptor. Moreover, the three major MAPK signaling molecules (ERK1/2, JNK and p38) play a role in mediating the effects of IL-13 in NHBE cells in the presence of epinephrine. Our data also suggests that a cAMP activated signaling cascade may be involved in mediating the inflammatory effect of IL- 13. The present report supports the notion that use of selective β2AR antagonists could be of value in the treatment of mucus overproduction in asthma and other similar disorders such as COPD [55]. MUC5AC expression, intracellular mucin 5AC protein levels and mucin content induced by IL-13 and epinephrine were not significantly inhibited by 50 μM Rp-cAMPS (IC50 ~10μM for inhibiting PKA in vitro), but were inhibited by 100 μM Rp-cAMPS (Fig 5A–5C). We then examined the effects of raising cAMP levels by treating NHBE cells with forskolin, a direct activator of adenylate cyclase, combined with the non-spe- cific phosphodiesterase inhibitor (PDE), IBMX. This treatment resulted in significant increases in MUC5AC transcripts in response to IL-13 (Fig 6). Taken together the data support a role of cAMP in regulating MUC5AC transcription as induced by IL-13. However, this data must be taken in the context of the high concentration of Rp-cAMPS required for the inhibition of MUC5AC expression, and that we used a compound (forskolin) that causes a ‘global’ intracel- lular increase in the activity of all adenylate cyclases, and an inhibitor of all PDE subtypes (IBMX) that regulate the breakdown of cAMP. Given the emergence of of data demonstrating distinct subcellular compartmentalization of cAMP associated with specific PDE subtypes [42] we cannot conclusively assert that epinephrine-induced cAMP increases cause the induction of MUC5AC. There are also studies suggesting that, at least with prolonged agonist exposures, other Gs-coupled receptors such as the adenosine A2B receptor can enhance lung injury and damage epithelial cell integrity [43, 44]. While our experiments could have eliminated a role for cAMP mediating mucin production, the data instead only suggest a role, and additional experiments using more refined pharmacologic tools that are PDE subtype specific, and genetic approaches will be needed to further investigate the possible role of β2AR-mediated cAMP increases in regulating mucin production. Given that MAP-kinases can be activated by cAMP mediated mechanisms, it would be interesting to test whether the effect of forskolin + IBMX is negated by inhibition of a MAP kinase, such as ERK or JNK, each of which show increased PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 13 / 18 Supporting Information S1 Fig. Epinephrine is required for mucin production in response to IL-13 in NHBE cell grown with 50 nM retinoic acid. NHBE cells were grown in the presence or absence of 3 μM epinephrine. At ALI, the cells were treated with 20 ng/ml IL-13 for 14 days, total RNA was har- vested and then MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to the corresponding treatment control (in the absence of IL13). , indicates p<0.05 significance as compared to + epinephrine,—epinephrine and −epinephrine + IL-13 treated cells respectively. N = 3. (PNG) S2 Fig. Formoterol also potentiates mucin production in response to IL-13. NHBE cells were cultured as described in S1 Fig, except that 10 nM formoterol was used in place of epi- nephrine. (PNG) S2 Fig. Formoterol also potentiates mucin production in response to IL-13. NHBE cells were cultured as described in S1 Fig, except that 10 nM formoterol was used in place of epi- nephrine. Acknowledgments The authors thank Gloria Forkuo for reviewing the manuscript. S2 Fig. Formoterol also potentiates mucin production in response to IL-13. NHBE cells were cultured as described in S1 Fig, except that 10 nM formoterol was used in place of epi- nephrine. 14 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0132559 July 10, 2015 β2-Adrenoceptor Signaling Required for Mucin Production S3 Fig. β2ARs are required for mucin production in response to IL-13 in NHBE cells. Rep- resentative images of data quantified in Fig 2. A: Intracellular MUC5AC content. B: Intracellu- lar mucin glycoproteins. (PNG) S4 Fig. Agonist induced β2AR signaling is required for mucin production in response to IL-13 in NHBE cells. Representative images of data quantified in Fig 3. A: Intracellular MUC5AC content. B: Intracellular mucin glycoproteins. (PNG) S5 Fig. MAPK signaling is required for mucin production in response to IL-13 in NHBE cells. Representative images of data quantified in Fig 4. A: Intracellular MUC5AC content. B: Intracellular mucin glycoproteins. (PNG) S6 Fig. Inhibiting PKA signaling reduced mucin production in response to IL-13 in NHBE cells. Representative images of data quantified in Fig 5. A: Intracellular MUC5AC content. B: Intracellular mucin glycoproteins. (PNG) S7 Fig. cAMP potentiates mucin production in response to IL-13 in NHBE cells. Represen- tative images of data quantified in Fig 6. A: Intracellular MUC5AC content. B: Intracellular mucin glycoproteins. (PNG) Conceived and designed the experiments: BJK RAB NS. Performed the experiments: NS IP AH OO HK. Analyzed the data: NS IP RAB BJK VT. Wrote the paper: NS VT RAB BJK. Conceived and designed the experiments: BJK RAB NS. Performed the experiments: NS IP AH OO HK. Analyzed the data: NS IP RAB BJK VT. Wrote the paper: NS VT RAB BJK. Author Contributions Conceived and designed the experiments: BJK RAB NS. Performed the experiments: NS IP AH OO HK. Analyzed the data: NS IP RAB BJK VT. Wrote the paper: NS VT RAB BJK. 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https://openalex.org/W2135289302	https://publications.goettingen-research-online.de/bitstream/2/44975/2/aa3829%5b1%5d.pdf	English	null	2MASS J0516288+260738: Discovery of the first eclipsing late K + Brown dwarf binary system?	Astronomy & astrophysics	2,003	cc-by	11,652	Received 8 April 2003 / Accepted 4 August 2003 Received 8 April 2003 / Accepted 4 August 2003 Abstract. We report the discovery of a new eclipsing system less than one arcminute south of the pulsating DB white dwarf KUV 05134+2605. The object could be identiﬁed with the point source 2MASS J0516288+260738 published by the Two Micron All Sky Survey. We present and discuss the ﬁrst light curves as well as some additional colour and spectral information. The eclipse period of the system is 1.29 d, and, assuming this to be identical to the orbital period, the best light curve solution yields a mass ratio of m2/m1 = 0.11, a radius ratio of r2/r1 ≈1 and an inclination of 74◦. The spectral anaylsis results in a Teﬀ= 4200 K for the primary. On this basis, we suggest that the new system probably consists of a late K + Brown dwarf (which would imply a system considerably younger than ≈0.01 Gyr to have r2/r1 ≈1), and outline possible future observations. Key words. ephemerides – stars: variables: general – stars: binaries: eclipsing – stars: low-mass, brown dwarfs – stars: individual: 2MASS J0516288+260738 and the Infrared Processing and Analysis Center / California Institute of Technology, funded by the National Aeronautics and Space Administration and the National Science Foundation. ⋆This paper uses observations made at the Bohyunsan Optical Astronomy Observatory of Korea Astronomy Observatory, at the South African Astronomical Observatory (SAAO), at the 0.9 m tele- scope at Kitt Peak National Observatory recommissioned by the Southeastern Association for Research in Astronomy (SARA), at Gunma Astronomical Observatory established by Gunma prefecture, Japan, at the Florence and George Wise Observatory, operated by the Tel-Aviv University, Israel and at Piszk´estet˝o, the mountain sta- tion of Konkoly Observatory of the Hungarian Academy of Science, Hungary. ⋆⋆⋆The Digitized Sky Survey was produced at the Space Telescope Science Institute under US Government grant NAG W-2166. The im- ages of these surveys are based on photographic data obtained us- ing the Oschin Schmidt Telescope on Palomar Mountain and the UK Schmidt Telescope. The plates were processed into the present com- pressed digital form with the permission of these institutions. † Visiting Astronomer, German-Spanish Astronomical Centre, Calar Alto, operated by the Max-Planck-Institute for Astronomy, Heidelberg, jointly with the Spanish National Commission for Astronomy. † Visiting Astronomer, German-Spanish Astronomical Centre, Calar Alto, operated by the Max-Planck-Institute for Astronomy, Heidelberg, jointly with the Spanish National Commission for Astronomy. 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system?⋆,⋆⋆,⋆⋆⋆ S. L. Schuh1,14,†, G. Handler2,3, H. Drechsel4, P. Hauschildt5, S. Dreizler1,14,†, R. Medupe3,6, C. Karl4,†, R. Napiwotzki4,†, S.-L. Kim7, B.-G. Park7, M. A. Wood8, M. Papar´o9, B. Szeidl9, G. Vir´aghalmy9, D. Zsuﬀa9, O. Hashimoto10, K. Kinugasa10, H. Taguchi10, E. Kambe11, E. Leibowitz12, P. Ibbetson12, Y. Lipkin12, T. Nagel1,†, E. G¨ohler1,†, and M. L. Pretorius13 S. L. Schuh1,14,†, G. Handler2,3, H. Drechsel4, P. Hauschildt5, S. Dreizler1,14,†, R. Medupe3,6, C. Karl4,†, R. Napiwotzki4,†, S.-L. Kim7, B.-G. Park7, M. A. Wood8, M. Papar´o9, B. Szeidl9, G. Vir´aghalmy9, D. Zsuﬀa9, O. Hashimoto10, K. Kinugasa10, H. Taguchi10, E. Kambe11, E. Leibowitz12, P. Ibbetson12, Y. Lipkin12, T. Nagel1,†, E. G¨ohler1,†, and M. L. Pretorius13 1 Institut f¨ur Astronomie und Astrophysik, Universit¨at T¨ubingen, Sand 1, 72076, T¨ubingen, Germany e-mail: schuh@astro.uni-tuebingen.de e-mail: schuh@astro.uni-tuebingen.de 2 Institut f¨ur Astronomie, Universit¨at Wien, T¨urkenschanzstraße 17, 1180 Wien, Austria 3 South African Astronomical Observatory, PO Box 9, Observatory 7935, Cape, South Africa 4 4 Dr.-Remeis-Sternwarte, Astronomisches Institut der Universit¨at Erlangen-N¨urnberg, Sternwartstr. 7, 96049 Bamber Germany 5 Hamburger Sternwarte, Universit¨at Hamburg, Gojenbergsweg 112, 21029 Hamburg, Germany 6 6 Department of Physics, University of the North-West, Private Bag X2046, Mmabatho 2735, South Africa 7 Korea Astronomy Observatory, 61-1, Whaam, Yuseong, Daejeon, 305-348, Korea 8 Department of Physics and Space Sciences and SARA Observatory, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL 32901-6975, USA 9 Konkoly Observatory, Box 67, 1525 Budapest XII, Hungary 10 Gunma Astronomical Observatory, 6860-86 Nakayama Takayama-mura Agatsuma-gun Gunma-ken, Postal Code: 377-0702, Japan 11 Department of Earth and Ocean Sciences, National Defense Academy, Yokosuka, Kan 12 11 Department of Earth and Ocean Sciences, National Defense Academy, Yokosuka, Kanagawa 239-8686, Japa e Observatory, Sackler Faculty of Exact Sciences, Tel Aviv ackler Faculty of Exact Sciences, Tel Aviv University, Tel A 13 Department of Astronomy, University of Cape Town, Rondebosch 7700, South Africa 14 Universit¨atssternwarte G¨ottingen, Geismar Landstraße 11, 37083 G¨ottingen, Germany Received 8 April 2003 / Accepted 4 August 2003 Astronomy & Astrophysics Astronomy & Astrophysics A&A 410, 649–661 (2003) DOI: 10.1051/0004-6361:20031241 c⃝ESO 2003 Send oﬀprint requests to: S. L. Schuh, e-mail: Schuh@astro.uni.tuebingen.de ⋆This paper uses observations made at the Bohyunsan Optical Astronomy Observatory of Korea Astronomy Observatory, at the South African Astronomical Observatory (SAAO), at the 0.9 m tele- scope at Kitt Peak National Observatory recommissioned by the Southeastern Association for Research in Astronomy (SARA), at Gunma Astronomical Observatory established by Gunma prefecture, Japan, at the Florence and George Wise Observatory, operated by the Tel-Aviv University, Israel and at Piszk´estet˝o, the mountain sta- tion of Konkoly Observatory of the Hungarian Academy of Science, Hungary. ⋆⋆This publication makes use of data products from the Two Micron All Sky Survey, a joint project of the University of Massachusetts 1. Introduction Detached eclipsing binaries provide precise fundamental stel- lar parameters like mass and radius and are thus the prerequisite for the validation of stellar evolutionary models. The empirical constraints from over four dozen systems have shown that for main sequence stars between 1 and 10 M⊙the agreement is ac- ceptable, i.e. better than 2% (Andersen 1991, 1998), while at the lower main sequence the situation is far less satisfying. Up to now, only three eclipsing systems with M-type primaries are known, despite the fact that low mass main sequence stars dom- inate the stellar population by number. The ﬁrst such system to be discovered was YY Gem (Joy & Sanford 1926; van Gent 1926), followed by CM Dra (Eggen & Sandage 1967, and ref- erences therein) and CU Cnc (Delfosse et al. 1999); ﬁrst mass determinations came from Leung & Schneider (1978), Lacy (1977) and Delfosse et al. (1999), respectively. While Metcalfe et al. (1996) ﬁnd the slope of the mass-radius relation derived from the M dwarf binary system CM Dra in agreement with model predictions, Delfosse et al. (2000) reported on a dis- agreement between empirical and theoretical mass-luminosity relations of 10–20% in the V band, and recent precise analyses of YY Gem (Torres & Ribas 2002) and CU Cnc (Ribas 2003) also revealed an underestimation (10–20%) of the radii of low mass stars from current evolutionary models. Additional con- straints for the empirical mass-radius relation are provided by the ﬁrst interferometric measurements of radii from lower main sequence stars (S´egransan et al. 2003). These results agree well with model predictions at the present accuracy level, with a possible discrepancy for stars with 0.5–0.8 M⊙. Such observa- tions do not provide an independent measurement of the stellar mass, however, so that eclipsing systems still are the primary source for a model-independent determination of fundamental parameters. Fig.1. Finding charts for 2MASS J0516288+260738 (DSS-2 red: left, DSS-2 blue: right). The side length is 4′ × 4′ for each image; north is up and east is to the right. signature of an eclipse in the Calar Alto 2.2m data set of 2001 Dec. 07 (see Table 1, only available in electronic form at http://www.edpsciences.org). Subsequent searches in the other data sets revealed that eight more eclipses had partly or fully been observed. A year later, 5 additional data sets were obtained, two of which covered the eclipse. 2. Positional information Future improvements of the theoretical mass-radius re- lation for the lower main sequence would strongly bene- ﬁt from a larger empirical database through an increased sample of eclipsing binaries. Recently, 137 eclipsing low- luminosity candidates were announced by the OGLE (Optical Gravitational Lensing Experiment) consortium (Udalski et al. 2002a,b, 2003), of which several of the secondaries turned out to be M-type stars (Dreizler et al. 2002). In this paper we re- port the discovery of another interesting eclipsing binary sys- tem, 2MASS J0516288+260738, whose components appear to bracket the M-star range, with the potential of extending the empirical mass-radius relation into the sub-stellar range. A search with SIMBAD yielded no catalogued object at or near the coordinates of the eclipsing object, but loading the Incremental Release Extended Source Catalog of the Two Micron All Sky Survey (2MASS) into ALADIN resulted in a match. We could clearly identify our object with the point source 2MASS J0516288+260738, and later with a point source in the USNO-B catalogue (cf. Sect. 4.1). We use the 2MASS catalog entry to give improved coordinates: RA = 05h16m28.s81, δ = +26◦07′38.′′8 (J2000). For a clear identiﬁ- cation, the object is marked with horizontal bars in the ﬁnding charts given in Fig. 1. The new eclipsing system has been discovered in obser- vational data taken during a coordinated photometric mon- itoring campaign in December 2001. This dataset has been obtained to monitor the light variations of the DB variable white dwarf KUV 05134+2605 (Grauer et al. 1989; Handler et al. in prep.). It consists of many individual light curves taken by either photomultiplier (PMT) or CCD instruments; the newly discovered object is included in 48 individual time series of images obtained with CCD cameras. While analysing ﬁeld stars for photometric stability to check whether they could be used as references, an object located a little less than one arcminute south of the DB was found to show the Received 8 April 2003 / Accepted 4 August 2003 ⋆⋆This publication makes use of data products from the Two Micron All Sky Survey, a joint project of the University of Massachusetts Article published by EDP Sciences and available at http://www.aanda.org or http://dx.doi.org/10.1051/0004-6361:200312 S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 650 S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? Fig.1. Finding charts for 2MASS J0516288+260738 (DSS-2 red: left, DSS-2 blue: right). The side length is 4′ × 4′ for each image; north is up and east is to the right. 1. Introduction The full time- resolved photometric data are compiled in Sect. 3. Archive searches contributed an identiﬁcation of the object as well as additional colour information (Sects. 2 and 4). Two months after the initial observations, an optical spectrum could be obtained, and in the following observing season, an infrared spectrum was taken (see Sect. 4). In the following, we compile the information that is cur- rently available on the object, report our results from the light curve solution and the spectral analysis, and propose a possible conﬁguration for this system. 3.2. Ephemeris Primary minima times were determined by ﬁtting parabolas to the eclipses. The results for the epochs 0 (two independent data sets), 3, 9, 10, 11, 12 (concatenated from two diﬀerent non- overlapping data sets), 259 and 262 are given in Table 1; no ﬁts could be obtained for epochs 5 and 7 since only parts of either ingress or egress had been observed there. A linear regression for the measured minima times then gives the linear elements and their 1σ errors for the primary minima as Photographic B, R and I magnitudes are published in the USNO-B catalogue (Monet et al. 2003), and infrared J, H and K magnitudes are available from the 2MASS catalog. They are compiled in Table 3. The USNO-B and 2MASS magnitudes m have been converted to Fλ using the relation Fλ erg s−1cm−2Å−1 = Fν0[Jy]·10−0.4m·3 × 10−13/λ0[µm]2. HJD = 2 452 251.d5173 + 1.d29395 · E. ±16 ±25 The photometric zero points Fν0 and central wavelengths λ0 used for the conversion are tabulated in Table 3 along with the results for Fλ. For the 2MASS ﬁlters, these quantities were ob- tained from Squires et al. (2002), while for the white light we used the values published for V by Campins et al. (1985) and Rieke et al. (1985) for the Johnson UBVRI+ system. This ephemeris was used to generate a folded proﬁle from the data taken in 2001. The folded proﬁle has also been carefully inspected to ver- ify that no secondary eclipse is apparent in the data. The proﬁle remains at the same relative ﬂux level outside of the primary eclipse with no signiﬁcant indication of ellipsoidal light vari- ations or reﬂection eﬀects. It was then used to obtain a light curve solution as discussed in Sect. 6. The Sloan Digital Sky Survey (SDSS) does not cover the ﬁeld in its EDR (Early Data Release, Stoughton et al. 2002) so that no further photometric information is available. Since the object, according to its infrared colours, is very red, we also checked the VLA FIRST survey at 20 cm, which cur- rently does not cover this ﬁeld either, and the NRAO/VLA Sky Survey (NVSS) at 1.4 GHz (Condon et al. 1998), which covers the ﬁeld but does not show a radio source in the vicin- ity. 3.1. Time-resolved photometric data All photometric data sets obtained with CCD cameras in the December 2001 KUV 05134+2605 campaign were compiled. Additional observations obtained in November 2002 were added later. A list of all data sets used is given in Table 1, with a complementary key to the observing sites involved in Table 2. All data were bias and ﬂatﬁeld corrected according to standard routines. Aperture photometry was performed on all of these frames using the TRIPP package (Schuh et al. 2003). S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 651 Table 2. Key to observatory sites. Site Telescope Observers CAHA Calar Alto Observatory, Centro Astron´omico Hispano Alem´an, Almer´ıa, Spain 2.2 m SD, SLS CAHA II Calar Alto Observatory, Centro Astron´omico Hispano Alem´an, Almer´ıa, Spain 1.2 m TN, EG BOAO Bohyunsan Optical Astronomy Observatory, Korea 1.8 m SLK, BGP SAAO South African Astronomical Observatory, Sutherland, South Africa 1.0 m GH, TM SARA Kitt Peak National Observatory, Tucson, Arizona, United States of America 0.9 m MW GAO Gunma Astronomical Observatory, Japan 1.5 m OH, KK, HT, EK WISE The Florence and George Wise Observatory, Tel-Aviv University, Israel 1.0 m EL, PI, YL Piszk´estet˝o Piszk´estet˝o, the mountain station of Konkoly Observatory, M´atra Mountains, Hungary 1.0 m MP, BS, GV, DZ Table 3. Johnson V, USNO-B B, R, I and 2MASS J, H, K magnitudes. Table 3. Johnson V, USNO-B B, R, I and 2MASS J, H, K magnitudes. B V R I J H K Magnitude 19.47 18.1 16.8 15.84 14.247 13.346 13.115 Error ±0.3 ±0.1 ±0.3 ±0.3 ±0.040 ±0.039 ±0.039 λ0 [µm] 0.43 0.55 0.70 0.90 1.25 1.65 2.17 Fν0[Jy] 4440 3810 2880 2240 1593 1089 713 Fλ [ ∗] 1.17 2.17 3.36 3.83 6.12 5.51 2.58 * erg s−1cm−2Å−1 × 10−16 . Two reference stars that are available on all frames (shown to be stable during the whole 2001 campaign) were chosen and used consistently for all data sets to produce relative light curves. 3.2. Ephemeris For completeness, we ﬁnally note that neither the ROSAT Bright Source Catalogue as compiled from the WFC All Sky Survey (Pounds et al. 1993) nor the ROSAT XUV Pointed 3.1. Time-resolved photometric data The total light curve was then scaled by a unique factor to pro- duce a light curve with a mean relative intensity of unity for the white light contributions outside eclipse. Finally, all times were converted from Julian date (JD) to heliocentrically cor- rected Julian date (HJD). The result is shown in Fig. 2. Two reference stars that are available on all frames (shown to be stable during the whole 2001 campaign) were chosen and used consistently for all data sets to produce relative light curves. The light curve shows a clear periodicity of 1.29 days. All observed eclipses are similar to each other, have a duration of about 0.10 days and exhibit a decrease in ﬂux of 15% (or 0.17 mag) at the deepest point. There is no indication of a sec- ondary eclipse in any of the eight 2001 data sets that partly or fully cover the phase where such an event would be expected. Furthermore, the three 2002 data sets covering that phase put a clear upper limit to the depth of any secondary eclipse: 0.49% (or 5.4 mmag) in white light and 0.70% (or 7.6mmag) in the Johnson I ﬁlter. Observatory’s 30′′ telescope using the UCT CCD Photometer. A series of 10 images was taken in a Johnson V ﬁl- ter on March 10 2003 starting at 18:19:03 UT (expo- sure time 100s). The measurements were thus made out of eclipse. We compare our results for KUV 05134+2605 and 2MASS J0516288+260738 to estimate that the mean V mag- nitude diﬀerence (KUV −2MASS) is 1.44m ± 0.02. Due to the rapid nature of the variations in the DB variable, it is not a problem to derive a good mean magnitude for that object. Using V = 16.70 (Wegner et al. 1990) for KUV 05134+2605, we obtain V2MASS = 18.1m ± 0.1. 4.2. Optical spectra Two medium resolution spectra of 2MASS J0516288+260738 were obtained in February 2002 at the Calar Alto 3.5 m tele- scope with the double beam spectrograph TWIN (see Table 4, ﬁrst part). Gratings # 5 and # 6 were used for the blue and red arm, respectively, with the dichroic set at 550 nm. Together with slit widths of 1.′′2 and 1.′′5 for the ﬁrst and second ex- posure, this resulted in spectral resolutions of 0.94 and 1.04 Å. Both spectra turned out later on to have been taken well out- side any eclipse, but only the spectrum taken on February 25 reaches an exposure level acceptable for further analysis: the signal-to-noise level per pixel for the ﬁrst spectrum is only 3, but reaches 8 for the second one. The frame and corresponding wavelength calibration frame were bias and ﬂatﬁeld corrected. Then the spectrum was extracted, sky corrected, subjected to a cosmic ray ﬁltering, corrected for the illumination function, and ﬁnally wavelength calibrated. Flux calibration was done by ﬁrst applying the same procedure to an exposure of the stan- dard star G191-B2B taken in the same night, then using tabu- lated ﬂux values to do the absolute calibration. The two result- ing optical spectra in the wavelength ranges of 3900–5000Å and 6000–7090Å of 2MASS J0516288+260738 are displayed By the start of the following observing season for 2MASS J0516288+260738 in autumn 2002, the compila- tion and reduction of the light curve was not only complete enough to allow the prediction of eclipse times, but also to attempt a ﬁrst light curve solution, based on an estimate of the spectral class obtained from the slope of the optical spectrum. This conﬁrmed the suspicion that the system might be made up from two low mass stars or a low mass star and a substellar object, and therefore justiﬁed taking infrared spectra during Director’s discretionary time at Calar Alto Observatory. An H and K band spectrum was observed in October 2002, and a J and H band spectrum in February 2003. Each time, a set of 24 spectra was obtained using the OMEGA-Cass instrument mounted on the 3.5 m telescope (see Table 4), well oﬀboth primary and secondary eclipse. Since the background is high for infrared observations, the set of 24 spectra was obtained in such a way that alternating ex- posures contain the source on two diﬀerent locations on the chip. 4.1. Colours To derive a visual magnitude for the analysed object, we have obtained further photometry of the ﬁeld. The ob- servations were made at the South African Astronomical S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system 652 2. Overview of the photometric observations; the ﬂux is given in fractional intensity units. Time is in days, with data binned into 0 s. Time increases from left to right and from bottom to top. The epochs (labelled #n) are displayed continuously up to the end of the paign, while for the 2002 observations only those epochs were included in the plot for which data points exist. The primary ecl layed at multiples of 0.97 d to place it at a phase of 0.75 in this plot, allowing convenient viewing of both phase 0.25 where the seco ima would be located as well as of the primary eclipses (both marked by horizontal dashed lines). The scatter in the individual light tributed by diﬀerent sites varies according to aperture, actual exposure time and weather conditions. Fig. 2. Overview of the photometric observations; the ﬂux is given in fractional intensity units. Time is in days, with data binned into units of 30 s. Time increases from left to right and from bottom to top. The epochs (labelled #n) are displayed continuously up to the end of the 2001 campaign, while for the 2002 observations only those epochs were included in the plot for which data points exist. The primary eclipse is displayed at multiples of 0.97 d to place it at a phase of 0.75 in this plot, allowing convenient viewing of both phase 0.25 where the secondary minima would be located as well as of the primary eclipses (both marked by horizontal dashed lines). The scatter in the individual light curves contributed by diﬀerent sites varies according to aperture, actual exposure time and weather conditions. . L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 653 S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 653 Fig. 3. 4.1. Colours Measured ﬂux calibrated optical and IR spectra (solid line), Johnson V, USNO-B and 2MASS colours converted to Fλ (diamond symbols), in comparison with the best ﬁt model spectrum (dotted). For details see text. Fig. 3. Measured ﬂux calibrated optical and IR spectra (solid line), Johnson V, USNO-B and 2MASS colours c symbols), in comparison with the best ﬁt model spectrum (dotted). For details see text. calibrated optical and IR spectra (solid line), Johnson V, USNO-B and 2MASS colours converted to Fλ (diamon on with the best ﬁt model spectrum (dotted). For details see text. Phase Source Catalogue as compiled from WFC observations during pointed phase (Kreysing et al. 1995) list sources at or near the object’s position. Phase Source Catalogue as compiled from WFC observations during pointed phase (Kreysing et al. 1995) list sources at or near the object’s position. in Fig. 3 (rescaling as described in Sect. 5). At the given S/N ratio, no lines or features, in particular no TiO bands, can be discerned. 4.2. Optical spectra For each observation, the two spectra for the standard star were then combined and compared to tabulated ﬂux values to obtain the factor for absolute ﬂux calibration, which was then applied to both the two object spectra and the two individual standard star spectra. optical spectra simultaneously, and a correction factor was ap- plied to each of the two independent infrared spectra (HK and JH). The uncertainty in the optical spectrum results since both object and ﬂux standard star were observed under non- photometric conditions. The same argument applies to the in- frared spectral observations, where observations from diﬀerent nights, although both nominally ﬂux calibrated, result in diﬀer- ent ﬂux levels for the overlapping H band. A consistent adjust- ment therefore seems justiﬁed. Residual errors may result from the transformation of magnitudes to F λ. In the following, it will be assumed that the observed spectral energy distribution consists of light from the pri- mary only; furthermore, for reasons detailed in Sect. 7.2, the primary will be presumed to be a late main sequence star. Since 2MASS J0516288+260738 is located close to the galac- tic plane, the eﬀect of interstellar reddening is not negligible even for low-luminosity and close-by objects. For the initial analysis of the observed data we use a grid of model atmospheres and synthetic spectra that is based on the models of Allard et al. (2001). We have extended the model grid to eﬀective temperatures of 10 000K for gravities from 5.5 ≤log g ≤−0.5 using spherical symmetry. The mixing length was set to twice the pressure scale height, this choice of the mixing length was calibrated on early M dwarfs (Ludwig et al. 2002). Comparing the results for the individual spectra for both stars leads to the conclusion that the error bars in the resulting combined spectra must be considered to be of the same mag- nitude as any “features” that one might be tempted to spot.The same conclusion results from a comparison of the H band parts of the spectra from the two diﬀerent observing dates, where most “features” are not reproduced. Furthermore, a ﬂux diﬀer- ence by a factor of about 1.5 between those two independent observations gives an estimate for the errors in the ﬂux cali- bration. The rescaled infrared spectra for 14 000–25000Å and 10 000–18000 Å are displayed in Fig. 3. 4.2. Optical spectra Synthetic spectra generated from the models were com- pared to the observed spectra using an IDL program. This step was restricted to the infrared spectra. First, the resolution of the synthetic spectra was degraded to that of each observed spec- trum by convolution with a Gaussian of the appropriate width, and the spectra were normalized to unit area for scaling. Next, for each observed spectrum the program calculated a quality function q, similar to a χ2 value, for the comparison with all synthetic spectra in the grid. The quality function is calculated by ﬁrst scaling the model spectrum to the observed ﬂuxes and then by mapping the synthetic spectrum (reduced to the resolu- tion of the observed data) onto the grid of observed wavelength points and then calculating 4.2. Optical spectra After bias and ﬂatﬁeld correction of the individual expo- sures, this allows to determine a mean background at the (dis- persed) location of the source for both types of images from the S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 654 Table 4. Spectroscopic observations. Object Instrument λ range [Å] Start time (UT) Exp. time HJD 2MASS J0516288+260738 TWIN 3900–5000, 6000–7090 2002 Feb. 23 21:34 1800 s 2 452 329.411 G191-B2B TWIN 3900–5000, 6000–7090 2002 Feb. 25 18:40 300 s 2 452 331.281 2MASS J0516288+260738 TWIN 3900–5000, 6000–7090 2002 Feb. 25 20:49 1800 s 2 452 331.380 2MASS J0516288+260738 OMEGA-Cass HK: 14 000–25 000 2002 Oct. 27 23:31 24 × 120 s 2 452 575.484 GD 71 OMEGA-Cass HK: 14000–25000 2002 Oct. 28 02:21 10 × 120 s 2 452 575.602 2MASS J0516288+260738 OMEGA-Cass JH: 10 000–18 000 2003 Feb. 03 21:49 24 × 120 s 2 452 674.412 GD 71 OMEGA-Cass JH: 10 000 – 18 000 2003 Feb. 03 23:12 24 × 120 s 2 452 674.470 respective subset of the complementary frames. These two measures of the mean background can then be used to sub- tract the appropriate background from all frames of the two subsets. To do this, the shift for each background row was ﬁrst determined by cross-correlating it along the dispersion direc- tion with the corresponding image row, and the overall run of the shift along the chip obtained by ﬁtting these row-by- row measurements with a low-order polynomial. To achieve the best possible subtraction in the vicinity of the source lo- cation on the chip, this ﬁt with sub-pixel accuracy was then used to shift the background onto the image before subtracting it. For the wavelength dispersions at the two source locations, no shifts could be detected during the course of the exposure series. Therefore, next these bias subtracted, ﬂatﬁeld corrected and background subtracted frames of each of the two sets were added to yield two summed images. The spectra were extracted from these two images using standard procedures for extrac- tion, sky correction, cosmic ﬁltering, illumination correction, and wavelength calibration. The same procedure was used for the set of 10 and 24 exposures of the standard star GD 71. 6. Light curve solution Hence it was important to use any available sec- ondary information from spectroscopy or stellar atmospheres’ theory to reduce the number of free light curve parameters and keep some of them at ﬁxed values. The resulting ﬁt is shown in Fig. 3. We have applied the reddening to the synthetic spectrum (dotted line) in order to fa- cilitate the comparison without modifying the data themselves. All available spectral and colour information is included in the ﬁgure. The ﬁt is in general acceptable, unfortunately data are missing in spectral regions where they would be extremely use- ful to test the resulting model parameters. No information at all is available for a possible eccen- tricity of the orbit, since the position of the unobserved sec- ondary eclipse cannot be determined, and radial velocity mea- surements do not exist so far. Therefore circular orbits (e = 0) and synchronously rotating components were assumed – as is mostly the case in close binary systems due to their very short synchronization time scales. According to the late spec- tral type, bolometric albedos A1 and A2 were ﬁxed at their usu- ally expected values of 0.5 for convective outer layers, and gravity darkening exponents g1 and g2 were set to 0.32 as predicted by Lucy’s law (1967). Linear limb darkening coef- ﬁcients are poorly known for very late spectral types. From an extrapolation of the grids of Wade & Rucinski (1985) and D´ıaz- Cordov´es et al. (1995) at their cool ends one obtains approxi- mate values of x1 = 0.5−0.6, which were used in the solutions. Values of x2 (and g2) are irrelevant due to the absence of any measurable secondary light. A consistency check of our solution can be performed by comparing our measured reddening with the model of the Galactic interstellar extinction constructed by Arenou et al. (1992). First we estimate the distance from the spectral type – absolute magnitude calibration of Schmidt-Kaler (1982). From their Table 13 we get an absolute magnitude of MV = 8.1 for spectral type K7 V. With E(B −V) = 0.9 as derived above, the dereddened V magnitude is 15.3 (adopting R = 3.1). Thus the resulting distance module is 7.2, corresponding to a distance of 280 pc. The reddening predicted from the Arenou et al. model and the position of 2MASS J0516288+260738 (l = 178.8, b = −6.9) amounts to E(B −V) = 0.48 ± 0.24. 6. Light curve solution and λobs the observed wavelength. For each model, this proce- dure was repeated for 0.0 ≤E(B −V) ≤2.5 in steps of 0.1 to independently determine the reddening. For this procedure, we used the reddening model of Cardelli et al. (1989). We then selected the models that resulted in the 3–10 lowest q values as the most probable parameter range for each individual star. The “best” value was chosen by visual inspection, at this point ad- ditionally considering the optical spectra to ensure a consistent ﬁt. This procedure allows a rough estimate of the uncertainty in the stellar parameters. Note that it does not eliminate system- atic errors in the stellar parameters due to missing, incorrect or incomplete opacity sources. The comparison was done for a total of 377 model atmospheres with solar abundances in the range 2000 K ≤Teﬀ< 5000 K and 5.5 ≤log g ≤0.0. Together with the search range in extinction this leads to 7539 combina- tions that were considered in the procedure. With the exception of allowing the extinction to vary this is the same procedure that was used in Leggett et al. (2001) and Leggett et al. (2002). and λobs the observed wavelength. For each model, this proce- dure was repeated for 0.0 ≤E(B −V) ≤2.5 in steps of 0.1 to independently determine the reddening. For this procedure, we used the reddening model of Cardelli et al. (1989). We then selected the models that resulted in the 3–10 lowest q values as the most probable parameter range for each individual star. The “best” value was chosen by visual inspection, at this point ad- ditionally considering the optical spectra to ensure a consistent ﬁt. This procedure allows a rough estimate of the uncertainty in the stellar parameters. Note that it does not eliminate system- atic errors in the stellar parameters due to missing, incorrect or incomplete opacity sources. The comparison was done for a total of 377 model atmospheres with solar abundances in the range 2000 K ≤Teﬀ< 5000 K and 5.5 ≤log g ≤0.0. Together with the search range in extinction this leads to 7539 combina- tions that were considered in the procedure. With the exception of allowing the extinction to vary this is the same procedure that was used in Leggett et al. (2001) and Leggett et al. (2002). 6. Light curve solution From the overall photometric data set, a subset of 23 contribu- tions was chosen to create the proﬁle used for the light curve solution. The subsets, marked “used for proﬁle” in Table 1, were selected according to their length, the coverage in phase they contributed to, the ﬁlter they were taken in (=none), and their reliability and quality with respect to trends. In contrast to the data shown in Fig. 2, each of the selected data sets was then cleaned from suspicious points and normalised at its max- imum. A folded proﬁle with 200 points, with a phase bin width of 0.005 units, and with phase zero set at the minimum of pri- mary eclipse, was then obtained from these data, and a few remaining clearly unreliable points were removed. This light curve, formed of 187 normal points (in in- tensity units), normalized to unity outside eclipse, was sub- jected to a numerical solution by the application of the MORO code (Drechsel et al. 1995). The code is based on the Wilson-Devinney (1971) logistical approach, but incorporates a modiﬁed Roche model to account for radiative interaction between the components and uses the SIMPLEX method as parameter optimization algorithm. The best ﬁtting model has an eﬀective temperature T eﬀ= 4200 K and a reddening of E(B −V) = 0.9. The low resolution of the data and the relative insensitivity of the spectral energy distribution to gravity prevent us from determining a value of log g, it is clear, however, that the object is a dwarf rather than a giant. The formal error in eﬀective temperature is about ±200 K and about ±0.2 for the extinction. The low resolution data also prevent detailed metallicity determinations, and so far only so- lar metallicities were considered. Overall, the spectral analysis results suggest a spectral type of about K7 V (±2 subclasses). The solution mode was chosen such that no a priori restric- tion of the system conﬁguration was imposed (equivalent to the original Wilson-Devinney mode 2). The total number of light curve parameters for a single passband curve amounts to 17. Since the observed eclipse minimum depth is only moderate (≈16% of maximum light), as no signature of the secondary ex- cept its light blocking eﬀect is evident, and because no colour information follows from the white light curve, solutions tend to be underdetermined, especially if the adjustable parameter set is too large. 5. Spectral analysis The ﬂux calibrated optical and infrared spectra as well as the broad band ﬁlter measurements converted to ﬂux values (dia- mond symbols) are all displayed together in Fig. 3. To obtain a consistent image of the spectral energy distribution, the mag- nitude measurements were used to rescale the spectra where necessary. A unique correction factor was applied to both q = i wi 0.5 f model i −f obs i f model i + f obs i  2 with wi = 0.5(f obs i+1 + f obs i )(λobs i+1 −λobs i ) where f model is the (mapped) ﬂux of the model spectrum, f obs is the observed ﬂux, 655 S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 6. Light curve solution d Fraction of third light at maximum. ∗Fixed. q ≈0.1, the secondary is of about the same size as the primary, and nearly ﬁlls its Roche lobe in a close to semi-detached con- ﬁguration. The photometric determination of T 2 and hence the temperature ratio must be considered very uncertain, because of the missing secondary eclipse and the extreme luminosity ratio. ratio T1/T2, and from a single unﬁltered curve no colour in- formation can be extracted. The remaining set of adjustable parameters therefore comprised inclination i, mass ratio q = M2/M1, secondary temperature T 2, surface potentials Ω1 and Ω2, primary luminosity L1, and third light l3. L2 was not in- dependently adjusted, but recomputed from T 2 and the sec- ondary surface area over the Planck law. Trial runs showed that the percentage of third light l3 attributable to a possible unre- solved ﬁeld star tended toward zero (except for solution g, see Table 5), so that this parameter was subsequently ﬁxed at l3 = 0 in the iterations of all other solutions. The overall representation of the observations by the theo- retical light curve is very good. Figure 5 (top) displays the nor- mal points in comparison with the synthetic curve (solid line). Especially the eclipse minimum is matched in detail. The stan- dard deviation amounts to only 7.5 mmag, which corresponds to the typical scatter of measurements binned to normal points. As shown in the bottom part of Fig. 5, most observations lie in a 1σ band, and all within a 3σ belt, with no apparent sys- tematic deviations. Figure 6 gives a 3-dimensional impression of the aspects of the system at diﬀerent phase angles as viewed under an inclination of 74◦; the conﬁguration corresponds to the parameters of solution c. Convergent solutions were achieved after numerous trial runs with a variety of start parameter sets (start simplices) and diﬀerent parameter increments as starting points of the auto- matic iteration process, which covered essentially the whole range of physically reasonable parameter values. For reasons discussed earlier the numerical process could not be expected to yield a single best and unique solution. Instead, for a cou- ple of comparably good solutions, there was no obvious way to qualify one of these as deﬁnitely best representation, as judged from the ﬁnal standard deviations of normal points from the synthetic curves. 6. Light curve solution The scatter results mostly from the patchiness of the interstellar medium in this region. Although this value is somewhat smaller than our measured reddening both values agree within the error limits. Note that the model of Galactic extinction provides an upper limit of E(B−V) = 0.72±0.36 for the reddening at the position of 2MASS J0516288+260738. This limit results from the fact that stars exceeding a certain distance are above the absorbing dust layers of the Galaxy. This allows us to rule out highly red- dened early type stars (cf. also the independent discussion of this aspect in Sect. 7.2 which leads to the same result). The primary eﬀective temperature was always ﬁxed at T 1 = 3000 K, typical for spectral type M5 V, since the result of Teﬀ= 4200 K from the spectral analysis has only become available recently, following the February 2003 OMEGA-Cass observations. This choice is however not critical, because the light curve solution only allows to derive the temperature S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 656 MORO solutions of the light curve of the eclipsing system 2MASS J0516288+260738. Table 5. MORO solutions of the light curve of the eclipsing system 2MASS J0516288+260738. Table 5. MORO solutions of the light curve of the eclipsing system 2MASS J0516288+260738. Parameter a b c d e f g h q (= M2/M1) 0.085 0.094 0.110 0.122 0.156 0.181 0.182 0.184 i 75.◦5 74.◦3 74.◦0 73.◦9 73.◦5 72.◦4 72.◦9 72.◦3 T1/T2 1.67 1.72 1.74 1.73 1.77 1.63 1.83 1.88 r1/r a 2 1.11 0.89 0.85 0.87 0.85 0.67 0.73 0.64 Ω1 5.138 5.350 5.510 5.520 5.598 6.123 6.001 6.220 Ω2 1.961 1.943 1.997 2.055 2.182 2.197 2.236 2.198 Lb 1 0.998 0.998 0.999 0.998 0.999 0.993 0.999 0.999 xc 1 0.60∗ 0.60∗ 0.50∗ 0.50∗ 0.60∗ 0.60∗ 0.488 0.60∗ x c 2 0.50∗ 0.50∗ 0.50∗ 0.50∗ 0.50∗ 0.50∗ 0.527 0.50∗ ld 3 0%∗ 0%∗ 0%∗ 0%∗ 0%∗ 0%∗ 1.5% 0%∗ 1σ deviation 0.00750 0.00750 0.00749 0.00749 0.00749 0.00755 0.00750 0.00749 a Ratio of mean Roche radii. b Relative luminosity L1/(L1 + L2); L2 is not independently adjusted, but recomputed from r2 and T2. c Linear limb darkening coeﬃcient; theoretical value for V band taken from D´ıaz-Cordov´es et al. (1995). 6. Light curve solution To give an impression of the typical scatter of ﬁnal parameters we present a subsample of 8 diﬀerent so- lutions with the relatively best sigma standard deviations in Table 5. These are sorted in a sequence of increasing q val- ues. It is obvious that one can identify two groups of solutions according to the value of the mass ratio: solutions a–d cluster around q ∼0.10±0.02,while cases e–h yielded q ∼0.18±0.01. 7. Discussion of alternate conﬁgurations The system is located only 6.9◦above the galactic plane (in an outward direction). This implies that the reddening through interstellar extinction is potentially very high. Although from spectral observations in conjunction with detailed Galactic ex- tinction models many stellar spectral and luminosity types other than late main sequence stars can be excluded, it is also instructive to make use of the independent information from the light curve solution alone. Through the geometry of the system, and fundamental stellar parameters that cannot be substantially altered even when a star resides in a close binary system, most of the following alternative combinations can be excluded. This in turn justiﬁes the restriction of the discussion in Sect. 5 to a late dwarf system. A common feature of all solutions are consistent values of inclination (i ∼72◦−75◦), temperature ratio (T1/T2 ∼ 1.6−1.8), ratio of radii (r1/r2 around 0.9), extremely low sec- ondary luminosity (L2/L1 ≈1−2 × 10−3), and similar system conﬁguration. As shown for solution c in Fig. 4, which can be considered as representative for the group of solutions with S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 657 S. L. Schuh et al.: 2MASS J0516288+260738: Discovery Fig. 4. Meridional intersection of surface and inner critical Roche equipotentials corresponding to the nearly semi-detached system con- ﬁguration of solution c (see Table 5); the substellar secondary compo- nent is close to contact with its Roche lobe. Fig.5. Top part shows the observed light curve in white light (dots are normal points formed by binning individual observations to phase intervals of width 0.005) together with the theoretical curve (solid line) corresponding to solution c of Table 5; maximum light (inten- sity) was normalized to unity, and phases were computed according to the ephemeris of Sect. 3.2; bottom part shows residuals of observa- tions (in intensity units) with 1σ and 3σ belts. Fig. 4. Meridional intersection of surface and inner critical Roche equipotentials corresponding to the nearly semi-detached system con- ﬁguration of solution c (see Table 5); the substellar secondary compo- nent is close to contact with its Roche lobe. 7.1. Reddened giant stars Up to spectral types G5 or earlier, luminosity class III red giants have radii larger than the orbital separation dictated by the mea- sured orbital period and a total mass sum of the system of up to twice their own mass. This is illustrated in Fig. 7: The solid line represents the orbital separation of the system components as a function of the total system mass for the given orbital period of 1.d29. It is therefore a strict upper limit to the radius of any single component of the system. The type III giant star spectral types are printed at the position of their radii, once over their corresponding stellar mass and once at twice that value. These overplotted radii for type III giants were taken from the mass- radius relation by Cox (2000). Using the stellar mass and the double of it means that in between those two values all possible mass combinations are covered, since the roles of primary and secondary would simply become reversed if an even greater fraction of the total mass were attributed to the presumed sec- ondary. The ﬁrst case represents the limit in which the mass of the companion is negligible, so that the total mass is solely made up of the giant’s contribution, while in the second case the mass of the giant amounts to half of the total mass in the system. Fig.5. Top part shows the observed light curve in white light (dots are normal points formed by binning individual observations to phase intervals of width 0.005) together with the theoretical curve (solid line) corresponding to solution c of Table 5; maximum light (inten- sity) was normalized to unity, and phases were computed according to the ephemeris of Sect. 3.2; bottom part shows residuals of observa- tions (in intensity units) with 1σ and 3σ belts. luminosity class for the more luminous object in the system must be V or higher. S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 658 Fig.7. Orbital separation a as a function of the mass sum M1 + M2 of the system at the 1.29 d period. Overplotted are radii for type III giants; explanation see text (Sect.7.1). Fig.7. Orbital separation a as a function of the mass sum M1 + M2 of the system at the 1.29 d period. Overplotted are radii for type III giants; explanation see text (Sect.7.1). Fig. 6. Aspects of the system at three diﬀerent phases; viewing angle is 74◦, and system parameters correspond to solution c of Table 5. Fig.7. Orbital separation a as a function of the mass sum M1 + M2 of the system at the 1.29 d period. Overplotted are radii for type III giants; explanation see text (Sect.7.1). sequence primary using the temperature and mass ratios of the respective photometric solutions c and g. For each square plot- ted, the corresponding error estimates from the typical disper- sion within each of the two groups are indicated by small dots which represent the end points of the associated error bars (not drawn as full lines to preserve more clarity in this complex representation). For the primary a variety of spectral types be- tween O5 V and M8 V were considered to cover the full zero- age main sequence (plus signs). When inspecting this ﬁgure and the following graph, note that the plot scale is logarithmic so that oﬀsets between curves can be much larger in regions of the plot that correspond to the upper main sequence than they might intuitively seem. The curve for the secondary corresponding to solution c only approaches and intersects the main sequence at its lower end and therefore excludes highly reddened hotter main se- quence stars as a possible primary, since the corresponding secondaries cannot exist. The other line corresponding to so- lution g starts oﬀclose to the main sequence and comes back to it earlier than the other one. As stated above, the combina- tion of two upper main sequence stars as a possible solution can be ruled out, because such extended stars could only reside within the given orbit if an appreciable distortion of the pri- mary is allowed for, which would inevitably result in an easily observable ellipsoidal light variation. 7.2. Reddened earlier main sequence stars Giant stars do not ﬁt within the prescribed orbit; but what about bright early main sequence stars that appear reddened by strong interstellar absorption? Very early main sequence stars have masses and especially radii similar to those of type III giants, so that, as above, geometry arguments can be brought forward to rule out a combination of two very early-type components. This is important, as results from stellar structure will be uti- lized to ﬁnd physically meaningful pairs in what follows. In a na¨ıve consideration, the early type giants could ﬁt within the orbital separation, even if it is clear that most of the time they would reach well over half of the total distance. But although these earlier types could just about ﬁt into the system, it can easily be shown that their deformation within the Roche potential would in all cases result in large ellipsoidal light vari- ations, which are not observed in the actual light curve. In addition, the width of the observed eclipse minimum would cover a much broader phase range. For these estimates, the bi- nary eclipse simulation program nightfall (R. Wichmann, Landessternware Heidelberg, Germany) which calculates syn- thetic light curves taking into account the distortion of the stars in Roche geometry was used. The light curve solutions constrain the mass ratio, the ra- dius ratio and the ratio of the eﬀective temperatures almost re- gardless of the absolute value of T 1. Using tabulated values for the masses, radii and eﬀective temperatures of stellar and sub- stellar objects, the possible components making up the system can be constrained by requiring that both of them have param- eters reasonably close to those of isolated main sequence stars or substellar objects. The stellar parameters used in the follow- ing were taken from Cox (2000), those for substellar objects Since these considerations equally apply to luminosity classes II and I, and even in a much stricter form there, the S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? Eﬀective temperature as a function of the stellar mass for zero-age main sequence stars and substellar objects (plus signs) and their secondaries according to Table 5 (squares); further explanations see text (Sect. 7.2). Table 6. Interpretation of Figs. 8 and 9; for explanations see Sects. 7.2 and 8. Possible primary spectral types for solution Group a–d Group e–h Fig. 8. Eﬀective temperature as a function of the stellar mass for zero-age main sequence stars and substellar objects (plus signs) and their secondaries according to Table 5 (squares); further explanations see text (Sect. 7.2). Table 6. Interpretation of Figs. 8 and 9; for explanations see Sects. 7.2 and 8. Possible primary spectral types for solution Group a–d Group e–h From mass-temperature relation (Fig. 8) upper limit G5 region G0–K0, or upper limit M2 From mass-radius relation (Fig.9) upper limit G5 upper limit M0 Combined constraints upper limit G5 upper limit M2 Consistency with spectroscopy (K7 ± 2) fully consistent marginally consistent at most Resulting secondary mass [M⊙] upper limit 0.11 upper limit 0.076 Resulting secondary mass [M⊙] for K7 0.062 ± 0.01 0.105 ± 0.01 upper limits can be even further constrained, as will be shown next. hard to claim consistency with the spectroscopy results. Table 6 nevertheless explores the mass range for the secondary in dif- ferent scenarios (entries in lines 5 and 6). The ratios of radius and mass for the binary components (also taken from Table 5) together with tabulated mass-radius relations from the same sources as above can be subjected to the same procedure. The designations in Fig. 9 are analogous to those in Fig. 8. The results are also compiled in Table 6. The primary can be constrained to be of spectral type G5 or later for solution c, or of spectral type M0 for solution g. The sec- ond line in Table 6 lists these limits without any additional age constraints that migth be present (see this discussion later). An additional constraint not taken into account so far is the age of the system, which for the more likely solutions a-d is re- stricted to below 0.01Gyr by the mass-radius relation. A young system is also allowed for by the mass-temperature relation. S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? Apart from this special case for g on the upper main sequence, solutions (discretised in, on average, 5-subclass steps!) were elected possible when- ever the error range for such a discrete secondary location in- tersected the stellar or substellar regime. Errors in parameter ratios are regularly smaller than the discretisation in spectral classes used, so the limits given can be regarded to be accurate to within roughly two subclasses. Fig. 6. Aspects of the system at three diﬀerent phases; viewing angle is 74◦, and system parameters correspond to solution c of Table 5. (for ages ranging from 1 M yrs to 10 G yrs) from Chabrier et al. (2000) and Baraﬀe et al. (2002). The ratios of eﬀective temperatures and masses of the bi- nary components for the light curve solutions c and g from Table 5, which can be considered representative for the two bulges of solutions clustering around q = 0.10 and 0.18, are used to ﬁnd the corresponding eﬀective temperatures and masses of the secondary as a function of primary mass. For all main sequence stars, their eﬀective temperatures and those re- quired by the two representative solutions for the secondary are plotted in Fig. 8. The zero-age main sequence objects and the youngest substellar objects (1 M yrs old) are marked by plus signs and are connected by a thick solid line; higher age sub- stellar models are also represented by plus signs, which re- main, however, isolated for clarity. For both solutions a row of squares connected by a solid line is shown. The squares cor- respond to the locations of the secondary in the T eﬀ−M dia- gram, which follow from the temperature and mass of a main On the lower main sequence, the earliest possible spectral types for the primary in the two cases are as listed in the ﬁrst line of Table 6. In case g, K5 and M0 primaries must be ex- cluded. When the mass-radius relation is taken into account in addition to the just invoked mass-temperature correlation, these . L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 659 S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 659 Fig. 8. S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? However, this corresponds to a lifetime of the system in which the K star will not have had enough time to attain the zero- age main sequence yet, and will hence not necessarily have the ZAMS parameters assumed to deduce these constraints in the ﬁrst place. This might well limit the overall usefulness of this discussion, and is a point that will have to be re-addressed once better data has become available for this object. Combining the constraints from both Figs. 8 and 9 yields an overall upper limit for each solution as listed in Table 6, line 3. Solution c allows for a primary no earlier than G5, while solution g restricts possible primaries to spectral types no ear- lier than M2. Spectroscopic results strongly favour the group a–d solution, since the overall constraint of G5 for the pri- mary spectral class is entirely consistent with the conclusion in Sect. 5. For an upper limit of M2, on the other hand, it would be 7.3. Nearly identical components A serious objection to the interpretation presented so far emerges if the orbital period is really twice as long as assumed S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 660 Fig. 9. Mass-radius relation for zero-age main sequence stars and substellar objects (plus signs) and their secondaries according to Table 5 (squares); further explanations see text (Sect.7.2). Fig. 9. Mass-radius relation for zero-age main sequence stars and substellar objects (plus signs) and their secondaries according to Table 5 (squares); further explanations see text (Sect.7.2). (see a similar initial ambiguity for CM Dra where this indeed turned out to be the correct interpretation in Eggen & Sandage 1967). The eclipse ephemeris given in Sect. 3.2 would then not correspond to the orbital ephemeris of the system any more, as assumed throughout the light curve analysis in Sect. 6. Hence, results obtained there are not applicable to the current discus- sion, where identical components could then produce undistin- guishable primary and secondary eclipses. In many binary sys- tems, the mass ratio is close to one, so this is not an altogether implausible, but from a statistical point of view highly unlikely conﬁguration. This possible complication can currently not be resolved, since the scenario could only be conclusively ruled out, or corrobated, with radial velocity measurements. the mass ratios given in Table 5 for each solution. All of these mass values, which are close to or below the substellar limit of 0.075 M⊙required for stable hydrogen burning, represent upper limits. Taking into account the additional information available from spectral analysis which favours a spectral type around K7 results in a value of ≈0.06 M⊙for the secondary’s mass. In this case, only solutions a-d are considered as likely since a spectral type of K7 would not be consistent with solutions e–h. A sub- stellar nature of the companion is therefore quite likely: The unusually low mass ratios in all solutions make the secondary a good Brown dwarf candidate. This interpretation should now be checked by trying to con- ﬁrm the spectral classiﬁcation via the detection of spectral lines in new high resolution, high signal-to-noise optical and/or in- frared spectra. These lines could then also be used to obtain ra- dial velocity measurements for the system which should even- tually provide absolute masses. 7.4. Two old white dwarfs A further scenario that requires consideration is a system con- sisting of two old and therefore very red white dwarfs (the eﬀect of interstellar reddening cannot contribute signiﬁcantly here due to the low intrinsic luminosity of these objects). It is however not very probable that the mass ratio in a double de- generate system is as low as q ≈0.1. For the given period, the duration of eclipse would be of order 10 −3 phase units, com- pletely incompatible with observations. Together with the extensive light curve available, the sys- tem has the potential to provide a new high-quality point for the mass-radius relation of the lower main sequence (or for pre- main sequence evolutionary tracks), and the ﬁrst one obtained from eclipse measurements for a sub-stellar object. Acknowledgements. The authors would like to thank K. Werner and H. Mauder for helpful discussions and friendly support, and P. A. Woudt for his assistance in obtaining a V magnitude for 2MASS J0516288+260738. We also would like to thank R. Gredel for allocating Director’s discretionary time and U. Thiele for car- rying out the OMEGA-Cass observation at Calar Alto observatory in service mode. We acknowledge the use of the nightfall program for light-curve synthesis of eclipsing binaries (http:// www.lsw.uni-heidelberg.de/∼rwichman/Nightfall.html), written by R. Wichmann. Part of this work was supported by the German Deutsche Forschungsgemeinschaft under project grants 8. Conclusion Despite the remaining uncertainties, from the data presented in this paper it is plausible that the newly discovered eclipsing binary system 2MASS J0516288+260738 consists of a late K- type (pre-)main sequence star as a primary and a substellar ob- ject as a secondary. For the spectral class upper limits derived in Sect. 7.2, Table 6 also lists the secondary masses according to S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system? 661 S. L. Schuh et al.: 2MASS J0516288+260738: Discove Leggett, S. K., Hauschildt, P. 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Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system?, Online Material p 2 S. L. Schuh et al.: 2MASS J0516288+260738: Discovery of the ﬁrst eclipsing late K + Brown dwarf binary system?, Online Material p 2 Table 1 Photometric observations able 1. Photometric observations. Site Start time [UT] Length[h] Frames Epoch Minimum [HJD] O−C Comments SARA 2001 Dec. 06 05:01 7.1 857 used for proﬁle SARA 2001 Dec. 07 03:14 5.5 664 used for proﬁle SARA 2001 Dec. 07 10:14 1.9 233 CAHA 2001 Dec. 07 20:09 8.8 1032 0 2 452 251.5164 −0.0009 used for proﬁle Piszk´estet˝o 2001 Dec. 07 19:23 7.3 380 0 2 452 251.5164 −0.0009 BOAO 2001 Dec. 08 11:20 9.0 1029 Piszk´estet˝o 2001 Dec. 08 18:14 4.7 200 used for proﬁle WISE 2001 Dec. 09 01:12 2.4 690 used for proﬁle WISE 2001 Dec. 09 18:02 1.8 511 Piszk´estet˝o 2001 Dec. 09 18:26 7.5 370 used for proﬁle WISE 2001 Dec. 09 20:46 2.6 768 used for proﬁle WISE 2001 Dec. 09 23:43 2.0 564 used for proﬁle WISE 2001 Dec. 10 01:47 1.8 505 used for proﬁle BOAO 2001 Dec. 11 16:51 1.6 181 used for proﬁle BOAO 2001 Dec. 11 19:17 0.9 101 used for proﬁle SAAO 2001 Dec. 11 22:22 3.0 1080 3 2 452 255.4007 +0.0016 GAO 2001 Dec. 12 11:33 1.4 308 SAAO 2001 Dec. 12 21:41 1.7 627 GAO 2001 Dec. 13 13:47 5.3 1000 SAAO 2001 Dec. 13 19:57 5.4 1821 GAO 2001 Dec. 14 12:37 6.7 1301 5 BOAO 2001 Dec. 14 16:35 2.5 257 used for proﬁle Piszk´estet˝o 2001 Dec. 14 18:08 6.7 328 used for proﬁle SAAO 2001 Dec. 14 20:11 5.1 1821 SAAO 2001 Dec. 15 22:01 1.9 643 GAO 2001 Dec. 16 16:25 3.2 700 used for proﬁle SAAO 2001 Dec. 16 19:58 5.3 1901 7 GAO 2001 Dec. 17 10:51 0.9 203 GAO 2001 Dec. 17 13:19 0.6 125 GAO 2001 Dec. 17 15:50 3.2 700 SAAO 2001 Dec. 17 20:02 5.1 1827 GAO 2001 Dec. 18 10:22 8.6 1750 SAAO 2001 Dec. 18 20:03 4.8 1710 GAO 2001 Dec. 19 12:22 6.7 753 9 2 452 263.1615 −0.0013 used for proﬁle SAAO 2001 Dec. 19 20:04 4.8 1738 SARA 2001 Dec. 19 04:54 7.2 817 used for proﬁle GAO 2001 Dec. 20 10:54 6.2 650 used for proﬁle SAAO 2001 Dec. Online Material 20 19:58 4.9 1752 10 2452264.4599 +0.0031 SARA 2001 Dec. 20 02:37 9.7 1107 used for proﬁle SAAO 2001 Dec. 21 19:53 5.0 1804 GAO 2001 Dec. 22 12:50 6.0 625 used for proﬁle SAAO 2001 Dec. 22 21:22 3.0 1074 SARA 2001 Dec. 22 04:31 3.3 404 11 2 452 265.7487 −0.0019 SARA 2001 Dec. 23 05:12 0.8 100 SARA 2001 Dec. 23 10:53 1.0 125 12 GAO 2001 Dec. 23 12:37 5.8 700 12 2 452 267.0450 +0.0004 used for proﬁle SAAO 2001 Dec. 23 19:54 4.9 1663 GAO 2001 Dec. 24 12:37 6.0 720 used for proﬁle CAHA II 2002 Oct. 31 23:11 3.6 50 used for proﬁle CAHA II 2002 Nov. 04 22:43 1.8 27 used for proﬁle CAHA II 2002 Nov. 07 02:14 2.4 17 259 2 452 586.6506 +0.0010 CAHA II 2002 Nov. 09 23:02 6.9 99 Johnson I ﬁlter data CAHA II 2002 Nov. 11 22:59 5.2 75 262 2 452 590.5304 −0.0010 Johnson I ﬁlter data
W2606815349.txt	https://periodicos.ufba.br/index.php/crh/article/download/20022/Andrea%20Borges	fr		FAZER DO VELHO UMA NOVIDADE: as reinvenções dos best-sellers juvenis	Caderno CRH	2,017	cc-by	9,111	FAZER DO VELHO UMA NOVIDADE: as reinvenções dos best-sellers juvenis1 DOSSIÊ Andréa Borges Leão Andréa Borges Leão* O artigo organiza um debate sobre a produção literária juvenil associada à venda massiva. Tem o duplo objetivo de conceituar os best-sellers e traçar as linhas históricas de conexão da produção contemporânea com a presença do livro europeu e americano na edição brasileira, desde o século XIX. A formação de um espaço literário nacional foi marcada pela importação de clássicos do patrimônio literário mundial, romances de viagem e de aventura, narrativas sentimentais e contos de fadas orientados pela circulação transatlântica da oferta e do consumo massivo. Os critérios estabelecidos para a análise da obra seriada da escritora Thalita Rebouças, que hoje objetiva o fenômeno best-seller nacional, embasam o argumento de que o livro juvenil, quando pego na lógica da circulação mundial da cultura, lança mão de um retorno temático ao passado, reinventando-se ao fazer do velho sempre uma novidade. INTRODUÇÃO: os fluxos transnacionais da cultura nacional 1 Já publiquei sete livros em Portugal e, em 2012, se tudo correr bem, meus livros serão editados em outros países da Europa e na América Latina. Depois de tantos anos correndo atrás, atingi um milhão (!!) de livros vendidos em 2011. Mas eu ainda quero mais, muito mais. Thalita Rebouças Até a década de 1980, os escritores, editores e estudiosos da literatura juvenil brasileira buscavam articular um projeto nacional comum alicerçado nas diferenças regionais.2 Não se pode desconhecer que os esforços na busca de uma marca nacional como marca identitária correspondiam a um momento de1 2 * Universidade Federal do Ceará – UFC. Departamento de Ciências Sociais. Programa de Pós-Graduação em Sociologia. Av. da Universidade, 2995. Cep: 60.020-181. Benfica – Fortaleza – Ceará – Brasil. aborgesleao@gmail.com 1 Devo essa expressão a Jean-Marc Gouanvic, que a publicou no texto “Traduire/adapter les classiques de la littérature ‘populaire’ américaine en français, ou de l’art de faire ‘du neuf avec du vieux’”. In: Sapiro, Gisèle (sous la direction). Les Contradictions de la Globalisation Éditoriale. Note-se que, no Brasil, os estudos críticos da literatura infantil e juvenil são tardios e dispersos. Marco inicial é o livro de Leonardo Arroyo, Literatura Infantil Brasileira, publicado Melhoramentos em 1968. 2 http://dx.doi.org/10.1590/S0103-49792016000300004 cisivo na luta pela constituição e autonomia de um espaço para o livro infantil e juvenil. Monteiro Lobato, Orígenes Lessa e toda a geração de escritores surgida nos anos 1970 e 1980, a exemplo de Ruth Rocha, Ana Maria Machado, Ziraldo, Marina Colasanti, entre outros, foram importantes artífices desse movimento. Só assim os caminhos seriam pavimentados para o amadurecimento do gênero e a profissionalização do trabalho literário. Hoje, os infantojuvenis − recriados muito mais como um gênero da edição do que da literatura – são as melhores vendas das editoras, além de objetos de políticas públicas, apostas e investimentos comerciais em traduções para o mercado mundial. Os produtores já não se movem mais em função das disputas simbólicas em torno do livro nacional. As oposições entre o nacional e o importado, o popular e o canônico, perderam forças ou, ao menos, foram levadas a se redefinir, e a presença das traduções se impõe como modelo, não provocando mais tanto conflito e tensão3 (Fundação…, [20--]). 3 A Fundação Nacional do Livro Infantil e Juvenil (FNLIJ), importante instância de mediação crítica, promove concursos e atribui selos de qualidade e excelência literária aos livros produzidos no Brasil. Sua atuação em prol da 463 Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 Palavras-chave: Best-sellers juvenis. Circulação transnacional da cultura. Série literária. Autoria literária. Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 FAZER DO VELHO UMA NOVIDADE ... Desde o século XIX, a formação de um espaço literário nacional foi marcada pela importação de clássicos do patrimônio universal, romances de viagem e de aventura, narrativas sentimentais e contos de fadas europeus orientados pela circulação transatlântica da oferta de países como França, Portugal e Inglaterra, e pelo consumo massivo incrementado pelos novos leitores das ex-colônias americanas. Por sua vez, os escritores brasileiros também cruzaram fronteiras. A obra de José Mauro de Vasconcelos, autor do best-seller O Meu Pé de Laranja Lima, publicado em 1968, abre um profícuo debate sobre a dimensão transatlântica do que se constrói como ficção no plano local, além de reequilibrar os termos da oposição entre sucesso comercial e legitimidade literária. Em 1978, a editora francesa Stock forma a coleção de livros de bolso Mon bel oranger com as traduções dos romances de Vasconcelos, que conhecem um sucesso absoluto4 (Avignon off, 2015). A não equivalência linguística e as forças desiguais implicadas nas trocas e empréstimos entre espaços literários nacionais, a exemplo das que organizam, ainda hoje, a carreira internacional do escritor brasileiro na França, permitem observar processos de formação literária em constituição nos dois países. Não se pode perder de vista, no entanto, que as circulações dos livros no mercado mundial de bens simbólicos são fortemente assimétricas, como lembram Sapiro, Pacouret e Picaud (2015), com a tendência à hegemonia do mercado norte-americano, em geral, e o de língua inglesa, em particular. Neste ensaio, formulo o duplo objetivo de conceituar os best-sellers contemporâneos e formação da leitura literária a leva a conhecer uma parte dos livros publicados no Brasil. Esses conflitos e tensões, no entanto, aparecem em algumas diretrizes e tomadas de posição da FNLIJ. 4 Sobre o percurso longevo e atual da obra de José Mauro de Vasconcelos na França, gostaria de destacar a adaptação de O Meu Pé de Laranja Lima, traduzido como Mon Bel Oranger, para o teatro juvenil de marionetes, em 2015, da companhia de teatro para jovens Théâtre De La Courte Échelle. Sobre a apresentação festival “Avignon off 2015”. Disponível em: https://compagnieaffable.wordpress. com/2015/05/29/le-programme-du-festival-davignon-off2015-est-en-ligne/. traçar as linhas de suas conexões, na história da edição juvenil brasileira, com a presença do livro europeu. Nos dias que correm, cada vez mais os produtores se orientam por uma concepção de campo literário em escala internacional, sobretudo face à intensificação das trocas decorrentes da concentração de grupos editoriais. Pode-se afirmar que a circulação transnacional dos livros e impressos publicados na Europa é um traço comum entre a atualidade e o passado. No segundo tempo da discussão, a obra seriada da escritora Thalita Rebouças, que objetiva o fenômeno best-seller, é situada nas diferenças e continuidades das estratégias, alvos de conquista e trajetórias para a profissionalização literária. A autora de sucesso, que bateu a casa do milhão na venda de exemplares5 (Thalita, [20--]), circula desenvolta entre os mundos da literatura, do teatro, do cinema, da televisão, do futebol e do carnaval, inventando uma posição literária de fronteira, como forma de aquisição de valor. Observando suas estratégias de aquisição de valor e afirmação de visibilidade, pode-se dizer que as fronteiras do campo literário são reposicionadas. Os critérios metodológicos estabelecidos para a análise embasam o argumento de que os livros best-sellers, pegos na lógica da circulação mundial da cultura, lançam mão de um retorno temático ao passado, reinventando-se ao fazerem do velho uma novidade. O trabalho dos agentes da circulação do livro, vinculados ou não ao mundo oficial – agentes literários, tradutores, livreiros, editores, diretores de coleção, professores − ganha protagonismo, assim como a balança de trocas e apropriações entre as mais diversas línguas nacionais. A relação dessa produção ficcional com as instâncias de legitimidade e mediações da literatura também se redefine. Tudo leva a crer que os escritores em posição best-seller tendem a dispensar os mediadores que portam competências intelectuais específicas, caso da crítica especializada. 5 Thalita Rebouças atinge a marca de 1 milhão de livros vendidos em 2011, com a série Fala Sério. 464 Andréa Borges Leão QUANDO OS BESTS TORNAM-SE LONG-SELLERS Um livro best-seller destinado a jovens leitores pode transforma-se em long-seller, ou até em clássico, adquirindo estabilidade pelo êxito das vendas, a depender do teor do que se configura como legitimidade, leia-se visibilidade pela repetição do consumo, conquistado pelo texto e pelo autor face aos leitores-fãs (Heinich, 2012). Por isso, a questão mais importante para a definição do livro best-seller não pode ser reduzida ao êxito comercial. O prestígio literário que reveste ou não uma obra e a figura do autor é o critério fundamental na classificação desse gênero do impresso. Os índices de prestígio são alcançados pelo reconhecimento do público massivo e, em decorrência, emergem novas exigências de originalidade e singularidade, tanto no texto escrito como no estatuto da autoria literária. Nesse caso, os prêmios das instituições literárias e os selos de indicações de usos nas escolas são atributos apenas complementares de reconhecimento. Não que os best-sellers juvenis sejam livros publicamente condenáveis. Simplesmente inexistem em certos espaços de mediações críticas porque são desconsiderados como ficção literária. Uma pergunta se impõe: a que se deve o silêncio histórico dos estudiosos, mediadores tão importantes nas classificações da ficção juvenil? As melhores vendas apresentadas periodicamente nas listas dos infantojuvenis publicadas na grande imprensa e sites especializados não delimitariam as obras, sob o ângulo de análise da sociologia, a uma classificação meramente comercial, sobretudo porque dizem respeito à preferência de milhares de leitores. Uma autora na posição best-seller, situada nas fronteiras entre diversos setores da produção cultural, depende da conquista de visibilidade midiática proporcionada pelas estratégias de publicidade dos grupos editoriais representados por suas obras. Mas, fundamentalmente, depende da cumplicidade dos leitores. Por 465 Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 Outro ponto de deslocamento relacionado ao precedente concerne ao público de leitores. Situados em blogs na esfera digital, agrupados ou individualmente, os jovens leitores de best-sellers são chamados a exercer funções críticas, são autorizados e autorizam-se a interpretar e a comentar as narrativas a partir de suas próprias categorias de julgamento estético em formação, uma vez que as fronteiras entre o mundo do livro, o mundo do autor e seus próprios mundos se esgarçam a cada novo lançamento, a cada nova postagem dos editores nas redes sociais, a cada nova aparição pública de escritores celebridades. Valérie Beaudouin (2012) argumenta não se tratar propriamente da dispensa ou supressão de mediadores especializados, mas de uma redefinição nas formas de mediação e julgamento, quando autores, sobretudo os que atuam nos espaços de escrita da web, inscrevem os leitores − e até seus próprios pares − nos territórios da crítica profissional. Para esta autora, fica difícil uma simples projeção do campo literário, tal como definiu Bourdieu (1996), no espaço digital de produção da escrita. Se toda cultura nacional é um mundo de conexões históricas transnacionais, o estudo dos best-sellers contemporâneos juvenis requer cronologia mais alargada. Os elos com a tradição literária, convocados por uma sociologia processual da circulação simbólica (Elias, 1994), que privilegia operações lógicas e afetivas nas construções de repertórios de textos separados no tempo e no espaço, organiza metodologicamente a reflexão. Para estudar a literatura juvenil na perspectiva das conexões históricas, é preciso ir além da parte mais visível e atual do mercado: o dinamismo e as inovações dos produtores, que sabem tirar proveito da moda inaugurada pela série de sucesso Harry Potter, de J. K. Rowling. É preciso introduzir uma reflexão sobre as transferências, trocas e empréstimos culturais efetivados no longo prazo. Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 FAZER DO VELHO UMA NOVIDADE ... isso, os best-sellers são saudados, para ficar com a observação de Letourneux (2010), como signos do retorno dos jovens às práticas de leitura6 (Casa…, [20--]). Desse modo, conquistam respeitabilidade, ao menos no que concerne à educação do gosto de leitores massivos, enquanto seus autores vão perdendo posição aos olhos e julgamentos dos que falam em nome das convenções canônicas da literatura, sendo postos no lugar de educadores, formadores pela via do entretenimento. Do ponto de vista da crítica literária, o termo best-seller, observa Silvia Borelli (1996, p. 139), “preenche certo número de critérios e requisitos que o separam da literatura culta e erudita”. A classificação editorial, por sua vez, priorizaria critérios quantitativos, encarando o livro como mercadoria sujeita às regras do mercado, com “muito pouca, ou quase nenhuma, densidade literária”, observa a socióloga. Os elementos da linguagem e da narrativa só seriam levados em conta quando úteis para atrair o grande público, quer dizer, para mediar a relação de compra e venda do produto. O best-seller, sob esse ângulo, não passaria de um livro de melhor venda, oferecido a um mercado homogêneo e sem rumo, como se não importassem os agentes mediadores e as instituições culturais que orientam as escolhas − as livrarias, as editoras e as escolas − ou os projetos intelectuais que visam a garantir a qualidade dos produtos no mercado. Nessa linha de raciocínio, o mercado seria constituído apenas pela comercialização do livro “procurado e selecionado por livre escolha”, continua a socióloga (Borelli, 1996, p. 140). As listas dos best-sellers seriam montadas exclusivamente a partir dos dados de vendas que dispensam as mediações de especialistas. Ora, quando consultamos as listas dos livros infantojuvenis produzidos no país7 (Publish…, 2001), constatamos que boa 6 Ver trechos de entrevistas publicadas no site da escritora Thalita Rebouças, que ela sugestivamente nomeia de “casa virtual”. Disponível em: http://thalita-reboucas.blogspot. com.br/2009/01/que-cena-mae.html. 7 Foram consultadas, entre os anos de 2013 e 2015, as seguintes fontes: jornal Folha de São Paulo, revista Veja e site Publishnews. Disponível em: http://www.publishnews.com.br parte se destina à compra e distribuição pelo governo federal e pelos governos estaduais, a exemplo do Programa Nacional do Livro Didático (PNLD), e do Programa Nacional Biblioteca da Escola (PNBE). Não se pode perder de vista, nesta discussão, que as escolhas das agências não poderiam ser aleatórias, uma vez que são guiadas por comissões de especialistas. Ao invés da pura e simples condenação dos best-sellers como gêneros menores, de escritas ligeiras e mecânicas, um caminho possível seria inseri-los nos regimes de historicidade nos quais adquirem sentido, o que permite compreender tanto seus sucessos e suas relações com as instituições de consagração e dominação simbólica como a crítica literária que os condena. O importante, sugere Jean-Yves Mollier (2003), é indagar a história dos sucessos literários do ponto de vista dos produtores e dos leitores: o que se lê em determinados períodos, quais são os usos dos livros na vida cotidiana (distração, instrução, passatempo) e se os best-sellers são fontes de distinção ou de estigmatização para quem os lê ou possui. Deslocada em relação ao que se poderia chamar longevo paradigma Monteiro Lobato – ainda em uso para definir o escritor de excelência, que representa, a um só tempo, o moderno e a nação –, a geração de escritoras teen, composta por Thalita Rebouças, Paula Pimenta, Patrícia Barboza, Babi Dewet, Bruna Vieira, entre outras, é produto da internacionalização da cultura. As trajetórias dessas jovens mulheres cristalizam o investimento da edição juvenil brasileira no mercado latinoamericano e europeu. E as consequências são ainda maiores. Se Thalita Rebouças passa a ser editada em Portugal, fecha contratos com editoras espanholas e italianas8 (Pimenta, 2010), e Paula Pimenta segue em direção aos países de língua espanhola e inglesa, pode-se vislumbrar a constituição de comunidades transnacionais de leitores (em inglês, espanhol, italiano e português), aproximando, por exempo, os tra8 Matéria publicada na revista eletrônica Educar para Crescer, da Editora Abril, reproduzida da Veja Rio. 466 balhos de Paula Pimenta aos da norte-americana Meg Cabot, só para ficar no gênero que mistura romances de sensibilidade do século XVIII, no estilo da inglesa Jane Austen, com recriações dos contos de fadas de Charles Perrault e até das fábulas morais dos irmãos Grimm. As trocas entre os românticos Diário da Princesa (primeiro volume da coleção de Meg Cabot) e o Livro das Princesas (em que colaboram com capítulos Meg Cabot, Paula Pimenta, Lauren Kate e Patrícia Barboza), publicado na coleção Galera Record, da editora Record, supõem a aproximação no gosto, na preferência e na competência, entre categorias nacionais de leitores. Essa pode ser uma revolução simbólica, operada pelas novas mulheres de letras, confirmando a hipótese de que, na lógica da formação dos espaços mundiais, as novidades se fazem com a circulação de velhas fórmulas. Uma das estratégias da internacionalização da cultura, não se pode desconsiderar, é a extensão dos espaços da recepção, lembra Gisele Sapiro (2013). Do ponto de vista dos nexos transnacionais, as autoras best-sellers, que transbordam as próprias obras e circulam pelas fronteiras das produções culturais nacionais, inaugurando um novo sistema de autoria, apresentam-se, sobretudo, como elásticas. É importante destacar, na dinâmica das circulações, a emergência de autores elásticos, que permitem, com maior facilidade, as transferências entre tradições culturais. Os autores de grande elasticidade ou de grande alcance tornam-se estratégicos por transmitirem valores morais sumários, normas de conduta estabelecidas, e mobilizarem um repertório de sentimentos e emoções previsíveis, complementa Pierre Bourdieu (2009). Se as obras preferidas do público não costumam alimentar projeto de nação, tampouco costumam fixar temporalidades, tanto no que toca aos enredos, que podem se passar em qualquer época, quanto no que diz respeito às suas carreiras editoriais de longa duração. Permanecem por meses e até por anos nas listas dos mais vendidos, acabando por se tornarem long-sellers. Vejamos como isso foi possível, no Brasil. A vocação exportadora da edição europeia, francesa e inglesa e, em consequência, o intenso movimento das trocas culturais iniciado pela circulação internacional das obras no século XIX, ocupam o centro das atenções dos estudiosos da formação cultural brasileira. As influências, imitações, assimilações ou dependências da cultura brasileira em relação à presença do produto europeu e americano ainda hoje animam as discussões e pontuam as agendas dos pensadores sociais.9 Se vistas por outro ângulo, percebe-se que as importações de livros populares franceses, portugueses e ingleses, na sociedade brasileira do século XIX, permitiram decisiva transferência de capital literário, formas narrativas e uma estrutura de aprendizagem a ser adaptada por escritores que destinavam suas obras a um incipiente público juvenil, a exemplo de Figueiredo Pimentel, Júlia Lopes de Almeida, Olavo Bilac, Coelho Neto, Viriato Correa e Paulo Barreto, o João do Rio (Leão, 2012). No caso específico do Brasil, o que poderia ser um projeto de colonização cultural, de pura e simples imposição de modelos, imagens e bens de consumo, permitiu o acúmulo de capital simbólico necessário à autonomia da literatura nacional. Importador de artefatos culturais desde fins do século XIX e por todo o século XX e, agora, no XXI, o Brasil assume um lugar na circulação mundial dos bens simbólicos, de tal modo que os horizontes de expectativas do leitorado, que se ampliaram ainda nos tempos do Império, lançam as bases para o que viria a ser um público de massa. Os livreiros estrangeiros que imigraram para o Rio de Janeiro a partir de 1850, os irmãos Garnier, os Aillaud, Garroux, Briquiet e Laurrane, investiram no trabalho de difusão de textos europeus clássicos, já de largo sucesso comercial em seus países de origem, apostando na durabilidade de diversos gêneros, vendendo, editando e reeditando, por longos anos, 9 Gilberto Freyre consagra a esse assunto boa parte da discussão do segundo livro da sua trilogia sobre a formação da sociedade brasileira, Sobrados e mucambos. Consultar: Leão, Andréa Borges, 2014. 467 Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 Andréa Borges Leão Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 FAZER DO VELHO UMA NOVIDADE ... romances para a juventude, a obra de escritores de sucesso, como os franceses Jules Verne e Sophie de Ségur, uma vasta literatura de viagens inglesas, todas as variações do Robinson Crusoé, dos contos de Perrault, dos irmãos Grimm e das viagens de Gulliver. Anos após, empresas nacionais, como as editoras Melhoramentos, Globo e Edições de Ouro, seguem a mesma linha e estratégia, publicando clássicos como Pinóquio, Alice no País das Maravilhas e O Pequeno Príncipe, elenca Laurence Hallewel, em O livro no Brasil (2012). Tudo indica que a contingência das trocas e empréstimos no vasto espaço transatlântico prosseguiu na formação de uma indústria cultural e ainda se faz notar nas reinvenções do século XXI. Assim, torna-se evidente que um dos princípios da formação da esfera literária juvenil brasileira são as incorporações de traduções e adaptações dos clássicos universais. Vale observar que a categoria universal diz respeito aos conteúdos morais das narrativas, fazendo com que suas aplicações práticas sejam um complemento da leitura. No primeiro momento, a criação de um circuito de importação de obras estrangeiras causou reação adversa. A principal delas foi o movimento de formação de um mercado de obras populares nacionais, escritas, impressas e comercializadas no país, protagonizado pelos livreiros-editores Pedro da Silva Quaresma e Francisco Alves de Oliveira, no final do século XIX e início do século XX.10 O livro Para Crianças, uma categoria criada para formar coleções populares e baratas, assumiu o projeto de simbolizar a nação. Não se pode afirmar que toda e qualquer produção ampliada seja situada nas margens do sistema literário, conduzindo ao descarte metodológico das dualidades entre os domínios do popular e do não popular, aí incluindo os bens produzidos em larga escala e que criam ou atendem públicos ampliados. Uma sociologia dos processos de circulação acaba enfrentando o modelo da divisão de uma mesma esfera literária em dois 10 Consultar: Leão, Andréa Borges. Brasil em Imaginação: livros, impressos e leituras infantis (1890-1915), 2012. polos,11 que podem ou não coincidir sem ser obrigatoriamente excludentes. Ora, uma obra, quando circula de um país ou continente a outro, adverte Pierre Bourdieu (2009), leva consigo o campo de sua produção. As transferências entre espaços nacionais se fazem por meio de uma série de operações entre agentes sociais, a exemplo da seleção do que merece ser traduzido e publicado e dos que reúnem competência e legitimidade para verter os textos de uma tradição linguística a outra. Tais operações de leitura estão baseadas em categorias de percepção e problemáticas próprias a um campo nacional específico. O trabalho da tradução atribui novos sentidos aos textos. Na lógica das escolhas, as afinidades de gosto de linhas editoriais e títulos podem ser explicadas por homologia de posições entre campos nacionais. As tramas de imposição simbólica ditadas pelas altas posições de escritores e editoras não reconhecem fronteiras, podendo se tornar um dos pontos de convergência entre as esferas nacionais e uma das propriedades do que se poderia chamar de espaços transnacionais de circulação literária. Por exemplo, gigantes e potentes grupos editoriais empreendem negócios e fusões com os grupos brasileiros já estabelecidos no mercado. Por outro lado, as estratégias de anexação de obras traduzidas ao repertório das coleções e séries juvenis brasileiras têm como objetivo nacionalizar os produtos estrangeiros. A circulação de modelos literários acaba por firmar pactos de leitura que auxiliam a compreensão do sucesso local de aventuras que se passam no Egito antigo, das fantasias medievais com a explosão da magia de fadas, elfos e anões em J. R. R. Tolkien, autor de Senhor dos Anéis, das tramas de Harry Potter, de J. R. Rowling, da novidade dos velhos diários na série Diário de um Banana, de Jeff Kinney, das atualizações da mitologia greco-romana nas traduções de Rick Riordan, entre outros universos e tempos médios 11 No livro As regras da Arte: gênese e estrutura do campo literário (1996), Pierre Bourdieu estuda a dinâmica da vida literária francesa de meado do século XIX. As adaptações, usos e abusos que se fazem para as mais diversas esferas literárias nacionais escapam ao projeto do sociólogo. 468 vividos por guerreiros, príncipes e princesas, anões e dragões. Não por acaso, o livro Pequeno Príncipe, do francês Antoine Saint-Exupéry, publicado em 1943, é um dos mais traduzidos no Brasil e vem ocupando posição de destaque nas listas dos tops nacionais. Da safra dos bestsellers destaca-se o já citado Meu pé de Laranja Lima, de José Mauro de Vasconcelos, publicado em 1968 e hoje traduzido em 52 línguas, o que aponta para o movimento de internacionalização dos produtos nacionais. Mais recentemente, o mesmo tem acontecido com os sucessos de Thalita Rebouças, Fala sério, na editora Rocco, a partir de 2003, e de Paula Pimenta, Fazendo o meu Filme, lançado pela Gutenberg a partir de 2008. O que é efetivamente necessário compreender é que as publicações em séries criam um amplo e cativo mercado receptivo em torno de heróis e heroínas recorrentes e estão longe de significar apenas obras mecânicas e apressadas. Situando a posição best-seller de Thalita Rebouças na longa história da publicação dos gêneros contos de fadas e romances populares sentimentais, fica evidente a permanência de velhos modelos literários em um universo cada vez mais orientado pela circulação transnacional de bens simbólicos, sob o domínio acelerado da concentração de grupos editoriais e de grandes redes de livrarias. THALITA REBOUÇAS, AUTORIA E FRONTEIRAS DA PRODUÇÃO SIMBÓLICA Na sessão “Perguntas e Respostas” da “casa virtual” de Thalita Rebouças, o site oficial da escritora, entre várias cartas de leitores perguntando como conseguir a primeira editora e como tornar-se escritor, além dos pedidos de leitura de originais, aparece uma garota perguntando como fazer para entrar no mundo da moda: “Você pode me ajudar a virar modelo?”12 (Rebouças, [20--]). Thalita responde: 12 As notas e passagens biográficas de Thalita Rebouças foram tomadas do site. Como não existe uma fortuna crítica “Não, meninas, não conheço absolutamente ninguém do mundinho fashion. Melhor procurar agências de modelos no Google. Boa sorte!”. Para quem ocupa uma posição de fronteira entre os espaços da produção simbólica e não estabelece interlocução com os mediadores especializados, a atenção do público é atraída mais pela distribuição massiva de sua imagem, que modula opiniões e dicas de escrita, observa Nathalie Heinich (2012), do que pelas obras publicadas. A conversa epistolar entre a autora e seus leitores, via internet, não poderia girar em torno de estética, simplesmente porque os jovens estão ainda sendo formados em literatura em escolas que talvez nem os preparem para o trabalho crítico. Para um autor, o que importa, continua a socióloga, no regime de visibilidade contemporânea, não é propriamente a assinatura do nome, mas o rosto e o corpo tornados visíveis em grande escala. O mundo dos livros pode levar às passarelas, já que ambos são parte do mesmo universo fashion construído pelo mundo da autora. Vejamos a seguir. Ser uma fada pop, emancipada e consciente de suas emoções, sem coações externas para realizar magias, necessitando apenas da boa leitura de um manual, corresponde ao horizonte de expectativas de uma leitora-modelo. É o que expressa a personagem do livro Uma Fada veio me visitar. Publicado pela editora Rocco, na coleção Jovens Leitores, o livro conta a aventura de uma fada de 857 anos, um ser atemporal que atualiza a moda da década de 60 em pleno ano de 2007. Tatu, a fada, surge em um apartamento da zona sul do Rio de Janeiro com a missão de ajudar a adolescente Luna a dar uma lição de moral na orgulhosa Lara, que vê sua rica família desmoronar com a prisão da irmã, Milena, acusada de participar de roubos de obras de arte no exterior. A moral é a de que não se deve julgar os outros pela aparência. Milena caiu no golpe por ter da obra ou estudo biográfico da escritora, o caminho possível foi recorrer a essa fonte. Tomei todo o cuidado para não cair nas armadilhas da produtora do site, a própria autora, na construção de sua autoimagem. Data de Acesso: 18 de março de 2016. 469 Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 Andréa Borges Leão Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 FAZER DO VELHO UMA NOVIDADE ... acreditado na conversa de um grupo de bandidos que a convidou para uma festa num castelo. Eternamente jovem, aparentando ter 20 anos, a fada Tatu frequenta academia, vê o oprograma “Fadástico” aos domingos, flutua e reaparece, nos capítulos finais, à moda antiga: com uma estrela lilás “carimbada na bochecha”. A mesma estrela que traz a autora na foto que ilustra a orelha do livro, embaraçando o mundo da ficção e a vida de Thalita Rebouças. Essas aproximações entre os dois mundos contribuem para a seguinte estratégia de profissionalização do autor best-seller: a busca de engajamento e cumplicidade do leitor para o exercício de uma função crítica singular, a de comentador nos blogs e redes sociais. Outra estratégia de afirmação da autoridade literária é o encontro com o público nos blocos de carnaval, em torcidas de futebol e séries de televisão, espaços de reconhecineto de Thalita Rebouças. O episódio da série Fala Sério!, Fala Sério, Mãe!, composto de crônicas humoradas, foi adaptado para o seriado As Brasileiras, da Rede Globo, com o nome de A Mãe da Barra. Thalita assinou o roteiro e atuou como atriz. Jornalista de formação, a carioca Thalita Teixeira Rebouças é a única filha de pai dentista e mãe dona de casa. Cursou a faculdade de direito por dois anos, antes de entrar para o curso de jornalismo, onde se titulou. Participou da Oficina de Atores da Rede Globo, uma porta de entrada na emissora, e iniciou a carreira de jornalista no jornal Gazeta Mercantil. Na autorrepresentação oferecida na sua casa virtual, vê-se a imagem de uma mulher empreendedora que tem o projeto intelectual de divertir e cativar os fãs, com a marca do humor e da ironia, praticando a crônica como “romances em pílulas”, aproximando seus livros das séries de sucesso da televisão. A publicação seriada define uma literatura em sequencia (em pílulas), cuja função é atrair o leitor com dramas e aventuras de personagens recorrentes em vários episódios de uma mesma e longa narrativa. A depender da ocasião, adverte Letourneux (2010, p. 91), as séries são descritas como um sintoma do triunfo da industrialização da cultura. Em Fala Sério!, iniciada em 2004, no selo Rocco Jovens Leitores, cada episódio da vida da personagem Maria de Lourdes, a Malu, corresponde a um volume. O leitor acompanha o aprendizado da heroína na seguinte ordem de relações: Fala Sério, Mãe!, Fala Sério, Pai!, Fala Sério, Filha!, Fala Sério, Amor!, Fala sério, Amiga!, Fala Sério, professor!. No blog da autora, o público é chamado a intervir nos textos ainda não publicados no suporte impresso, comentando as tramas precedentes, enviando sugestões para os próximos episódios e para a construção de novos personagens. Forma-se um triângulo amoroso entre autor, leitor e personagem. Além de comentadores, os leitores inspiram a elaboração de novos enredos. Uma visita à “casa virtual” acaba fidelizando-os. Em função dos e-mails recebidos após as visitas, o que traduz o nível de prestígio alcançado, Thalita resolve oferecer um curso online sobre “como iniciar uma carreira de escritor”,13 conferindo a si mesma a legitimidade para falar em nome do que é e de como praticar a literatura. No compartimento Quem sou da casa, Thalita propõe o seu lugar no sistema de fronteira simbólica da autoria, onde se lê: Sou fofa. Pelo menos é o que dizem as boas línguas. Nasci no dia 10 de novembro de 1974, sou carioquésima (daquelas que louvam o Rio e agradecem diariamente por ser de uma cidade tão linda e especial), empolgada, teimosa, escorpiana, portelense, Fluminensesesê!, abracenta, sorridente, chata à beça na TPM, chorona (do tipo ridícula, choro até vendo comercial de detergente), alucinada por sambas e marchinhas de Carnaval, louca por brigadeiro (para comer de colher) e adrenalina − já saltei de pára-quedas e asa-delta algumas vezes − e viciada em algumas séries de TV (Friends, Seinfeld, Sex and The City, Big Bang Theory e Brothers and Sisters são minhas preferidas) (Thalita..., 2016). A vontade de escrever, declara, nasceu da paixão material pelos impressos. Aos 10 13 Disponível em: http://www.iedb.com.br/. 470 anos, sonhava em ser “fazedora de livros”. Depois de corrigir os textos, preocupava-se com os mínimos detalhes da produção − desenhava, encadernava, grampeava a capa e as folhas. Com esses ingredientes autobiográficos, conquista um lugar de inserção ou paixão na cultura literária juvenil e vai preparando, via internet, a recepção de sua obra. A carreira literária de Thalita Rebouças começou mesmo em 2001, na Bienal do Livro do Rio de Janeiro, em frente ao estande de sua primeira editora, Ao Livro Técnico. Foi assim, narra a escritora: Vários autores consagrados estavam presentes, como eu poderia competir com eles? Meu Traição entre amigas era apenas mais um livro naquele universo de títulos disponíveis na Bienal. O estande da minha editora, apesar de bonitinho e bem localizado, era um entre muitos espalhados em dois imensos pavilhões do Riocentro. Se eu quisesse vender livros teria que inventar uma forma de chamar atenção, de aparecer, de me destacar e rápido (Thalita..., 2016). Nessa tarde, lembrou do tempo em que fazia teatro e começou “a bater palmas, a brincar com quem passava na frente do estande e a anunciar o livro em altos brados, como um vendedor empolgado com o seu produto”. Comerciante dela mesma, Thalita Rebouças prepara uma cena literária fora da literatura, dando um rosto a um nome que começaria a circular. O problema por vir seria, lembra Heinich (2012, p. 159), converter o reconhecimento do grande público em valor literário. No relato da atuação, fica evidente o cuidado especial com a didatização de uma imagem que se voltaria para o público juvenil: “logo juntou gente ao meu redor, rindo e escutando, e o livro começou a vender como água no deserto”. Ao mesmo tempo em que recupera a “arte das vovós” contadoras de histórias e transmissoras orais, animando rodas de leitores, encarnava outras figuras populares, os mágicos e os comerciantes de feiras, tornando a Bienal um espaço de construção da autoria, como se ela mesma escrevesse um prefácio elogioso a seu livro. Para Thalita Rebouças, a indentidade de uma auto- ra não se restringiria ao mundo da escrita. A partir daí, começa um trabalho independente de divulgação do livro. Dispensou intermediários como mais tarde dispensaria as mediaçãos críticas, e saiu batendo nas portas das grandes redes de livrarias para anunciá-lo, como um caixeiro viajante ou um vendedor de enciclopédias. Não tardou para que a experiência da escritora pop star se transformasse em ficção. Em março de 2003, assina contrato com a editora Rocco para a publicação do seu primeiro best-seller, Tudo por um Pop Star. Esse livro conta as aventuras de três amigas da cidade de Resende, Manu, Gabi e Ritinha. O trio parte em busca dos ídolos de uma banda internacinal que está vindo ao Rio de Janeiro fazer um show, no estádio do Maracanã. O cantor pop Júnior Lima assina um texto de apresentação aos leitores, que provavelmente fazem parte da mesma comunidade de fãs de suas canções. O universo cor-de-rosa das meninas pop sugere, como hipótese, a filiação atualizada da escritora carioca aos romances populares e sentimentais da série Rosa da coleção Harlequin, que publicava, nos anos 40 e 50 do século XX, histórias açucaradas conhecidas como “romances para moças”. Esses romances formavam a sensibilidade das leitoras numa linha de continuidade à recepção feminina de histórias românticas no século XIX. Tudo sem perder o frescor. As interdependências entre o novo e o velho indicam que uma etapa de formação da esfera literária juvenil contém, simultaneamente, sua antecedência e sucessão. Norbert Elias (1998) descortinaria, nesses processos, tendências de civilização que orientariam estruturas de aprendizagem. A obra de Thalita Rebouças conduz a sua interpretação, retrospectivamente, como uma assimilação dos diversos gêneros da literatura popular, desde os contos de fadas, os romances-folhetins do século XIX, os clássicos universais traduzidos e toda a produção destinada ao grande público que marcou a indústria editorial e a cultura de massa no Brasil. Para Daniel Compère (2011), o romance 471 Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 Andréa Borges Leão Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 FAZER DO VELHO UMA NOVIDADE ... popular nasce no ano de 1836, com o aparecimento do romance-folhetim publicado em capítulos nos jornais franceses. Gênero menor diante da poesia e do teatro, o romance popular voltava-se para a satisfação das expectativas de novos leitores que emergiam com o progresso da alfabetização, as mulheres, os jovens e os trabalhadores. É amplamente difundido graças ao jornal, um meio acessível a todos. Do ponto de vista dos estudos literários, o romance popular inicia a sua carreira por uma não recepção, quer dizer, pela exclusão do repertório das obras legítimas e reconhecidas por instâncias como a crítica e a escola. Assim, continua Compère, os romances populares estão vinculados a um público ampliado, formado pelos progressos da educação e pelo desenvolvimento de novas formas de publicação. O exemplo são as coleções de livros de aventuras e viagens do editor francês Louis Hachette, baratos e de pequenos formatos, feitos para a venda nas estações de trem de Paris. A partir daí, o romance popular foi se diversificando em subgêneros: a ficção-científica, os policiais e westerns, as fantasias, o horror e demais formas sobrenaturais, os sentimentais, os eróticos e a pornografia. Os subgêneros, voltados ao entretenimento dependem dos suportes de difusão e da distribuição comercial ampliada. Pode-se, então, elaborar a hipótese de que as escritoras em posição best-seller utilizam os blogs e redes sócias da internet do mesmo modo que os escritores de romace-folhetim do século XIX utilizavam o jornal impresso e os das novelas utilizavam as rádios, evidenciando uma linha de sucessão também midiática (Ortiz, 1988). Vale lembrar, com Marlyse Meyer (1996), a velocidade com que o romance folhetim oitocentista atravessa o Atlântico e passa a circular no Brasil. As traduções das novelas francesas para o Jornal do Comércio eram feitas imediatamente após aparecerem em Paris. Por essas e outras estratégias de circulação, os best-sellers são constantemente reinventados, e a literatura de entretenimento continua um sucesso. Do mesmo modo, a coletânea de contos O livro das princesas – Novos contos de fadas, publicada pela Galera, do Grupo Editorial Record, em 2013, e na qual colabora a escritora Paula Pimenta, deve muito aos compiladores dos contos populares de fins do século XVII, em especial a Charles Perrault. O livro, que já nasceu duplamente clássico e best-seller, e deve muito à posteridade e às transformações do gênero. Uma chave de compreensão da carreira de Thalita Rebouças é a que situa sua obra nas reinvenções retrospectivas de uma cultura literária para a juventude. Ou melhor, a que apreende os processos de informalização14 (Wouters, 2007) dos comportamentos que investem os livros alinhados em uma dinâmica da produção que atualiza velhas fórmulas editoriais, temas e modelos literários. Os romances de princesas com a nostalgia das aventuras e regressos do heroísmo, as narrativas intimistas de conselhos, diários e cartas, definem uma literatura sentimental, aparentada aos melodramas dos séculos XIX e XX. Não faltam regras e padrões de etiqueta. A permanência, na atualidade, de temas já gastos, como as fadas e outros seres sobrenaturais, evidencia o papel da literatura nos processos de refreamento dos afetos e controle das emoções, conforme identificou Norbert Elias (1994) em estudo clássico baseado no corpus dos livros de boas maneiras. Tudo leva a crer que a civilidade contemporânea é um ponto de encontro com o passado. Os sucessos juvenis, desse modo, ocupam tanto lugares de inserção nos sistemas literários nacionais como se inscrevem no patrimônio universal. O que explica o sucesso de venda e a reação apaixonada são os pactos de leitura firmados entre autoras e leitores-fãs e um maior controle da produção e distribuição dos livros por parte dos escritores. O meio digital abre ca14 Considero processos de informalização,emancipação das emoções nas sociedades contemporâneas. Com a maior flexibilidade e diferenciação dos códigos sociais, as pessoas tornam-se menos rígidas e mais conscientes das restrições sociais. 472 Andréa Borges Leão 15 Norbert Elias (1994), na teoria da civilização, incorpora a ideia de mudança na estrutura psíquica e cognitiva dos indivíduos, atribuindo a esse fenômeno o termo “psicogênese”. do computador, até mesmo a leitura escolar de apoio às “práticas da educação básica”16 (Fundo Nacional do Desenvolvimento da Educação, 2012). Diante disso, os usos dos best-sellers juvenis trazem para o debate o problema das novas recepções e modalidades de apropriação (nos blogs de escritores e nas redes sociais), que supõem um gosto estético comum às conexões literárias das séries contemporâneas (aos mesmos heróis e tramas). O leitor entra num universo que se torna familiar e não consegue mais sair dele, passando a viver a continuação dos episódios na pele dos heróis. Esta lógica pode fazer de um best um long seller, traçando os destinos da série Fala Sério! por gerações de leitores ainda por vir. CONCLUSÕES A primeira conclusão é a de que um aspecto comum aos livros eleitos pelo público, dos mais populares aos canonizados, é o desconhecimento de nacionalidades. A força de representação dos best-sellers parece seguir a lógica inversa dos discursos da identidade nacional. Se seus autores escapam à categoria de gênios da nação, talvez isso não se deva unicamente à força da interlocução estabelecida com os leitores. Thalita Rebouças encarna a autoria contemporânea por insistir em encontrar seus leitores nos diversos espaços da produção cultural, prolongando o convívio entre as festas de carnaval e tardes de partidas de futebol com a leitura de textos fáceis de ler e de interessar. A segunda conclusão é a de que os heróis, heroínas e tramas dos sucessos juvenis continuam seguindo temas abstratos, como amizade, amor, traição, inveja, o que os torna ainda clássicos, por deixarem as temporalidades e espaços nacionais em suspenso. Por isso, prestam-se tão bem a representar processos de 16 A esse respeito, consultar PNBE. 473 Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 minho para uma nova invenção autoral e, por conseguinte, para a redefinição de interesses e reposicionamento dos agentes nos circuitos de produção e recepção. A nova geração de escritoras, sem ambivalências, concebe os livros simultaneamente como obras literárias e produtos à venda. Tudo indica que o acesso à visibilidade como uma marca de reconhecimento converte o sucesso comercial do livro em valor literário. Para elas, o importante é corresponder aos horizontes de expectativas de meninas fãs de princesas e de meninos românticos. Suas atenções não se prendem aos paradoxos do que seja ou não a verdadeira literatura, do que seja o livro nacional ou as fronteiras que separam o centro e as margens na esfera literária brasileira. O dilema do duplo, intelectual e negociante, que marcou a carreira de gerações de escritores em busca de autonomia no interior de um campo, passa longe delas. O desafio da análise é apreender a psicogênese15 do leitor previsto nos best-sellers e, em consequência, seguir as pistas explicativas das identificações e preferências de longo prazo. Essas pistas nos levam não apenas às variações no gosto literário, mas também às censuras e proibições veiculadas nas narrativas, à assimilação das regras e ao progressivo relaxamento no manejo das emoções nas práticas de leitura. Como lembra Roger Chartier (2000), o livro impresso é o melhor lugar para o encontro entre a forma material, o texto e o leitor. Com relação aos leitores, uma última palavra. Deve-se levar em conta que o gênero jovem adulto, no qual boa parte dos best-sellers juvenis são classificados, relaciona-se tanto às injunções das políticas públicas do livro e da leitura, a exemplo do Programa Nacional Biblioteca na Escola (PNBE), como às transformações do mercado editorial transnacional. Os livros destinados a um público leitor que vai dos 12 aos 20 e poucos anos preveem leituras tanto no suporte impresso como na tela Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 FAZER DO VELHO UMA NOVIDADE ... nacionalização literários diversificados (afinal, quem conseguiria localizar o planeta de O Pequeno Príncipe?). Também se tornam clássicos por força da estabilidade das vendas, permanecendo por anos a fio nos catálogos das editoras. Em consequência, os best-sellers são facilmente traduzidos e adaptados, circulando com desenvoltura de uma língua a outra, de uma dinâmica literária a outra, por entre espaços nacionais, continentes e épocas. Os temas, potencialmente universais, resumiu muito bem Jean-Marc Gouanvic (2009), possuem o condão de fazer do velho sempre uma novidade. O que muda, com a passagem do tempo, são as práticas de leitura de acordo com os modos de vida e as percepções dos leitores. A terceira conclusão segue aquela a que chegou o sociólogo Renato Ortiz (1988), em análise sobre a consolidação de uma sociedade de mercado no Brasil nos anos de 1970: em uma indústria popular de massa, o contato histórico com os produtos estrangeiros por meio de importações e traduções, diversas vezes pensado como dependência e colonialismo cultural em relação aos países de economias centrais, atesta uma mudança de posição do país periférico no contexto internacional. Esse reposicionamento coincide com a progressiva autonomia da esfera cultural brasileira. Sem desconhecer, é claro, que os espaços internacionais de circulação dos textos não estão orientados por livres trocas, mas por relações de força hierárquicas e desiguais entre agentes e instituições. A quarta e última conclusão aponta para a necessidade de se fazer um inventário histórico dos sucessos juvenis, considerando as especificidades de cada conjuntura editorial e cada espaço literário, o que exige o exame de uma sequência temporal alongada e capaz de evidenciar as permanências e mutações nos processos de formação do mercado de bens simbólicos. A própria noção de sucesso muda com as preferências de cada público, do mesmo modo que, hoje, as boas vendas devem muito ao alargamento dos circuitos de difusão da publicidade na internet. A leitura dos novos contos de fadas não é simples experiência de retorno ao passado. As mutações da sociedade contemporânea afetam a estrutura das narrativas e a concepção dos personagens, aproximando o universo atemporal da fantasia à sociedade midiática, à tecnologia e à tradição. Tudo leva a crer que existam gêneros ficcionias em estado de latência; de tempos em tempos, com a emergência das indústrias editoriais, eles vêm à tona. O importante é considerar os gêneros literários, sobretudo, como construções do mercado editorial, o que explica os dispositivos de classificação e controle dos textos utilizados pelos agentes da produção. Reunir textos e inventar coleções juvenis também diz respeito às gradações do aprendizado e às formas de divertimento que podem oferecer (Cf. Olivero, 1999). Os critérios de classificação do cânone literário em padrões de excelência, que operam distinções e gradações de literalidade, o que é ou não a boa ficção, pouco ajudam a elucidar o problema. Sendo assim, a compreensão do que sejam obras populares e eruditas, best ou long-seller, é apropriadamente alcançada por meio de uma sociologia processual da circulação simbólica (Elias, 1994), que privilegia operações lógicas e afetivas nas construções de repertórios de textos separados no tempo e no espaço. Recebido para publicação em 04 de maio 2016 Aceito em 05 de setembro de 2016 REFERÊNCIAS ALMEIDA, P. Surge uma nova categoria de leitores: o new adult. 2013. Disponível em: http://www.publishnews.com. br/materias/2013/05/28/73226-surge-uma-nova-categoriade-leitores-o-new-adult. Acesso em: 26 mar. 2016. ARROYO, L. A literatura infantil brasileira. São Paulo: Companhia editora melhoramentos, 1988. AVIGNON Off 2015. 2015. Disponível em: http://www. larevueduspectacle.fr/Avignon- Off-2015-Mon-beloranger-d-apres-Jose-Mauro-de-Vasconcelos_a1378.html. Acesso em: 25 fev. 2016. BEAUDOUIN, V. Trajectoires et réseau des écrivains sur le web: construction de la notoriété et du marché. Réseaux: industries culturelles et internet: les nouveaux instruments de la notoriété, Paris, v. 30, n. 275, sept./oct. 2012. 474 Andréa Borges Leão LEÃO, A. B. Brasil em imaginação: livros, impressos e leituras infantis (1890-1915). Fortaleza: INESP: UFC, 2012. BOURDIEU, P. As regras da arte: gênese e estrutura do campo literário. São Paulo: Companhia das letras, 1996. ______. Nós e os franceses: Gilberto Freyre à prova de Adèle Toussaint-Samson. Etnográfica, Lisboa, v. 18, n. 3, p. 625-647, 2014. ______. Les conditions sociales de la circulation internationale des idées. In: SAPIRO, G. L’espace intellectuel en Europe: de la formation des États-nations à la mondialisation XIXe-XXIe siècle. Paris: La découvert, 2009. LE PROGRAMME du Festival d’Avignon OFF 2015 est en ligne! 2015. Disponível em: https://compagnieaffable. wordpress.com/2015/05/29/le-programme-du-festivaldavignon-off-2015-est-en-ligne/. Acesso em: 20 mar. 2016. CASA virtual. [20--]. Disponível em: http://www.thalita. com/site/quem-sou.html. Acesso em: 20 mar. 2016. LETOURNEUX, M. Séries, collections et sérialité en littérature pour la jeunesse. La revue des livres pour enfants, Paris, n. 256, dec. 2010. CHARTIER, R. La culture de l’imprimé, avant-propos. In: ______. Les usages de l’imprimé. Paris: Librairie artheme fayard, 1987. MEYER, M. Folhetim: uma história. São Paulo: Companhia das letras, 1996. ______. As práticas da escrita: da renascença ao século das luzes. São Paulo: Companhia das letras, 1991. (História da vida privada, 3). MOLLIER, J.-Y. Histoire culturelle et histoire littéraire. Revue d’histoire littéraire de la France, v. 103, n. 3, p. 597612, 2003. ______. A Ordem dos livros, autores e bibliotecas na Europa entre os séculos XIV e XVIII. Brasília: UnB, 1994. OLIVERO, I. L’invention de la collection: de la diffusion de la littérature et des savoirs à la formation du citoyen au XIXe siècle. Paris: Éditions de L’IMEC, 1999. ______. Lectures “populaires”: culture écrite et société. In: ______. L’ordre des livres (XIV – XVIII siècle). Paris: Albin Michel, 1996. ______. Entre poder y placer: cultura escrita y literatura en la edad moderna. Buenos Aires: Ediciones cátedra, 2000. ______. Cultura escrita, literatura e história: conversas de Roger Chartier com Carlos Aguirre Anaya, Jesús Anaya Rosique, Daniel Goldin e Antonio Saborit. Porto Alegre: Artmed editora, 2001. ORTIZ, R. A moderna tradição brasileira: cultura brasileira e indústria cultural. São Paulo: Editora brasiliense, 1988. PIMENTA, L. Ela atende as meninas. 2010. Disponível em: http://educarparacrescer.abril.com.br/leitura/thalitareboucas-574173.shtml. Acesso em: 5 jan. 2016. PIMENTA, P. Princesa pop. In: CABOT, M. O livro das princesas: novos contos de fadas. Rio de Janeiro: Editora record, 2013. COMPÈRE, D. Les romans populaires. Paris: Presses sorbonne nouvelle, 2011. PUBLISH News. 2001. Disponível em: www.publishnews. com.br. Acesso em: 23 mar. 2016. ELIAS, N. O processo civilizador: uma história dos costumes. Rio de Janeiro: Jorge Zahar, 1994. v. 6. REBOUÇAS, T. Uma fada veio me visitar. Rio de Janeiro: Editora rocco, 2007. ______. Como pueden las utopías científicas y literarias influir sobre el futuro? In: WEILER, V. Figuraciones en proceso. Santafé de Bogotá: Fundación Social, 1998. ______. Que cena, mãe! 2009. Disponível em: http:// thalita-reboucas.blogspot.com.br/2009/01/que-cena-mae. html. Acesso em: 20 mar. 2016. FREYRE, G. Sobrados e mucambos: decadência do patriarcado rural e desenvolvimento do urbano. São Paulo: Global, 2003. ______. Quem sou. [20--]. http://www.thalita.com/site/ quem-sou.html. Acesso em: 18 mar. 2016. FUNDAÇÃO Nacional do Livro. [20--]. Disponívelem: http://www.fnlij.org.br/. Acesso em: 15 fev. 2016. FUNDO NACIONAL DO DESENVOLVIMENTO DA EDUCAÇÃO. Apresentação. 2012. Disponível em; http://www.fnde.gov.br/programas/biblioteca-da-escola/ biblioteca-da-escola-apresentacao. Acesso em: 15 fev. 2016. GOUANVIC, J.-M. Traduire adapter les classiques de la littérature “populaire” américaine en français, ou de l’art de faire “du neuf avec du vieux”. In: SAPIRO, G. (Dir.). Les contradictions de la globalisation éditoriale. Paris: Nouveau monde éditions, 2009. p. 303-313. HALLEWELL, L. O livro no Brasil: sua história. São Paulo: EDUSP, 2012. HEINICH, N. De la visibilié: excellence et singularité en régime médiatique. Paris: Éditions gallimard, 2012. IEDB EDUCAÇÃO interativa online. 2009. In: http://www. guiaead.com.br/instituicao/ver/219. Acesso em: 26 mar. 2016. SAPIRO, G. Le champ est-il national? La théorie de la différenciation sociale au prisme de l’histoire globale. Actes de la Recherche en Sciences Sociales, Paris, p. 71-85, déc. 2013. ______. (Ed.). Translatio: le marche de la traduction en France à l’heure de la mondialisation. Paris: CNRS éditions, 2008. ______; PACOURET, J.; PICAUD, M. Transformations des champs de production culturelle à l’ère de la mondialisation. In: La culture entre rationalisation et mondialisation. Actes de la Recherche en Sciences Sociales, Paris, p. 5-13, mars, 2015. THALITA Rebouças. [20--]. Disponível em: http://www4. thalitareboucas.net. Acesso em: 19 mar. 2016. ______. 2016. Disponível em: http://editoras.com/thalitareboucas/. Acesso em: 19 mar. 2016. WOUTERS, C. Informalization: manners & emotions since 1890. London: Sage Publications, 2007. 475 Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 BORELLI, S. H. S. Ação, suspense, emoção: literatura e cultura de massa no Brasil. São Paulo: EDUC: Estação liberdade, 1996. FAZER DO VELHO UMA NOVIDADE ... MAKING THE OLD A NOVELTY: reinventions of children and young adult literature best-sellers C’EST AVEC DU VIEUX QUE L’ON FAIT DU NEUF: les ré-inventions des best-sellers juvéniles Andréa Borges Leão Andréa Borges Leão This article organizes a debate about the young adult literary production associated to mass production. Thus, the article has the double objective of conceptualizing the best-sellers and tracing the historic lines of connection of the contemporary production with the presence of European and American books in Brazilian edition since the 19th century. The formation of a Brazilian national literary space was marked by the importing of classics of world literary heritage, travel and adventure romances, sentimental narratives and fairy-tales guided by the transatlantic circulation of the offer and the mass consumption. The criteria established for the analysis of the writer Thalita Rebouças’ series, which focus on the national bestseller phenomenon today. These criteria supports the argument that the young adult book when following the logic of world culture circulation makes use of a thematic return to the past, reinventing itself by making the old a novelty. L’article propose un débat sur la production littéraire juvénile associée aux ventes massives. Il a comme double objectif de conceptualiser les best-sellers et de retracer le lien entre les tendances historiques de la production contemporaine, en prenant en considération la présence des livres européens et américains dans les éditions brésiliennes depuis le XIXe siècle. La formation d’un espace littéraire national a été marquée par l’importation des classiques du patrimoine littéraire mondial, romans de voyage et d’aventure, narratives sentimentales et contes de fées, suivant la tendance de la circulation transatlantique de l’offre et de la demande massives. Les critères établis pour l’analyse de l’oeuvre de l’écrivaine Thalita Rebouças, qui fait actuellement l’objet d’un phénomène national de best-seller, se basent sur l’argument selon lequel les livres juvéniles, lorsqu’ils entrent dans la logique de la circulation mondiale de la culture, ont recours à des thèmes du passé et se réinventent en faisant toujours du vieux quelque chose de neuf. Caderno CRH, Salvador, v. 29, n. 78, p. 463-476, Set./Dez. 2016 Keywords: Young adult best-sellers. Transnational Mots-clés: Best-sellers juvéniles. Circulation circulation of culture. Literary series. Literary transnationale de la culture. Série littéraire. Auteur authorship. littéraire. Andréa Borges Leão – Doutora em Sociologia pela Universidade de São Paulo. Professora do Departamento de Ciências Sociais e do Programa de Pós-Graduação em Sociologia da Universidade Federal do Ceará. Pós-doutorado em História Cultural, na École des Hautes Études en Sciences Sociales, Paris, sobre as coleções juvenis da Livraria Garnier, e no Centre d’Histoire Culturelle des Sociétés Contemporaines, da Université de Versailles Saint-Quentin-en-Yvelines, sobre as traduções brasileiras dos livros juvenis de Sophie de Ségur e Jules Verne. Integra os grupos de pesquisa CMD (Cultura, Memória e Desenvolvimento) e GECCA (Grupo de Estudos em Cultura, Comunicação e Arte). Desenvolve pesquisas na área da edição e circulação da literatura infantil e juvenil. Atua nos seguintes temas: Sociologia e História Editorial, Práticas Editoriais, Circulação da Cultura e da Literatura, Representações Literárias e Sociologia Histórica da Cultura. Publicações recentes: O popular no Brasil numa fábula de costume francesa: estéticas e mediações transatlânticas. Sociedade e Estado (UnB. Impresso), v. 31, p. 631-650, 2016; Fazer do velho uma novidade: as reinvenções dos best-sellers juvenis. Caderno CRH (UFBA. Impresso), v. 29, p. 463-476, 2016; Ceará, lado moleque (as letras e a sociogênese do humor). Arquivos do CMD, v. 3, p. 1-16, 2015. 476
https://openalex.org/W2900629493	https://jyx.jyu.fi/bitstream/123456789/60506/1/teacherpsyckalajamantyla.pdf	English	null	3. ‘The English Class of My Dreams’: Envisioning Teaching a Foreign Language	Multilingual Matters eBooks	2,018	public-domain	8,405	This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Kalaja, Paula; Mäntylä, Katja Author(s): Kalaja, Paula; Mäntylä, Katja Title: "The English Class of My Dreams!" : Envisioning Teaching a Foreign Language Title: "The English Class of My Dreams!" : Envisioning Teaching a Foreign Language Year: Version: Copyright: Rights: Rights url: Please cite the original version: In Copyright http://rightsstatements.org/page/InC/1.0/?language=en © Multilingual Matters, 2018. Accepted version (Final draft) Kalaja, P., & Mäntylä, K. (2018). "The English Class of My Dreams!" : Envisioning Teaching a Foreign Language. In S. Mercer, & A. Kostoulas (Eds.), Language Teacher Psychology (pp. 34-52). Multilingual Matters. Psychology of Language Learning and Teaching, 1. https://doi.org/10.21832/9781783099467-007 2018 Chapter 3 ‘The English Class of My Dreams’: Envisioning Teaching a Foreign Language PAULA KALAJA and KATJA MÄNTYLÄ The psychology of the second (or foreign) language learner has been extensively researched since the mid-1950s. Unfortunately, the same cannot be said of the psychology of the teacher. Of the psychological make-up of the learner, motivation has been found to be one of the key issues affecting not only the outcome but also the process of learning second (or foreign) languages. Much less is known of the motivation of the teachers involved in these efforts. The teaching of English as a foreign language is faced with new challenges because of the rapid spread of the language in different parts of the world, including Finland. Here, this has meant reconsidering the status of the language, with consequent revisions in the curricula of both schools and teacher education. In this chapter, our concern is with university students of English and how they keep up their motivation as future teachers. More specifically, our concern is the development of their pedagogical knowledge in teaching the language before they start their careers as qualified teachers in Finland and their ability to turn this knowledge into principles and practices that they could imagine applying in their future classes of English. Related to their motivation and identities as future professionals, it is their ability to envision, or the visions of these students, which the study will look into. For this purpose, a group of pre-service teachers were asked to visualise and draw an image of their ideal class of English to be given in the not-so-distant future and elaborate on the picture in writing: where would the class take place, what would be taught and how? This chapter is organised as follows. First, some background is provided for the present study by reviewing such key issues as teacher motivation, envisioning and narratives, and by summarising some related studies. After this, the study that was carried out is reported in more detail, including its aims, data collection and analysis, and findings. Finally, the implications of the study are discussed. Please cite the original version: Kalaja, P., & Mäntylä, K. (2018). "The English Class of My Dreams!" : Envisioning Teaching a Foreign Language. In S. Mercer, & A. Kostoulas (Eds.), Language Teacher Psychology (pp. 34-52). Multilingual Matters. Psychology of Language Learning and Teaching, 1. https://doi.org/10.21832/9781783099467-007 Teacher motivation and envisioning The motivation of learners of foreign languages has been studied extensively and from a number of different theoretical starting points in the past few decades (for a review, see e.g. Dörnyei & Ushioda, 2010). Recently, it has been suggested that learner motivation be viewed in terms of a motivational self-system (e.g. Dörnyei & Ushioda, 2011), consisting of learners’ possible selves, including Ideal self and Ought-to self, and related to their past experiences of learning a foreign language. This system taps into two major aspects of learning a foreign language: firstly, the learners’ own aspirations, fears and ideals (in comparison to their actual self or -selves); and, secondly, the expectations of, or social pressure from, others around them (e.g. teachers, parents, friends, school and society at large), each playing a role in, and having an effect on, the process and outcomes of learning a foreign language. Even more recently, it has been suggested that the motivational self-system could be applied to teachers of foreign languages, too (e.g. Dörnyei & Kubanyiova, 2014): to both pre-service teachers and in-service teachers. Teacher motivation – in all its current complexity – is thus a crucial issue to address in teacher education: in the course of their studies, student teachers acquire pedagogical knowledge of the various aspects involved in learning and teaching foreign languages, while at the same time improving their proficiency in the language they will be teaching. In addition, their identities and Ideal or Ought-to selves are bound to develop. It is important for student teachers to compare the current reality in schools with their ideals: how do they see themselves and their practices now and in the future, and how far or how close are these from each other? The ability to envision their teaching in the years to come is thus related to their motivation. However, as pointed out by Dörnyei and Kubanyiova (2014: 125, emphasis original), the purpose is not to identify some kind of idealised fantasy image of a language classroom that may never exist, but, rather, to develop a personally meaningful possible vision that is integral to who the teacher is and that is sensitive to the context in which his/her work is located. Teacher motivation and envisioning The point is to make student teachers aware of their beliefs about their professional future, and to reaffirm or reconsider these, which will possibly have consequences for the principles and practices that they will apply in their teaching once they enter working life as qualified teachers. Visual narratives looking forward in time In teacher education, narratives have been acknowledged to be a means for pre- and in-service teachers to make sense of themselves and their profession (e.g. Johnson & Golombek, 2011, 2013); more specifically, in constructing their identities and reflecting on their experiences of teaching English or other foreign languages. One way of describing narratives is to say that they consist of a series of events: Briefly, in everyday oral storytelling, a speaker connects events into a sequence that is consequential for later action and for the meanings that the speaker wants listeners to take away from the story. Events perceived by the speaker as important are selected, organized, connected, and evaluated as meaningful for a particular audience … (Riessman, 2008: 3) Briefly, in everyday oral storytelling, a speaker connects events into a sequence that is consequential for later action and for the meanings that the speaker wants listeners to take away from the story. Events perceived by the speaker as important are selected, organized, connected, and evaluated as meaningful for a particular audience … (Riessman, 2008: 3) Another way is to define them by function. Narratives can be used not only in looking back but also in looking forward: ‘[Stories] assist humans to make life experiences meaningful. Stories preserve our memories, prompt our reflections, connect us with our past and present, and assist us to envision our future’ (Kramp, 2004: 107, as quoted in Barkhuizen, 2013: 4). Furthermore, narratives can be recounted in more than one mode: they can be verbal (oral or written), visual, or multimodal, e.g. text complemented with pictures, tables, figures, etc. or sound and video clips. Depending on the mode, narratives have different possibilities and limitations (Kress & van Leeuwen, 2006: 46): what can be described verbally may not be possible to do visually, and vice versa. The present study is an attempt to explore further the possibilities of visual narratives (e.g. Kalaja et al., 2013; Kalaja, 2016), this time in looking forward in time and addressing visions of future teachers of English. Review of related studies A small number of studies have been carried out that have focused on visions of teachers or future teachers or made use of narratives. In her studies, Hammerness (e.g. 2003, 2006) asked novice teachers in the United States to imagine their ideal classroom. Data were collected by administering a survey (N = 80) and conducting interviews with some of the teachers (N = 16) involved in which they were asked to imagine taking the interviewer round their ideal classroom and to give details in response to a set of questions. Basically, they were asked what they would teach, how and why. The teachers’ visions (reported verbally) were analysed along three dimensions: focus, range and distance. In other words, the images or visions of future classrooms were viewed in terms of either being clear or vague in focus; broad or narrow in range; or close to or distant from the teachers’ current practices or daily experiences. Although these two studies are interesting in their research topic, design and methodology, they do not focus on language teachers or their ideal visions. In contrast, a follow-up study to a longitudinal project entitled From Novice to Expert (e.g. Kalaja et al., 2013; Kalaja, 2016) asked student teachers of foreign languages, including English (N = 61), who were just about to graduate from an MA degree programme in Finland, to envision a future class. This was done not only verbally but also visually by having the participants draw a picture and comment on it briefly in writing. This study made use of visual narratives to look forward in time and asked the teachers to imagine what would be involved in the teaching of foreign languages in the future. In another two studies (Borg et al., 2014; Clarke, 2008), student teachers were asked to recollect some positive examples of what it was like to teach English to small children in two very different contexts: in Spain and the United Arab Emirates. In the first study, the students were expected to describe events like this visually in two different ways: first of all, by drawing a picture before and after a teaching methodology course, and then orally, in an interview. In other words, visual narratives were used to look back in time in order to recollect what had happened before, when teaching English. Review of related studies In the second study, data were collected by asking students to submit postings to an internet discussion forum. The findings of the three studies with student teachers of English or other foreign languages are in fact quite comparable, despite their different foci, type of data or contexts. In all the studies, two discourses could be identified: one was based on the participants’ past experiences of learning English; this was in contrast to the other discourse, which was based on their current understanding of what the teaching of English involves. In the latter, such principles and practices were recycled as student-centredness, the Teacher as Guide, a focus on meanings or oral/real communication, the use of modern IT, and authentic materials. These were complemented with comments on how their classrooms would be organised and what their classrooms would be equipped with. The principles and practices seem to stand in sharp contrast to their past experiences as learners of English, when they were still at school. The present study The present study is a continuation of previous research carried out in Finland, but with a more recent group of student teachers and some refinements in research methodology. Overall, this is an attempt by us to make this group aware, firstly, of their ideal teacher selves (i.e. what they would expect of themselves once they enter working life as professionals in a few years’ time), and secondly, of their current teaching practices and ideals, and how close or far apart these are at this point in their studies. These issues are closely related to how motivated they feel about entering the profession in the next few years. While the students in the previous study reviewed above (Kalaja et al., 2013; Kalaja, 2016) were about to complete their teacher education, the students in the present study were only half-way through. We used visual narratives to look forward in time, and carried out their collection in a more focused and structured way than in the previous study by using a set of prompt questions. The study seeks to answer the following research question: What would an ideal class of English be like, and more specifically, where would the class take place, what would be taught there, and how? In order to answer the questions, the pre-service teachers were asked to envision a future class of English, but a class that it would still be feasible to give after their graduation, and to describe it in two modes, visually and verbally. Findings of the analysis will be reported in the form of case studies to illustrate the overall variation in the classes of English envisioned. Context of the study English is the most popular foreign language studied and taught in the Finnish educational system. Until now, its study started in Year (or Grade) 3, i.e. from the age of nine years, but following the latest revisions to the national curricula, it can be offered even earlier in Years 1 and 2. The teaching of English or any other foreign language is regulated to varying degrees by a number of guidelines. Some of these are European (The Common European Frame of Reference for Languages, Council of Europe, 2001), some national and others local (e.g. a town, a local authority or a school might have curricula of its own). The Finnish National Core Curricula for compulsory education (Years 1-9) and for post-secondary education (Years 10-12, or Sixth Form), where students take the matriculation examination, a nationally administered high-stakes test, before graduating, have just been revised, and so have the local curricula, all effective as of August 2016. The previous curricula were drawn up in the early 2000s. In the new curricula, the order of importance of the three main aims for the teaching of foreign languages has been reversed and their emphasis and scope have been revised. Now the first aim is to raise learners’ awareness and appreciation of multilingualism and multiculturalism, and of languages in general. The second aim is to provide students with practice in learning-to-learn skills, and the third is to develop their proficiency in English in three skills, namely, in the ability to interact, interpret and produce oral, written and multimodal texts. Importantly, it is now acknowledged that the status of English has changed to Lingua Franca or global language, even though the language does not as yet have an official status in Finland. However, young people in Finland learn and use English not only in formal school contexts but also, and increasingly, in a variety of informal contexts such as hobbies, spare time activities, travelling, using modern IT with all its applications, including the Internet, and later on in working life as well. The teaching of English in Finland is thus faced with new challenges in terms of its aims, classroom practices, teaching materials, roles of teachers and learners and assessment (for details, see Kalaja et al., in press a). The data and their processing For the purposes of the study, a task sheet was designed. It drew on ideas from the studies by Hammerness (2003, 2006), and was our attempt to explore further the possibilities of visual narratives for the purpose of envisioning (e.g. Kalaja et al., 2013; Kalaja, 2016). There were two tasks (for details, see Appendix A). Task 1 asked the participants to produce a picture entitled ‘An English class of my dreams’, in which they depicted a class that they could imagine giving after graduating from the five- year MA programme. The images could be drawn by hand or done on a computer, or produced by compiling a collage out of magazine clippings. In addition, the participants were asked to comment on the picture, writing a few sentences in response to the question ‘What would be taking place in your class – and why?’. Task 2 (on the reverse side of the task sheet) asked the participants to consider the envisioned English class in greater detail (and in a more systematic way than before, e.g. Kalaja et al., 2013; Kalaja, 2016). This gave the students a chance to elaborate on the target group that they would like to teach, the roles of those involved (i.e. the teacher and students), what they would teach and how, where their teaching would take place and what equipment would be available to them. The students completed the task sheet in Finnish (with the exception of the few exchange students attending the course) as the last home assignment of the introductory course. The pictures were compared and contrasted in discussions (first in small groups and then as a whole group) in English during the very last session of the course – either in late November 2015 or in April 2016. The pictures as such were not assessed, and they did not count towards the overall grade for the course. Permission was requested from the students (in writing) to use the pictures and descriptions anonymously for research purposes. The contents of the visual narratives, complemented by their verbal commentaries, were first examined to get an overall idea of what the pool of data contained. In addition, the written explanations and elaborations of the pictures were analysed to gain a more thorough and accurate understanding of their visual narratives. Participants Teacher education is a joint effort of two departments on the campus where this study was conducted in Finland. The Department of Teacher Education offers studies in Pedagogy, including teaching practicum in cooperation with local schools. Completing a Bachelor’s degree in Pedagogy qualifies a student for any teaching post in the Finnish educational system. In contrast, the Department of Language and Communication Studies is responsible for providing all pre-service teachers with two compulsory introductory courses on learning and teaching any foreign language (or Finnish). The English Section of the department continues from there by offering a third course called Current Issues in Teaching English (CITE) and some other more advanced courses. The participants in the study were a group of pre-service teachers of English (N = 35), mainly second- or third-year university students. The majority, whether majoring or minoring in English, were studying to become foreign language teachers at the secondary or post- secondary level. However, a few students wanted to become elementary school teachers, getting a qualification to teach in English or offer courses in Content and Language Integrated Learning (CLIL). In addition, there were a couple of exchange students taking the course. All the participants had some pedagogical studies behind them. Some had also completed their practical teacher training, and/or worked as supply teachers. The participants formed a heterogeneous group in that the amount of studies they had already completed varied, as did their teaching experience. The students attended CITE, which is a compulsory course for future teachers of English, in the academic year 2015–2016. The data for this study were collected at the end of the course. The course is a five-credit (ECTS) course and part of the BA programme, that is, second- or third-year studies, in English. The students had weekly reading assignments from an introductory textbook by Hummel (2014), and the topics were discussed in class. The course focused on the teaching of English in the context of Finland, and the topics addressed included a review of key issues in research on second language acquisition over the past few decades, contexts of learning, the development of learner language, individual learner differences, teaching methodology, syllabus design, assessment and bi- or multilingualism. Participants Considering that the field is full of controversies, it was interesting to see how this group of student teachers made sense of the pedagogical knowledge they had acquired so far during their studies and how they managed to turn it into a set of principles and practices that they could imagine applying in their teaching of English after entering the profession. Findings A total of six contextualised case studies (as defined by Duff, 2007) will be reported below to illustrate the overall variation in the pictures, or in the ways the pre-service teachers envisioned giving a class of English in the years to come. Two cases from each of the three groups, physical environment, the roles of the teachers and the students, and the contents and means of teaching were selected. The cases per group illustrate the range of visions in the data. The data and their processing In the written texts, the participants gave, for instance, explanations for their choices or clarified their drawings and their contents. After this, the focus was narrowed down for the purposes of this study, and the visualisations of ‘The English class of my dreams’ were grouped according to: 1) what the physical environment for the class would be, 2) what roles the future teacher and his or her students would adopt, and 3) what would be taught and how in the future English class. The cases are presented below using pseudonyms to guarantee the anonymity of the participants. Environment (1): Omnipresent language In Mikko’s picture, the idea was of a language learning environment that was omnipresent (Figure 3.1), and this placed the focus on the learner. There was no classroom and there were no walls, but language could be learnt everywhere. There were books, and the learner’s curiosity was emphasised, as was the variety of language learning contexts and, to an extent, also interaction with others. Mikko was quite advanced in his studies and had completed his pedagogical studies and teacher training. In his commentary, he explained that the lessons were designed to meet the interests of the students. The idea of integrating languages with other subjects was strongly present. This mirrors the ideas of the new National Core Curricula, which emphasise language awareness and the integration of school subjects. Also, Mikko would like to teach in ways that the students themselves want and need, taking different learners into account. This reflects the ideals of teacher education in Finland in recent years: future teachers have been made aware of the variety of learning styles. He thought it was important, however, that whatever the students learn should be written down, whether the equipment is a blackboard or a tablet computer. This urge to write down is perhaps in slight contradiction to his willingness to take teaching to a new level, steering away from the classroom and textbooks and emphasising real language use and communication. Mikko said he was teaching life. Figure 3.1. Mikko – teacher training and pedagogical studies completed Environment (2): A classroom with sofas Tiina’s picture offered a more traditional view of language learning and its environment (Figure 3.2). There was a classroom with desks for the students and a chair for the teacher at the front of the classroom. The teacher’s desk had been placed to the side. There was also a smartboard for doing and checking exercises. What made the setting different from a traditional classroom was that the desks were in a horseshoe arrangement, and there were cushions, sofas, games, books and magazines. Tiina had done some pedagogical studies but not the practical training, and she did not have any teaching experience at that time. Her views are therefore likely to have reflected quite heavily how she herself had been taught languages at school. What was done in this rather traditional classroom was interesting. Environment (1): Omnipresent language Unlike in Mikko’s picture, where the focus had been on language usage, culture and interaction, in Tiina’s description, the emphasis was on practising vocabulary and going through texts, and taking advantage of mnemonics when learning grammar. Tiina regarded games, mnemonics and the like as innovative methods that motivate students to learn. Thus, her view of language is traditional, as to a large extent are also the ways of teaching it. Furthermore, in her commentary, Tiina talked about going through issues, implying that the teacher has a list of curricular content points to cover and this is then done using games and other activities, employing different learning and teaching strategies. Figure 3.2. Tiina – some pedagogical studies Figure 3.2. Tiina – some pedagogical studies Figure 3.2. Tiina – some pedagogical studies Roles of the teacher and students (1): Student in the centre Roles of the teacher and students (1): Student in the centre On the other hand, the teacher was also claimed to be a transmitter of information, which is a more traditional view and conflicts with the picture and its buzzwords. As for the methods, Sanna mentioned a wide range of options, from the use of a smartboard and the Internet to games and using drama. However, in her explanation, she made no reference to the pedagogical opportunities offered by these different methods; they were rather seen as a necessary variation to keep students interested. All in all, interaction and communication were once again highlighted. Sanna, too, emphasised the central role of the learner as an active participant in learning; the teacher’s role was to support learning. On the other hand, the teacher was also claimed to be a transmitter of information, which is a more traditional view and conflicts with the picture and its buzzwords. As for the methods, Sanna mentioned a wide range of options, from the use of a smartboard and the Internet to games and using drama. However, in her explanation, she made no reference to the pedagogical opportunities offered by these different methods; they were rather seen as a necessary variation to keep students interested. All in all, interaction and communication were once again highlighted. Roles of the teacher and students (1): Student in the centre Figure 3.3. Sanna – training for elementary school teachers completed Figure 3.3. Sanna – training for elementary school teachers completed Sanna attempted to present a fairly comprehensive and complex picture of language learning (Figure 3.3), with many different roles for the teacher and student. The students were in the centre, surrounded by the teacher and the context. Different factors affecting language learning and teaching were mentioned, such as the Zone of Proximal Development (ZPD), atmosphere, and authenticity. Sanna also mentioned quite a few teacher tasks, from scaffolding and motivating students to dynamic assessment, thus expanding the role of the teacher. Sanna was a second-year student of English but she was about to graduate as an elementary school teacher. She had not done pedagogical studies targeted specifically at language teachers. However, her previous pedagogical studies and work experience as an elementary school teacher showed in the concepts she used. These concepts, such as life-long learning, learner agency, ZPD, and dynamic assessment are at the very core of current thinking in teacher education in Finland. Also, authenticity has been a frequently discussed theme in language teaching in Finland for the past few years. In her verbal commentary, Sanna attempted to present a fairly comprehensive and complex picture of language learning (Figure 3.3), with many different roles for the teacher and student. The students were in the centre, surrounded by the teacher and the context. Different factors affecting language learning and teaching were mentioned, such as the Zone of Proximal Development (ZPD), atmosphere, and authenticity. Sanna also mentioned quite a few teacher tasks, from scaffolding and motivating students to dynamic assessment, thus expanding the role of the teacher. Sanna was a second-year student of English but she was about to graduate as an elementary school teacher. She had not done pedagogical studies targeted specifically at language teachers. However, her previous pedagogical studies and work experience as an elementary school teacher showed in the concepts she used. These concepts, such as life-long learning, learner agency, ZPD, and dynamic assessment are at the very core of current thinking in teacher education in Finland. Also, authenticity has been a frequently discussed theme in language teaching in Finland for the past few years. In her verbal commentary, Sanna, too, emphasised the central role of the learner as an active participant in learning; the teacher’s role was to support learning. Roles of the teacher and students (2): Modern roles Figure 3.4. Päivi – no pedagogical studies or teaching experience Päivi expressed a similar idea to Sanna’s in her picture (Figure 3.4), but worded it more simply, or rather, in layman’s terms. This probably reflects the fact that Päivi was at the beginning of her pedagogical studies and as yet had no teaching experience. She mentioned that the classroom could be a traditional one, organised in a traditional way, and that books, games and other traditional means would be useful as equipment. However, her ideas of the Figure 3.4. Päivi – no pedagogical studies or teaching experience Päivi expressed a similar idea to Sanna’s in her picture (Figure 3.4), but worded it more simply, or rather, in layman’s terms. This probably reflects the fact that Päivi was at the beginning of her pedagogical studies and as yet had no teaching experience. She mentioned that the classroom could be a traditional one, organised in a traditional way, and that books, games and other traditional means would be useful as equipment. However, her ideas of the roles of the teacher and the learner were following more recent ideas of teaching and learning: the teacher’s role is to listen, while students are expected to listen and also speak. The setting, then, was irrelevant: what was more important was what happened in the lesson and the classroom and what kind of roles the teacher and students took. Päivi’s picture includes a list of qualities such as being motivated, curious, and self-confident, but it is not quite clear whether these apply to the teacher, students, or both. Authenticity, equality, visuality, practice, many-sidedness, and enthusiasm were also mentioned. In her verbal commentary, Päivi also stressed a pleasant atmosphere in the classroom. Ways of teaching and learning (1): Project work in groups Figure 3.5. Noora – pedagogical studies completed Instead of a traditional classroom, or even the more modern one of the local teacher training school that the students were familiar with, Noora opted for something different (Figure 3.5). Figure 3.5. Noora – pedagogical studies completed Instead of a traditional classroom, or even the more modern one of the local teacher training school that the students were familiar with, Noora opted for something different (Figure 3.5). In her picture, the lesson was taking place in a grand house, offering ample opportunities for group work, which formed the core of her teaching. Interestingly, the teacher was the only one standing, and she seemed to be keeping an eye on things and being available when needed. The students were shown as working in small groups, using English and working on their projects, related to culture. Also activities outside the classroom were mentioned. The teacher was seen as a facilitator and guide, and the focus was on the learners. Even though the setting was an old-fashioned mansion, modern technology, including tablet computers, was present. Noora also talked about projects and the integration of different school subjects, for instance, biology and languages. This is very much in accordance with the National Core Curricula, which highlight language awareness and reaching over subject boundaries at school. Noora had completed her pedagogical studies, and as part of her studies, she had also acted as a supervisor in a language camp for children, which had relied heavily on action- based learning. Ways of teaching and learning (2): Each item in the classroom has a purpose Finally, let’s consider Iris’s picture (Figure 3.6). Iris was an exchange student, and she had completed some pedagogical studies and teacher training in her home country. Her lesson could have taken place anywhere, but the classroom was organised so that each area in it and each object had a specific function. In Iris’s picture, it was irrelevant whether there were chairs or cushions, but the classroom had various items that each served a specific language usage function. A carpet was there to share experiences on, a window was a window to the imagination, etc. The blackboard or smartboard was not used to check the correct answers to exercises but, for instance, to share ideas about what the students wanted to learn. Ways of teaching and learning (1): Project work in groups In her commentary, Iris explained that the teacher would give out the theme of the week, and the students could then decide what they wanted to learn about the theme and carry out a small- scale project to that end. The students shared information among themselves, and the teacher was a facilitator in the students’ construction of their own knowledge. Hence, the ways of learning were varied but always student-centred. Figure 3.6. Iris – pedagogical studies and teaching practice abroad Figure 3.6. Iris – pedagogical studies and teaching practice abroad Comparison of cases As far as the environments of learning are concerned, the cases reviewed above illustrate the student teachers’ underlying beliefs that more novel ways of learning are possible even in classroom settings that have been found in schools for centuries, that is, teacher’s desk facing neatly arranged rows of student desks. On the other hand, when given a free rein to depict language learning and teaching settings, the participants were willing to visualise life outside the school building and classroom walls. Languages are not learnt in a vacuum, and not from textbooks. The absence of textbooks was indeed interesting, as a fairly recent survey shows that language teachers in Finland in fact rely heavily on textbooks in their teaching (Luukka et al., 2008). In all the pictures, there was at least an attempt at student-centred learning. The idea of professional selves shows not only in the terminology used by, for instance, Sanna, but also in the ideals in the pictures: students are at the centre of what is going on, the methods of teaching and learning are varied and encourage co-operation and interaction, and authenticity and integrating languages with other school subjects were seen as significant. This seems to be a reflection of what has been highlighted in pedagogical studies, but also in the guidelines of the National Core Curricula. One common trait in all the pictures and their commentaries was the highlighting of a relaxed, friendly atmosphere in the classroom. Finnish schools have often been criticised for their atmosphere, and even though the learning results may be good internationally speaking, children do not feel comfortable at school (Harinen & Halme, 2012). This is something that has been discussed a lot in Finnish society, and this discussion was also reflected in the pictures: the students were presented as being given a say in what and how they learn, in a positive and relaxed atmosphere. Discussion This study set out to discover how a specific group of pre-service teachers would envision future classes of English. Their visions turned out to vary in a number of respects. Firstly, there was variation in the environments where the teaching and/or learning of English would take place. More specifically, the English class would take place either in a regular classroom, with some further variation in how the classroom would be furnished or equipped, or outside the classroom. In other words, it is assumed either that the teaching and learning of English is confined within the walls of a traditional classroom, or alternatively, that these activities can take place anywhere, not only in formal but also in informal contexts (for an even more complex account of contexts, see Benson & Reinders, 2011). Some pictures described the classroom in minute detail, indicating that where the teaching or learning of English would take place was important, while others paid little attention to this aspect, even claiming that the environment was of little or no relevance at all. Secondly, there was variation in the activities and therefore also in the roles of those involved. Some of the visions highlighted some aspects of teaching by a teacher as the main activity in a future class of English; others the learning by learners; and yet others teaching and learning as a joint activity, involving negotiation between two parties, that is, the teacher and students. The role(s) of the teacher varied accordingly: from that of a transmitter of information, in control of what takes place in the classroom, to that of a guide, ensuring learning opportunities for everyone, and as one partner in negotiating what would take place in the class and how. The role(s) of learners varied, too: from passive recipients of information to active seizers of learning opportunities and an equal partner in the negotiations. In other words, there was variation in how teacher- or student-centred the visions were. Similarly, there was variation in the responsibilities the teacher would have in the classroom: not only teaching, but also motivating students, assessing learning outcomes, fostering life- long learning and ensuring a positive atmosphere. Thirdly, there was further variation in the focus of teaching and learning of English. On the one hand, the focus could be on the actual teaching of English. Discussion There was then variation in the aspects addressed in a future English class: was English to be addressed in terms of an abstract system, including vocabulary and grammar, or in terms of its uses in specific contexts, or as a means of communication? On the other hand, the focus could be on teaching in English. In other words, English would be used as a medium of instruction in teaching other school subjects (or in CLIL). The pictures and their explanations reflected both the Ideal – and Ought-to selves of the pre- service teachers. While the participants, for instance, wanted to teach life and saw language and opportunities for its learning to be everywhere, they at the same time, regarded it necessary to write down important things in the class. Hence, their ideals went hand-in-hand with what they were used to in their own school days and maybe considered to be something expected of them. Conclusion To sum up, this study made use of visual narratives and their commentaries, or multimodal data, in looking forward in time, that is, asking pre-service teachers to envision an ideal class of English to be given in the years to come after they have graduated from their MA programme. Envisioning, in turn, is an aspect of feeling motivated in the profession in the years to come. In addition, the study was an attempt by us to refine our research methodology, that is, making use of visual data but collecting data also in another mode (written commentaries) to ensure a more systematic comparison of the cases than before (e.g. Kalaja, 2016). The findings are basically comparable to those of the previous studies reviewed above, despite the partly different contexts and student bodies. There is, however, a clear difference in that in this study there seems to be much greater variation in the pools of data, and so the group of pre-service teachers is much more heterogeneous in their visions of giving a future English class: some classes are envisioned as being not very different from the classes the students themselves attended when they were at school, while other classes have been inspired by more recent developments in the fields of Applied Linguistics (or Foreign/Second Language Learning and Teaching) or Foreign Language Education. In addition, they seem to be well informed about the innovations in the new National Core Curricula (effective from August 2016). The awareness of all this is already reflected in some of the principles or practices the students wish to apply in their future teaching, albeit to varying degrees and with some contradictions (of which they themselves appeared to be unaware). It seems that this variation is at least partly dependent on the stage on the MA degree programme the students found themselves in, and perhaps even more importantly, on their experience of teaching English. As was explained earlier, the group was heterogeneous in this regard, as some students had taught under the supervision of teacher trainers as part of their pedagogical studies, and others had worked as supply teachers at some point in their studies – for a shorter or longer period of time. The more advanced the students were in their studies and/or the more teaching experience they had had, the more sophisticated or complex their visions of an ideal English class tended to be. Conclusion The exercises or tasks that we have described here were an attempt to make the students on our course aware of their beliefs about teaching and learning English, to compare and contrast their own beliefs with those of their classmates, and reflect on possible discrepancies between their current understanding of the issues involved and their ideals. Becoming aware is, however, only the first stage in possible changes in thinking or behaviour, or in the professional development of pre- or in-service teachers of foreign languages (e.g. Kalaja et al., in press b; Kubanyiova, 2012). For us as teacher educators, the study is an indication of how long and complex for students the process is of acquiring new pedagogical knowledge and turning it into a systematic and justified set of principles and practices to be applied in future English classes. We must still wait some time before we can see whether the students in this present study manage to keep up their motivation and hold onto their ideals or visions, especially if these were very different from their past school experiences, once they start their careers as teachers of English in the Finnish educational system (for a few longitudinal studies on the fate of previous groups of novice teachers of foreign languages in Finland, see e.g. Kalaja, 2017; Nyman, 2009; or Ruohotie-Lyhty, 2011, 2016a, 2016b). The exercises or tasks that we have described here were an attempt to make the students on our course aware of their beliefs about teaching and learning English, to compare and contrast their own beliefs with those of their classmates, and reflect on possible discrepancies between their current understanding of the issues involved and their ideals. Becoming aware is, Barkhuizen, G. (2013) Introduction: Narrative research in applied linguistics. In G. Barkhuizen (ed.) 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https://openalex.org/W3131389624	https://link.springer.com/content/pdf/10.1007/s10533-021-00765-z.pdf	English	null	Role of external inputs of nutrients to aquatic ecosystems in determining prevalence of nitrogen vs. phosphorus limitation of net primary productivity	Biogeochemistry	2,021	cc-by	9,863	Biogeochemistry (2021) 154:293–306 https://doi.org/10.1007/s10533-021-00765-z (0123456789().,-volV)(0123456789().,-volV) Role of external inputs of nutrients to aquatic ecosystems in determining prevalence of nitrogen vs. phosphorus limitation of net primary productivity R. W. Howarth . F. Chan . D. P. Swaney . R. M. Marino . M. Hayn Received: 4 November 2020 / Accepted: 3 February 2021 / Published online: 17 February 2021 The Author(s) 2021 aquatic systems and how the balance of N to P inputs inﬂuences nutrient limitation. For ocean subtropical gyres, a relatively balanced input of N and P relative to the Redﬁeld ratio from deep ocean sources often leads to near co-limitation by N and P. For lakes, the external nutrient inputs come largely from watershed sources, and we demonstrate that on average the N:P ratio for these inputs across the United States is well above that needed by phytoplankton, which may contribute to P limitation in those lake that experience this average nutrient loading. Watershed inputs are also important for estuaries and coastal marine ecosystems, but ocean sources of nutrients are also signiﬁcant contributors to overall nutrient loads. The ocean-nutrient sources of N and P are very often at or below the Redﬁeld ratio of 16:1 molar, and can be substantially so, particularly in areas where the continental shelf is wide. This large input of coastal ocean nutrients with a low N:P ratio is one factor that may make N limitation more likely in many coastal marine ecosystems than in lakes. Abstract Whether net primary productivity in an aquatic ecosystem is limited by nitrogen (N), limited by phosphorus (P), or co-limited by N & P is determined by the relative supply of N and P to phytoplankton compared to their elemental require- ments for primary production, often characterized by the ‘‘Redﬁeld’’ ratio. The supply of these essential nutrients is affected by both external inputs and biogeochemical processes within the ecosystem. In this paper, we examine external sources of nutrients to Responsible Editor: Kate Lajtha. This paper is an invited contribution to the 35th Anniversary Special Issue, edited by Sujay Kaushal, Robert Howarth, and Kate Lajtha. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10533-021-00765-z. R. W. Howarth (&) D. P. Swaney R. M. Marino M. Hayn Department of Ecology & Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA e-mail: howarth@cornell.edu Keywords Nutrient limitation Nitrogen limitation Phosphorus limitation Aquatic ecosystems Estuaries Coastal marine ecosystems Redﬁeld ratio NANI NAPI Nutrient stoichiometry R. W. Howarth R. M. Marino M. Introduction Aquatic scientists have long noted major differences in nutrient control of net primary productivity across different types of ecosystems, with phosphorus (P) limitation in many lakes (Schindler 1974; Vollen- weider 1975), nitrogen (N) limitation in many estuar- ies and coastal marine ecosystems (Ryther 1954), and co-limitation by N and P in many open ocean systems such as the subtropical gyres (Redﬁeld 1958). Excep- tions to these generalities exist, and some coastal marine ecosystems are limited by P rather than N while some lakes are limited by N (Howarth 1988; Bergstro¨m and Jansson 2006; Elser et al. 2009; Hayes et al. 2018). Time scale matters, and ecosystems can switch between N and P limitation seasonally, includ- ing both lakes and estuaries (Fisher et al. 1992; Dolman et al. 2016). The focus of this paper is to examine the role of external nutrient inputs to aquatic ecosystems as one of the factors that may tend to make primary production more likely to be limited by N, P, or both. We and others have written extensively about the role of biogeochemical processes such as N ﬁxation, denitriﬁcation, and phosphorus sorption and desorp- tion as factors that can lead more toward limitation of N or P in an aquatic ecosystem (see for example Howarth and Marino 2006 and references therein). However, the ratio of N:P for the external nutrient inputs to the ecosystem sets the stage against which these internal biogeochemical processes can act. For example, N ﬁxation by planktonic cyanobacteria can help maintain P limitation in lakes when the N deﬁcit is relatively small (Schindler 1977; Flett et al. 1980; Schindler et al. 2008). But in lakes where the N:P ratio of external inputs is sufﬁciently low, N ﬁxation may not be able to make up the N deﬁcit compared to P availability, and N limitation can occur (Moss et al. 2013; Grantz et al. 2014; Hayes et al. 2018). Signif- icant planktonic N ﬁxation is largely absent from saline estuaries, which is one of the reasons we believe coastal marine ecosystems are more likely than lakes to be N limited (Howarth et al. 1988; Marino et al. 2002; Marino and Howarth 2016). The topic of nutrient limitation in aquatic ecosys- tems is controversial, with long-standing debates about how one best determines which nutrient is most limiting. Role of external inputs of nutrients to aquatic ecosystems in determining prevalence of nitrogen vs. phosphorus limitation of net primary productivity Hayn The Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA F. Chan OSU-NOAA Cooperative Institute, Oregon State University, Corvallis, OR 97331, USA 123 Biogeochemistry (2021) 154:293–306 294 ratio of dissolved inorganic nutrient concentrations is an imperfect indicator of nutrient limitation, particu- larly when near the Redﬁeld ratio of 16:1 or in ecosystems where inorganic N and P are extremely low, the ratio near 1:1 in West Falmouth Harbor and the average ratio of more than 1000:1 in Cayuga Lake clearly indicate N and P limitation on primary production, respectively (NRC 2000). Introduction This balance between N and P cycling in open ocean waters is at the center of the Redﬁeld (1958) concept. United States by region and over time, presenting 5-year averages from 1987 to 2012. The approach is based on the Net Anthropogenic Nitrogen Inputs (NANI) and Net Anthropogenic Phosphorus Inputs (NAPI) to the landscape (Howarth et al. 1996, 2006, 2012; Hong et al. 2011, 2012, 2013; Han et al. 2011). United States by region and over time, presenting 5-year averages from 1987 to 2012. The approach is based on the Net Anthropogenic Nitrogen Inputs (NANI) and Net Anthropogenic Phosphorus Inputs (NAPI) to the landscape (Howarth et al. 1996, 2006, 2012; Hong et al. 2011, 2012, 2013; Han et al. 2011). Most lakes receive the vast majority of their nutrient inputs from their watersheds, although atmo- spheric inputs of both N and P can be important, particularly in lakes with relatively pristine landscapes and consequently small rates of watershed loading. Estuaries and coastal marine ecosystems also receive substantial inputs of nutrients from watersheds and atmospheric deposition, but they additionally receive inputs of nutrients from ocean sources (Nixon et al. 1996; Howarth et al. 2011). This major difference in the external nutrient sources between lakes and coastal marine systems often escapes attention and is one of the major foci of this paper. In this paper, we ﬁrst explore the average inputs of nutrients to aquatic ecosystems from their watersheds in the context of the United States, using a mass balance modeling approach at the regional scale, and then we examine dissolved N and P data from across the global oceans as sources of nutrients to the coastal zone. NANI is the sum of N inputs to a region from synthetic N fertilizer, N ﬁxation by agricultural crops, atmospheric deposition of N, and the N in food and feed for humans and livestock. If there is a net export of N in food and feed, this is treated as a negative input and is subtracted from the other inputs (Howarth et al. 1996; Hong et al. 2011). The approach for NAPI is very similar, but the net import of P in detergents and other non-food uses is included, and atmospheric deposition of P is assumed to be negligible (Han et al. 2011; Hong et al. 2012). We present data for several large regions of the United States based on county- scale data (Fig. Introduction Our goal in this paper is not to further debate the evidence by which one determines which nutrient is more limiting, which we have previously discussed in some detail (Howarth 1988; Howarth and Marino 2006); see also NRC (2000). We agree with the suggestion of Moss et al. (2013) that limitation by a single nutrient is more likely in human-impacted, more nutrient-enriched systems, and that co-limitation by N and P is more likely in oligotrophic ecosystems (see also Begon et al. 2014). In our view, P limitation is more prevalent than N limitation in mesotrophic and eutrophic lakes, and N limitation is more prevalent in temperate-zone estuaries. Our personal research expe- rience informs this belief, including our long-term study of a coastal marine lagoon that is clearly N limited (Howarth et al. 2014) and research on a lake that is clearly P limited (Peterson et al. 1974; Roberts and Howarth 2006). The coastal system, West Fal- mouth Harbor on Cape Cod, MA, is characterized by low dissolved inorganic N concentrations (generally\ 1 lM) and moderate concentrations of soluble reac- tive P (0.25–1 lM; Hayn et al. 2014; Howarth et al. 2014). The lake, Cayuga Lake in the Finger Lakes region of central NY, is characterized by similar concentrations of soluble reactive P (SRP) but concentrations of dissolved inorganic N that are more than two magnitudes of order greater (mean value of 380 lM; Halfman 2017). Although the molar N:P For the surface waters of much of the world’s oceans away from shore and continental shelves, including the subtropical gyres that cover half of the area of the oceans and 35% of the total surface of the Earth, the largest source of nutrient inputs by far is mixing of deeper ocean waters across the thermocline (Michaels et al. 1996). The mixing rate is low, but the nutrient content of this water is quite high, so the input to the surface oceans is substantial. As we present below, the ratio of N to P in these deep waters is quite close to the Redﬁeld ratio (Karl et al. 1997; Michaels et al. 1996). This leads to co-limitation of net primary productivity by both nutrients in the subtropical gyres, consistent with the thought that co-limitation by N and P may be the norm in oligotrophic ecosystems (Begon 12 123 Biogeochemistry (2021) 154:293–306 295 et al. 2014). Introduction 1a). As of the writing of this manu- script, county-scale data for fertilizer sales after 2012 are still not available from the USDA agricultural census (Slater, AAPFCO, pers. comm.). Since this paper is an invited contribution for the special issue of Biogeochemistry celebrating more than 35 years of the journal, we note that the watershed nutrient inputs we discuss next are based on our 1996 paper published in Biogeochemistry as part of another earlier special issue (Howarth et al. 1996). This paper won the John Martin ASLO award in 2018 in recognition of being a highly inﬂuential paper. The 1996 special issue focused on papers developing an N budget for the North Atlantic Ocean as part of the International SCOPE Nitrogen Project. Other papers from that issue (Nixon et al. 1996; Michaels et al. 1996; Seitzinger and Giblin 1996) are also central to the development of the logic behind this current paper. The input terms for NAPI are as described in Swaney and Howarth (2019). For NANI, the input terms are as described in Swaney et al. (2018) except for N deposition. In our previously published work, atmospheric N deposition estimates for NANI were based on the U.S. Environmental Protection Agency CMAQ model. Here, we use updated estimates of atmospheric N deposition from a hybrid approach now favored by the US EPA (Schwede and Lear 2014), combining CMAQ output with National Atmospheric Deposition Program observational data to make deposition estimates (NADP 2018). The reﬁnement to the approach shows lower values of NOy deposition for 2012 than our previous estimates (Howarth et al. 2012; Hong et al. 2013), consistent with observed declines in emissions (Butler et al. 2011). Nutrient inputs from the landscape Net Anthropogenic Nitrogen Inputs per watershed area are the highest in the Mid-Atlantic region but are also high in California, the Great Lakes area, the Mississippi River basin, and the South Atlantic/Gulf Coast (Fig. 1b). Values are considerably lower in New England, the Paciﬁc Northwest, and the Texas Gulf coast area. In all of the regions, changes in NANI over time have been relatively small, sometimes increasing and sometimes decreasing. Net Anthropogenic Phos- phorus Inputs per area of watershed are highest in the The rate of nutrient loading from the landscape to lakes and estuaries has been estimated for thousands of systems globally. However, there is no uniformly accepted methodology for estimating N and P loading (NRC 2000), complicating comparisons across sys- tems. Here, we build on our previous work and use a regional nutrient accounting approach to estimate the average N and P loads from watersheds across the 12 3 3 Biogeochemistry (2021) 154:293–306 Mid-Atlantic and South Atlantic/Gulf Coast regions, and are far lower in most other regions, particularly New England and the Paciﬁc Northwest (Fig. 1c). As with NANI, time trends for NAPI over time have been relatively small, with the exception of the Great Lakes region which saw a steady decline from 1987 to 2012. Fig. 1 a Regions of the United States for which we report data on NANI and NAPI. b NANI for each major region of the United States over time from 1987 to 2017. c NAPI for each major region of the United States over time from 1987 to 2017. d Molar ratio of nitrogen to phosphorus exported from each major region of the United States over time from 1987 to 2017. The Redﬁeld ratio of 16:1 is shown as a dashed red line for context 296 Biogeochemistry (2021) 154:293–306 296 d Molar ratio of nitrogen to phosphorus exported from each major region of the United States over time from 1987 to 2017. The Redﬁeld ratio of 16:1 is shown as a dashed red line for context Fig. 1 a Regions of the United States for which we report data on NANI and NAPI. b NANI for each major region of the United States over time from 1987 to 2017. c NAPI for each major region of the United States over time from 1987 to 2017. Nutrient inputs from the landscape Most of the N that is not exported downstream is denitriﬁed or stored in the watershed (van Breemen et al. 2002). g It is important to reiterate that NANI and NAPI have several input terms and generally are not driven by any particular term such as fertilizer use (Howarth et al. 1996, 2012; Hong et al. 2011, 2012). This is illustrated by contrasting two different regions, New England and the Mississippi River basin. For NANI in New England, the biggest input terms are atmospheric deposition and net import of food and animal feeds, with fertilizer use and N ﬁxation by agricultural crops being considerably smaller (Fig. 2a). On the other hand, the largest N inputs in the Mississippi River basin are from fertilizer use and agricultural N ﬁxation. Further, the food and animal feeds term is negative in the Mississippi River basin, indicating a net export rather than an import, as in New England. In fact, more N leaves the Mississippi River basin via export of food and feed than ﬂows down the Missis- sippi River (Fig. 2a). In both New England and the Mississippi River basin, it is NANI that determines riverine N ﬂows, not any single input term. For NAPI, fertilizer is the largest input for both New England and the Mississippi River basin, followed by import in food and feeds (Fig. 2b). While the import of P in food and feeds in the Mississippi River basin is small, it is a positive value on average, in contrast to the net export of N from the basin. Mean values of the input terms for NANI and NAPI for the 1987–2012 period for each of the regions are shown in tables in on-line only supplemental material. For NAPI, we assume that 5.9% is exported downstream as total P. This is the average observed for 57 moderately large watersheds in the United States (Zhang et al. 2015). Somewhat lower export percentages are observed in China (2.9%) and Europe (3.9%), and Zhang et al. (2015) report an overall average export of 3.4% for 158 watersheds in the United States, China, and Europe. For this paper, we are using the 5.9% value since we are considering only regions in the United States. We recognize that this may bias our P export numbers towards the high end. Nutrient inputs from the landscape Using NANI and NAPI export percentages of 24% and 5.9% respectively, we can calculate the molar N:P ratio of nutrients leaving each region of the United States over time (Fig. 1d). In all regions and at all times since 1987 this N:P ratio is high, well above the Redﬁeld ratio of 16:1. As we state in the paragraph above, our P export values may be biased high, and if that is the case and actual P ﬂuxes are less than we estimate, the N:P ratios shown in Fig. 1d would be even higher. The N:P ratios of exports from the landscape are particularly high in the Great Lakes, the Mississippi River basin, New England, and the Paciﬁc Northwest, ranging from somewhat [ 50:1 to well over 100:1. The N:P ratio is lowest in the Texas Gulf region. Changes in the N:P ratio of exported nutrients over time have been small in most regions, but the Great Lakes region saw a large increase in this N:P ratio from 1987 to 2012 (Fig. 1d), largely driven by the decrease in NAPI in this region over time (Fig. 1c). The regional-scale watershed ﬂuxes we estimate are for total N and total P. Not all of this N and P will be biologically available to primary producer organ- isms in recipient ecosystems. Of the organic N and P, some will be mineralized to readily available forms on ecologically meaningful time scales, but some will simply be buried and not become available, particu- larly for total N (Howarth et al. 1996; Nixon et al. 1996). It is important to note that as human activity increases overall nutrient loads, inorganic N and P increasingly come to dominate total N and P ﬂuxes (Meybeck 1982; Howarth et al. 1996; Seitzinger et al. 2005). All of the inorganic N that enters an aquatic ecosystem remains biologically available, but for inorganic P, much can be adsorbed to soil and sediment particles. Some of this may become avail- able in recipient ecosystems but some will not. Adsorbed P is much more likely to desorb and become There is some debate over when and if nutrients stored in the landscape (‘‘legacy nutrients’’) are ultimately released, thereby enriching receiving waters, and how signiﬁcant this is as a proportion of total nutrient loading. Some modeling efforts suggest current nutrient loads are due in part to release of legacy nutrients (Van Meter et al. Nutrient inputs from the landscape Mid-Atlantic and South Atlantic/Gulf Coast regions, and are far lower in most other regions, particularly New England and the Paciﬁc Northwest (Fig. 1c). As with NANI, time trends for NAPI over time have been relatively small, with the exception of the Great Lakes region which saw a steady decline from 1987 to 2012. with NANI, time trends for NAPI over time have been relatively small, with the exception of the Great Lakes region which saw a steady decline from 1987 to 2012. 12 3 To examine the ratio of total N to total P likely exported from each region to aquatic ecosystems, we paper (Howarth et al. 1996). While there is some variation in the percentage of NANI that is exported Fig. 1 continued Biogeochemistry (2021) 154:293–306 297 Biogeochemistry (2021) 154:293–306 297 Fig. 1 continued Fig. 1 continued Fig. 1 continued To examine the ratio of total N to total P likely exported from each region to aquatic ecosystems, we ﬁrst calculate the portions of NANI and NAPI that move downstream. For NANI, an average of 24% is exported from large watersheds as total N, based on a data set for 154 watersheds in the United States, Europe, and the United Kingdom (Howarth et al. 2012; Hong et al. 2013). Note that this is virtually identical to the 26% export of NANI that we estimated from the analysis of large regions in our original NANI paper (Howarth et al. 1996). While there is some variation in the percentage of NANI that is exported across these watersheds, it is relatively small. Surpris- ingly, the variation does not seem tied to the speciﬁc N inputs, the intensity of agriculture, topography, soil types, or other such factors (Howarth et al. 1996, 2012; Hong et al. 2013). Most of the differences in export across regions are related to climate, with greater percentage exports in regions where precipitation and river discharge per area are higher. For simplicity in 12 3 3 298 Biogeochemistry (2021) 154:293–306 N relative to P, and the N:P ratio could be somewhat higher. this paper, we use the average export of 24%. Most of the N that is not exported downstream is denitriﬁed or stored in the watershed (van Breemen et al. 2002). this paper, we use the average export of 24%. Nutrient inputs from the landscape 2017; McCrackin et al. 2018). However, these legacy nutrient sources are ultimately the result of excess NANI and NAPI storage in the landscape, and are not an additional input to the system. Van Meter et al. (2017) suggest that current nutrient exports have a larger proportion of legacy N than legacy P, indicating that using the long-term average export proportions as in the calcu- lation above would produce a conservative estimate of 12 3 123 299 Biogeochemistry (2021) 154:293–306 Fig. 2 a Comparison on input terms for NANI in New England (bottom) and the Mississippi River basin (top) in kg N per square kilometer per year. Also shown are exports of N in water ﬂowing to coastal ecosystems based on 24% of NANI. See text. b Comparison on input terms for NAPI in New England (bottom) and the Mississippi River basin (top) in kg P per square kilometer per year. Also shown are exports of P in water ﬂowing to coastal ecosystems based on 5.9% of NAPI. See text Fig. 2 a Comparison on input terms for NANI in New England (bottom) and the Mississippi River basin (top) in kg N per square kilometer per year. Also shown are exports of N in water ﬂowing to coastal ecosystems based on 24% of NANI. See text. b Comparison on input terms for NAPI in New England (bottom) and the Mississippi River basin (top) in kg P per square kilometer per year. Also shown are exports of P in water ﬂowing to coastal ecosystems based on 5.9% of NAPI. See text b Comparison on input terms for NAPI in New England (bottom) and the Mississippi River basin (top) in kg P per square kilometer per year. Also shown are exports of P in water ﬂowing to coastal ecosystems based on 5.9% of NAPI. See text b Comparison on input terms for NAPI in New England (bottom) and the Mississippi River basin (top) in kg P per square kilometer per year. Also shown are exports of P in water ﬂowing to coastal ecosystems based on 5.9% of NAPI. See text Fig. 2 a Comparison on input terms for NANI in New England (bottom) and the Mississippi River basin (top) in kg N per square kilometer per year. Also shown are exports of N in water ﬂowing to coastal ecosystems based on 24% of NANI. See text. Nutrient inputs from the landscape What are the relative requirements of N and P for algae and cyanobacteria? Redﬁeld (1958) originally proposed that marine phytoplankton need N and P in a 16:1 molar ratio. There is some plasticity in this ratio for N:P needs across different primary producers, both in the oceans and in lakes (Hecky and Kilham 1988; Karl et al. 1997). Limnologists often use the N:P ratio of seston to infer whether a lake is more limited by N or P (Hecky and Kilham 1988; Dolman et al. 2016), although it is difﬁcult to tease apart how much of the sestonic particulate N and P is in living phytoplankton vs detritus, including that of terrestrial origin. Nonetheless, the N:P ratio of seston in lakes, while generally[16:1, is low compared to the loading ratios we show in Fig. 1d, ranging from 17:1 to 39:1 in a variety of lakes, with a mean of 23:1 for small lakes and 26:1 for large lakes (Sterner et al. 2008). biologically available in estuaries and coastal marine ecosystems than in lakes, due to higher ionic strength and more ions competing for sorption sites as salinity increases (Froelich 1988; Howarth et al. 1995). Nutrient inputs from ocean sources In many cases, oceanic inputs to estuaries are not measured and are not included in nutrient budgets (as discussed by Nixon et al. 1996). However, just as salt from coastal ocean systems is mixed and advected up into an estuary, nutrients from these coastal waters are also transported. Nutrient inputs from the ocean can be substantial, particularly for P, as can be seen for coastal systems with datasets rich enough to make good estimates. For example, an estimated 24% of the P inputs to Chesapeake Bay and 36% of P inputs to the Potomac River estuary come from ocean sources, with virtually no input of N from the ocean to these systems (Boynton et al. 1995). For both the Changjiang River estuary (Li et al. 2011) and the heavily N-loaded West Falmouth Harbor (Hayn et al. 2014; Howarth et al. 2014), P inputs from the ocean source are sufﬁcient to maintain N limitation. In both of these systems, P inputs were proportionally greater than N inputs relative to the Redﬁeld ratio of 16:1. y y This same construct of the N:P ratio for external nutrient inputs setting the stage for limitation of primary production by N vs P also should apply to estuaries and coastal marine ecosystems: unless inter- nal biogeochemical processes are sufﬁcient to com- pensate, nutrient inputs from the watershed with an N:P ratio substantially above the needs of the phyto- plankton would be expected to lead to P limitation. The N:P ratios shown in Fig. 1d are far above the Redﬁeld ratio of 16:1 or the seston N:P ratios commonly found in marine ecosystems, which tend to be below 20:1 in coastal waters and productive areas and range between 15:1 and 28:1 in oligotrophic ocean systems (Karl et al. 1997; Sterner et al. 2008; Martiny et al. 2013). Why then is N limitation commonly observed in most coastal marine ecosystems, in sharp contrast to so many lakes (Howarth and Marino 2006)? Part of the answer undoubtedly lies in the differences between the relative inﬂuence of the biogeochemical processes that occur within coastal ecosystems compared to lakes. These differences include a lack of planktonic N ﬁxation in saline estuaries (Howarth et al. 1988; Marino et al. 2002; Marino and Howarth 2016) and the desorption of P from particles as salinity increases in an estuary (Froelich 1988; Howarth et al. 1995). Inﬂuence of watershed loads on N vs P limitation For lakes, those that receive nutrient loads from their watershed with an N:P molar ratio for total nutrients above the relative N and P requirements of phyto- plankton would be expected to be P-limited, unless biogeochemical processes within the lake decrease the availability of N relative to P, lowering the N:P ratio for available nutrients below that needed by the primary producers. Of course, processes within the lake can lower the ratio of available N:P, for instance if denitriﬁcation rates are greater than rates of N ﬁxation. The rates of biogeochemical processes that control N and P availabilities vary as a function of lake size, water residence time, and other factors (Moss et al. 2013; They et al. 2017). Nonetheless, the watershed nutrient inputs set the stage for the relative availability of N and P that support primary production. The high N:P loading ratios shown in Fig. 1d would be expected to lead toward a tendency for P limitation in the receiving waterbodies, particularly considering that much of the particle-adsorbed P may not become available in a lake. This is consistent with the widespread prevalence of P limitation of net primary productivity across many North American lakes 12 12 3 3 300 Biogeochemistry (2021) 154:293–306 (Vollenweider 1975). We want to emphasize, though, that lakes undoubtedly vary greatly in the N:P ratio of their nutrient inputs, and no individual lake receives the average N and P loads indicated by this regional approach based on NANI and NAPI. Combined with biogeochemical processes within lakes that can alter N and P availability, it should come as no surprise that many lakes are N limited. Lakes in catchments that are totally forested will have lower total loads of both N and P, with no inputs from agricultural sources; in regions where atmospheric N deposition is low, lakes in forested catchments are often oligotrophic and N limited or co-limited by N and P, but high deposition rates over time can shift the N:P stoichiometry, leading to P limitation (Bergstro¨m and Jansson 2006; Elser et al. 2009). Note further than even lakes in largely forested catchments may have inputs of both N and P related to the food and feed term of NANI and NAPI, for instance due to nutrient inputs from onsite wastewater treatment systems from nearby homes. systems receive a signiﬁcant portion of their nutrient inputs from ocean sources, not just from their watersheds. Nutrient inputs from ocean sources near-bottom waters which are depleted in N relative to P would thus help maintain the deﬁcits of inorganic N in coastal waters that ﬂow into estuaries. The advec- tion of dissolved materials into an estuary from coastal waters with low N:P ratios will import relatively more P and strengthen the likelihood of persistent N-limitation. For both nitrate ? nitrite (Fig. 4a) and SRP (Fig. 4b), concentrations are quite variable in loca- tions where the continental shelves are narrow. For areas where the shelf is less than 30 km wide, nitrate ? nitrite varies from 0 to 33 lmol kg-1 and SRP varies from 0 to 2.4 lmol kg-1. In areas where the continental shelves are wider, concentrations of nitrate ? nitrite and of SRP are not as high as sometimes observed at sites on the narrower shelves. For sites from areas where the shelf is wider than 100 km, nitrate ? nitrite concentrations are generally at or near 0 lmol kg-1 (Fig. 4a), while SRP concen- trations are \ 0.5 lmol kg-1 (Fig. 4b). These low concentrations for nitrate ? nitrite and SRP reﬂect both uptake by phytoplankton and net exchanges with sediments. The shallower water depths of the conti- nental shelves can encourage phytoplankton blooms, with concomitant drawdown of both nitrate ? nitrite and SRP. Further, the sediments on continental shelves are the major site both for burial of P (Ruttenberg and Berner 1993; Howarth et al. 1995) and of denitriﬁca- tion (Falkowski 1997; Galloway et al. 2004) globally We observe that the N:P ratio for inorganic nutrients of bottom waters often appears to be lower along coasts that have a substantial continental shelf, such as the East and Gulf coasts of the United States, the European coast of the North Sea, and the coast of China (Fig. 3). Much of the research on nutrient limitation in estuaries has occurred in these areas, where N limitation has been found to be so common. Interestingly, oligotrophic lagoons along the French Mediterranean coast are P limited (Souchu et al. 2010), and the nearby bottom waters in the Mediter- ranean Sea have a very high N:P ratio for inorganic nutrients compared to most ocean regions (Fig. 3). Nutrient inputs from ocean sources Another impor- tant part of the answer, although one less discussed in the literature, is the N:P ratio of external inputs to the ecosystem: many estuaries and other coastal marine Figure 3 shows molar N:P ratios for inorganic nutrients (as nitrate ? nitrite and SRP) in the deep waters below the thermocline for the world’s oceans from the World Ocean Atlas 2018 (WOA18; Garcia et al. 2018). Values represent observations from the deepest bins within each 1 gridded cell. We present data on inorganic N and SRP, rather than total N and P as we did for the watershed loading estimates, simply because inorganic data are common and total N and P data rare in oceanographic studies and data bases. Note, though, that in deep ocean waters, concentra- tions of inorganic N and SRP are very high and greatly dominate both the total N and total P pools (Michaels et al. 1996). For most of the world’s oceans, bottom waters have molar N:P ratios near the Redﬁeld ratio of 16:1. Mixing of these deep-ocean waters into the surface waters of the subtropical gyres provides N and P in a ratio very near the Redﬁeld ratio, so it should not be surprising that these open ocean systems are nearly co-limited by N and P, as originally described by Redﬁeld (1958). On the other hand, the N:P ratio in the bottom waters near the coast are often well below the Redﬁeld ratio (Fig. 3). The ﬂux of nutrients from these 123 Biogeochemistry (2021) 154:293–306 301 Fig. 3 Molar ratio of nitrate ? nitrite to SRP in the bottom waters of the world’s oceans Fig. 3 Molar ratio of nitrate ? nitrite to SRP in the bottom waters of the world’s oceans Data Analysis Project (GLODAP) database (Olsen et al. 2016) which offers highly quality-controlled observations from precise sampling locations. The data are for individual samples from cruises over many years and are snapshots in time, not longer-term mean values. near-bottom waters which are depleted in N relative to P would thus help maintain the deﬁcits of inorganic N in coastal waters that ﬂow into estuaries. The advec- tion of dissolved materials into an estuary from coastal waters with low N:P ratios will import relatively more P and strengthen the likelihood of persistent N-limitation. Nutrient inputs from ocean sources To further examine the effects of continental shelf biogeochemical processes in structuring N and P inputs to estuaries, we plot the inorganic nutrient concentrations for surface waters on the continental shelves of the United States as a function of the width of the continental shelf at the sampling site. Since the 1 resolution of the WOA18 can conﬂate shelf and open ocean observations, we used the GLobal Ocean 12 3 3 Biogeochemistry (2021) 154:293–306 302 Fig. 4 a Nitrate ? nitrite concentration in surface waters for sites on the continental shelves of the United States plotted as a function of the width of the shelf at that site. East coast and west coast shown separately. b SRP concentration in surface waters for sites on the continental shelves of the United States plotted as a function of the width of the shelf at that site. East coast and west coast shown separately. c Molar ratio of nitrate ? nitrite to SRP in surface waters for sites on the continental shelves of the United States plotted as a function of the width of the shelf at that site. East coast and west coast shown separately Fig. 4 a Nitrate ? nitrite concentration in surface waters for sites on the continental shelves of the United States plotted as a function of the width of the shelf at that site. East coast and west coast shown separately. b SRP concentration in surface waters for sites on the continental shelves of the United States plotted as a function of the width of the shelf at that site. East coast and west coast shown separately. c Molar ratio of nitrate ? nitrite to SRP in surface waters for sites on the continental shelves of the United States plotted as a function of the width of the shelf at that site. East coast and west coast shown separately (Howarth 1998). In other regions, the deep-ocean nutrient source can be less important. The northern Gulf of Mexico receives only 5% of its nitrogen from the deep ocean, 0.14 Tg N year-1 compared to 2.1 Tg N year-1 from the Mississippi River basin and 0.28 Tg N year-1 from direct atmospheric deposition (Howarth 1998). (Howarth 1998). In other regions, the deep-ocean nutrient source can be less important. Nutrient inputs from ocean sources (2011) were able to infer the importance of the ocean source of P for maintaining N limitation in the Changjiang River estuary. However, we suggest that future ecosystem-scale budgets concentrate on ﬂuxes of total N and total P, as NANI and NAPI do for watershed ﬂuxes. This facilitates the construction of mass balances and also acknowledges that organic forms of N and P can be recycled and become available to primary producers within the ecosystem on ecologically meaningful time scales. the North Sea, than those near a narrow continental shelf such as the west coast of the United States. We want to emphasize that the data in Figs. 3, 4a–c are for inorganic N and SRP, not total N and P. In deep ocean waters, organic N and P are small compared to inorganic N and P (Michaels et al. 1996), but this is less true in surface waters or on the continental shelves, where inorganic N and SRP concentrations are lower. In a qualitative sense, we believe the inorganic data well represent the patterns of relative availabilities of N and P as sources of nutrients to surface waters and estuaries. If dissolved inorganic N remains low compared to SRP in the waters entering an estuary, it suggests a disproportionately high input of available P. Nonetheless, we urge more emphasis on patterns and ﬂuxes of total N and P in future studies, with routine measurement of total N and P by oceanographers working at continental shelf sites as well as by estuarine scientists, and more interaction of biogeochemists with physical oceanographers in esti- mating total N and P ﬂuxes in ecosystem budgets. The input of nutrients from coastal ocean waters into an estuary depends strongly on the physics of the estuary, as this controls the rate of upstream advection. All else being equal, the upstream advection is higher in systems that have a greater tidal amplitude, but the rate of freshwater discharge into the estuary is also important. We hypothesize that an estuary can be oligotrophic only in cases where not only are water- shed inputs of nutrients low but the advection into the system from coastal waters is also low, as in the micro- tidal lagoons along the French Mediterranean coast (Souchu et al. 2010). Nutrient inputs from ocean sources The northern Gulf of Mexico receives only 5% of its nitrogen from the deep ocean, 0.14 Tg N year-1 compared to 2.1 Tg N year-1 from the Mississippi River basin and 0.28 Tg N year-1 from direct atmospheric deposition (Howarth 1998). in the world’s oceans. Denitriﬁcation can have a substantial inﬂuence on the inorganic N availability in regions where the continental shelf is wide. For example, denitriﬁcation on the continental shelf along the east coast of the United States and Canada is estimated to be the major N sink there (Seitzinger and Giblin 1996; Fennel et al. 2006). The magnitude as well as the relative importance of deep-ocean nutrient inputs onto the continental shelves can vary from region to region. All along the east coast of the United States and Canada, the advection of deep-ocean, nutrient-rich waters is the largest supply of nutrients to the shelves (Nixon et al. 1996; Michaels et al. 1996; Howarth et al. 1996; Howarth 1998). For example, the continental shelf along the northeastern United States from Maine south through Delaware receives 75% of its N inputs from the deep-ocean source, 1.54 Tg N year-1 compared to only 0.27 Tg N year-1 from watershed sources and 0.21 Tg N year-1 from direct atmospheric deposition The input of nutrients from the ocean (continental shelf) source is only one factor that determines overall nutrient availability and limitation in an estuary, but in many cases, this ocean-sourced water can be substan- tial and may contain proportionately more SRP than nitrate ? nitrite, resulting in low N:P ratios (Fig. 4c). This is an important driver that can counterbalance the higher N:P ratio inputs from watersheds, and which may therefore make N limitation more likely in many estuaries than in lakes. Based on this factor, we suspect that N limitation is more likely in estuaries near a wide continental shelf in close proximity to deep-ocean waters, such as the East Coast of the United States or 123 Biogeochemistry (2021) 154:293–306 303 coastal systems, such as the LOICZ project in the 1990s and 2000s, only dissolved inorganic nutrients were considered and not total N and P (Crossland et al. 2005; Swaney et al. 2011). There is great value in examining patterns of inorganic N and P in estuaries and other coastal marine ecosystems. For example, it is from these patterns for inorganic nutrients that Li et al. Nutrient inputs from ocean sources The corollary is that estuaries such as those along the New England coast would have been naturally mesotrophic before European settle- ment (with concomitant increase in the watershed nutrient load) simply because of the large tidal inputs of nutrients into these systems from ocean sources. While the nutrient inputs to pristine lakes will almost always be very low, this is not the case for many estuaries because of the ocean-source nutrients. As a generality, we would expect primary production in pristine estuaries to be greater than in pristine lakes. Concluding thoughts and suggested future research Whether an estuary or other coastal marine ecosystem is N or P limited will be determined by the balance of N and P inputs from land, of N and P inputs from ocean sources, and of biogeochemical processes within the ecosystem that alter the availability of N and P. We suggest that the nutrient inputs from the deep ocean onto the continental shelf and into the coastal ocean and estuaries may be much more important in the overall balance than generally recognized, and we encourage more effort in characterizing these inputs and including them in ecosystem-scale N and P budgets. Further, whether primary productivity in any speciﬁc estuary is more limited by N or P should be evaluated with consideration of coastal ocean inputs, speciﬁcally considering the N:P ratio of the coastal waters and how these vary among regions partially due to differences in the width of the continental shelves and the rate of advection of deep-ocean waters onto the shelves. Our analysis suggests that the width of the conti- nental shelf may be one important factor regulating inorganic N and P concentrations as well as the N:P ratio of inorganic nutrients in water advected into estuaries and shallow coastal systems. However, the rate of advection of deep ocean water onto the continental shelf, the water residence time on the shelf, and the rate of advection of water and nutrients across the shelf to an estuary all seem likely to be important drivers controlling the nutrient inputs to As noted above, nutrient budgets for coastal marine ecosystems typically do not include the ocean source (Nixon et al. 1996). Of some of the previous efforts that did include ocean inputs in the nutrient budgets of 12 3 3 Biogeochemistry (2021) 154:293–306 304 individual systems. We therefore urge more collabo- rative studies between coastal oceanographers and estuarine biogeochemists, to evaluate how these factors affect the nutrient inputs to an estuary. In this context, we again suggest that both patterns of inorganic nutrients and ﬂuxes of total N and P be considered. sources of nutrients and wrote the related text for the ﬁrst draft. DS produced the data on trends of NANI and NAPI in space and time and wrote the supplemental material. RM and MH contributed the data on changes in total P and N in Buzzards Bay over time and contributed signiﬁcantly to writing the manuscript. References Begon M, Howarth RW, Townsend CR (2014) Essential of ecology, 4th edn. Wiley, Hoboken Bergstro¨m A-K, Jansson M (2006) Atmospheric nitrogen deposition has caused nitrogen enrichment and eutrophi- cation of lakes in the northern hemisphere. Glob Chang Biol 12:635–643 Boynton WR, Garber JH, Summers R, Kemp WM (1995) Inputs, transformations, and transport of nitrogen and phosphorus in Chesapeake Bay and selected tributaries. Estuaries 18:285–314 Butler T, Vermeylen FM, Rury R, Likens GE, Lee B, Bowker GE, McClune L (2011) Response of ozone and nitrate to stationary source NOx emission reductions in the eastern USA. Atmos Environ 45:1084–1094 Crossland CJ, Kremer HH, Lindeboom H, Crossland JM, Le Tissier MD (eds) (2005) Coastal ﬂuxes in the Anthro- pocene: the land-ocean interactions in the coastal zone project of the International Geosphere-Biosphere Pro- gramme. Springer, Berlin Dolman AM, Mischke U, Wiedner C (2016) Lake-type-speciﬁc seasonal patterns of nutrient limitation in German lakes, with target nitrogen and phosphorus concentrations for good ecological status. Freshw Biol 61:444–456 Acknowledgements Preparation of this manuscript was supported by a National Science Foundation Grant # 1654845 from the Long Term Research in Environmental Biology program, a grant from the Atkinson Center for a Sustainable Future at Cornell University, and by an endowment given to Cornell by David R. Atkinson to support a professorship held by RWH. We thank Kate Lajtha and Sujay Kaushal for inviting this submission. We also thank three anonymous peer reviewers for their very helpful comments. Doney SC, Ruckelshaus M, Duffy JE, Barry JP, Chan F, English CA, Galindo HM, Grebmeier JM, Hollowed AB, Knowlton N, Polovian J, Rabalais NN, Sydeman WJ, Talle LD (2012) Climate change impacts on marine ecosystems. Annu Rev Mar Sci 4:11–37 Elser JJ, Andersen T, Baron JS, Bergstrom A, Jansson M, Kyle M, Nydic KR, Steger L, Hessen DO (2009) Shifts in lake N:P stoichiometry and nutrient limitation driven by atmospheric nitrogen deposition. Science 326:835–837 Open Access This article is licensed under a Creative Com- mons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any med- ium 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. Concluding thoughts and suggested future research Finally, human-caused climate change has already altered circulation of the world’s oceans, including near the continents (Doney et al. 2012; Rahmstorf et al. 2015). We hypothesize that changes in these oceanographic dynamics may alter the ocean source of nutrients to estuaries, and we speculate that such changes may already be occurring. As an example from our long-term study site, the P concentrations in the water entering West Falmouth Harbor from the coastal waters of Buzzards Bay now appear to be lower than a decade or so ago, both for SRP and total P (Hayn et al., manuscript in preparation). The total N concentration in Buzzards Bay also appears to be lower now than observed a decade ago. This may reﬂect less advection of deep ocean water onto the continental shelf south of Cape Cod or a longer residence time of the water on this shelf, leading to lower concentrations of N and P. 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https://openalex.org/W4226218055	https://pure.rug.nl/ws/files/224479827/Rat_bite_fever_in_a_total_knee_arthroplasty_an_unusual_case_of_periprosthetic_joint_infection.pdf	English	null	Rat bite fever in a total knee arthroplasty: an unusual case of periprosthetic joint infection	Arthroplasty	2,022	cc-by	3,738	Rat bite fever in a total knee arthroplasty Rat bite fever in a total knee arthroplasty Fokkema, Anne Theunis; Kampschreur, Linda Martine; Pirii, Loredana Elena; Zijlstra, Wierd Pieter Published in: Arthroplasty DOI: 10.1186/s42836-022-00114-x IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2022 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Fokkema, A. T., Kampschreur, L. M., Pirii, L. E., & Zijlstra, W. P. (2022). Rat bite fever in a total knee arthroplasty: an unusual case of periprosthetic joint infection. Arthroplasty, 4(1), Article 13. https://doi.org/10.1186/s42836-022-00114-x Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). University of Groningen University of Groningen © 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 Background: Periprosthetic joint infection (PJI) is a serious complication of orthopedic arthroplasty surgery. Rat bite fever is a rare infection with Streptobacillus moniliformis (S. moniliformis). Rat bite fever can lead to serious complica- tions such as pyogenic infection of joints, bacteremia, endocarditis and even death. We hereby present the first case of a patient with a total knee arthroplasty, infected with S. moniliformis, successfully treated by surgical debridement, antibiotics and implant retention (DAIR). Case presentation: The patient was a 64-year-old female. S. moniliformis was isolated from blood cultures and an aspirate of the left knee by 16S rRNA gene polymerase chain reaction technique. It was assumed that the S. monili- formis had a systemic origin and secondarily infected the knee due to fever nine days before the onset of symptoms of the knee. The patient was successfully managed with DAIR and intravenous administration of ceftriaxone for six weeks and oral doxycycline for another six weeks. Conclusions: S. moniliformis is a rare pathogen and is difficult to culture. The 16S rRNA sequencing is helpful in the determination of a causative microorganism in the case of a culture-negative PJI. A DAIR procedure in combination with 12 weeks of antibiotics could successfully treat S. moniliformis prosthetic joint infection. Keywords: Total knee arthroplasty, Prosthetic joint infection, Rat bite fever, DAIR, Streptobacillus moniliformis contact with a rat [5]. The disease typically presents itself with fever, pharyngitis, headache, arthralgias and skin rash [2]. Typically there is minimal inflammatory reac- tion over the site of infection [2]. Various complications of RBF are known, such as reactive arthritis or pyogenic septic arthritis, bacteremia, endocarditis, pneumonia, spondylodiscitis, osteomyelitis and metastatic abscess [2, 6, 7]. Recommended treatment may consist of surgi- cal debridement and lavage of the septic joint. Based on limited experience, recommended antibiotic therapy should be five to seven days of intravenous penicillin, followed by seven days of oral medication [8]. S. monili- formis is known as an extremely fastidious bacterium that needs microaerophilic conditions to grow and this makes the diagnosis difficult [5, 9]. While arthralgia, pyogenic infection of the joint, antibiotic treatment of prosthesis infection and even arthroscopic treatment of an infected knee prosthesis have been reported before. We hereby Copyright strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of th or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. down policy believe that this document breaches copyright please contact us providing details, and we will remove access to the wo vestigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 Arthroplasty Fokkema et al. Arthroplasty (2022) 4:13 https://doi.org/10.1186/s42836-022-00114-x Background Periprosthetic joint infection (PJI) is a serious compli- cation of orthopedic arthroplasty surgery. Common causative bacteria are Staphylococcus aureus and coagu- lase-negative Staphylococci [1]. Rat bite fever (RBF) is a rare zoonotic infection caused by Streptobacillus mon- iliformis (S. moniliformis) or Spirillum minus [2]. S. mon- iliformis is a gram-negative rod that was first isolated in 1914 by Schotmüller [3]. It colonizes the nasopharynx of rats and other rodents [4]. Patients can be infected by either a rat bite or scratch, or oral ingestion of sub- stances soiled by rat feces and urine with S. moniliformis [2]. Approximately 30% of the patients cannot recall *Correspondence: anne.fokkema@mcl.nl 1 Department of Orthopedic Surgery, Medical Center Leeuwarden, Henri Dunantweg 2, Leeuwarden, The Netherlands 8934 AD Full list of author information is available at the end of the article Case presentation A 64-year-old female patient, with a medical history of axial spondylarthropathy, diabetes mellitus type II, hypertension, morbid obesity and laparoscopic Roux en Y bypass, received a left TKA in 2015 for end-stage osteoarthritis. The surgical procedure and follow-up were uneventful. In May 2021, the patient returned with pain in both shoulders, upper arms, the right wrist and, most prominently, in the left knee. She reported that she had felt ill for a day and had 39 degrees fever nine days before she visited the hospital. The physical examina- tion showed a non-acute ill patient with a large swelling of the left knee, and a heavily impaired function with a flexion of 50 degrees, and extension deficit of 30 degrees. The blood examination showed a C-reactive protein (CRP) of 156 mg/L and a white blood cell count (WBC) of 13.1 × 109/L per liter. A sterile puncture was per- formed and samples were taken and analyzed for gram stain, culture and uric crystals, and blood cultures were taken. A rheumatologist was consulted and the aspirate of the knee was analyzed for uric crystals, which were not found. The gram staining was negative but was positive for leukocytes. Initially, the clinical diagnosis was made of reactive arthritis due to systemic infection with an unknown primary location. The patient was hospitalized for close monitoring, while antibiotics were withheld. On the second day of admission, the blood cultures turned positive for a gram-negative rod. Intravenous cefuroxime was started at 1.5 g four times a day. Due to minor clinical improvement with a decrease in knee pain and a drop of CRP to 120 mg/L, no surgery was performed the second day. The third day after admission, the CRP, however, rose to 149 mg/L again, and the patient reported more pain in the knee. Therefore, the decision was made to perform a Debridement, Antibiotics and Implant Retention (DAIR) procedure. Preoperatively, two grams of cefazolin was administered to prevent secondary infections. An open arthrotomy was performed. Perioperatively, there was no collection of pus but cloudy synovium was observed. Four cultures were taken of synovial fluid, peripros- thetic tissue, joint capsule and interface tissue around the prosthesis, each with a clean instrument. The prosthesis insert could not be removed due to unavailability of the insert because this type of prosthesis was discontinued by the manufacturer. Fokkema et al. Arthroplasty (2022) 4:13 Page 2 of 4 performed, by using a diathermic knife. The prosthesis and surrounding tissues were then brushed manually with a scrubbing sponge and povidone-iodine for at least two minutes. This was followed by extensive flushing of the knee by pulsating lavage with six liters of 0.9% saline solution. Subsequently, a new extremity drape was placed around the knee, and gloves were changed and the knee was closed in layers using new instruments. Immediately after surgery, the patient improved clinically. A trans- esophageal ultrasound was performed on the request of the infectious disease specialist and showed no evidence of infectious endocarditis. The blood cultures (BD Bac- tec) became positive after two incubation days and they were cultured aerobically and anaerobically on solid media. The bacteria were seen after five days on blood agar with 5% sheep blood and on chocolate agar media. A Vitek MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDITOF) (bioMé- rieux) was used based on mass spectrometry principle for the identification of the grown colonies, but the iden- tification failed repeatedly. The identification with Vitek MS MALDITOF (bioMérieux) depends on the database of the analyzer which can impose limitations. For this reason, molecular diagnostics using the 16S rRNA gene polymerase chain reaction technique (PCR) were chosen to help the identification and eventually S. moniliformis was found. The knee aspirate was also cultured and after 7 days of incubation. S. moniliformis was also identi- fied in the knee aspirate by 16S rRNA gene sequencing. Unfortunately, the susceptibility of S. moniliformis could not be determined despite all the efforts to use micro- aerophilic conditions and Schaedler anaerobe agar with sheep blood with haemin and vitamin K1 as advised in the literature [5]. Knowing the etiological agent of bacte- remia, the antibiotics were switched to ceftriaxone based upon literature review [8]. Supplementary history taking was performed to trace the origin of the infection. The patient reported having no memory of a rat bite, nor liv- ing close to rats, nor living close to open water or having eaten spoiled food. She reported having two dogs. Pos- sibly the dogs could have been infected by S. moniliformis by either eating rats, being bitten by rats or by swimming in water polluted by rat feces. Therefore, both dogs were swabbed and samples were taken of their saliva. S. mon- iliformis could not be cultured and recovered from the dogs. Sixteen days after the DAIR, the patient could be discharged home with intravenous antibiotics in good clinical condition. The CRP at time of discharge was 20 mg/L and the WBC was normal at 3.9 × 109/L. A total antibiotic regimen of twelve weeks was planned accord- ing to clinical guidelines [12]. After multidisciplinary consultation with a medical microbiologist, an infec- present, to the best of our knowledge, the first case of a PJI caused by S. moniliformis in a patient who received a total knee arthroplasty (TKA) and was managed by sur- gical Debridement, Antibiotics and Implant Retention (DAIR) [10, 11]. Case presentation If it had been available, the insert would have been exchanged for a new insert. Extensive debridement, with all “suspicious” tissues excised, was Page 3 of 4 Fokkema et al. Arthroplasty (2022) 4:13 parts should be removed to provide optimal view and cleaning possibilities. However, this was not possible in our patient as the required insert was discontinued by the manufacturer. Parts of the prosthesis that remained in place were brushed with sponges to remove biofilm using povidon iodone solution. Finally, the joint was flushed with 6 L of saline using pulsed lavage. Gloves were changed and the knee was closed in layers. The surgical treatment was followed by antibiotic therapy, generally fourteen days of intravenous antibiotics, fol- lowed by ten weeks of oral antibiotics [12]. Based on multidisciplinary consultation with medical microbi- ologists and infectious diseases specialists, a course of intravenous ceftriaxone and oral doxycycline was chosen. following antibiotic regimen was agreed upon: six weeks intravenous ceftriaxone two grams once daily because of bacteremia and another six weeks of oral doxycycline 200 mg once daily to complete the treatment of the PJI [12]. Due to penicillin allergy confirmed with skin test- ing, the patient could not be treated with oral amoxicil- lin, which normally would be the oral antibiotic of choice. At three, six and thirteen weeks after surgery, the patient was seen for follow-up. At the most recent follow-up at thirteen weeks when the patient was one week after the last administration of doxycycline, the pain had dimin- ished. The function of the knee was improved to normal walking with a flexion of 120 degrees and full extension to zero degrees. Laboratory results showed CRP < 1 mg/L and WBC of 2.9 × 109/L. Two cases of rat bite fever PJI have been described before but with different treatment and outcomes. Stehle et al. (2003) have described a case of a patient with a history of bilateral TKA, who was successfully treated by oral antibiotics [10]. Smallbones et al. (2020) described a case of TKA with S. moniliformis infec- tion, who was unsuccessfully treated by arthroscopy and hence in need of two-stage revision surgery [11]. It remains debatable whether patients could be treated purely with antibiotic therapy. We believe a DAIR pro- cedure is superior to both antibiotic treatment, and arthroscopic lavage in the case of an infected arthro- plasty with S. moniliformis. Case presentation A DAIR procedure has a low morbidity and moderate to good chances of success if performed meticulously by experienced hands [12]. Discussion h b To the best of our knowledge, this is the first case of periprosthetic joint infection with S. moniliformis in a patient with a TKA, which was successfully treated by DAIR and a 12-week course of intravenous and oral anti- biotics. The use of 16S rRNA sequencing was vital for establishing the diagnosis. Rat bite fever is a rare zoonotic infection character- ized by fever, rash and arthritis caused by S. moniliformis [2]. Streptobacillus moniliformis is a pleomorphic, fila- mentous, gram-negative, non-motile and non-acid fast bacillus. The bacteria grow slowly (2 to 3 days) and may take as long as 7 days to yield the positive result. The bio- film-forming capacity of S. moniliformis is still unknown and future research is necessary. S. moniliformis is an extremely fastidious bacterium that needs microaero- philic conditions to grow. This makes microbiological diagnosis difficult. Optimal growth requires Trypticase soy agar or broth enriched with 20% blood and micro- aerophilic conditions [5]. Once bacterial growth takes place, it is worthwhile to try to identify it by using a MALDITOF MS analyzer. This was not successful in this case. Nonetheless, we expect that, in the future, the data- bases of the analyzers will be extended with new bacterial profiles and identification of S. moniliformis can become possible. Until then, the 16S rRNA sequencing remains the fundamental method for the identification of S. mon- iliformis [11]. A possible limitation in this case was that no insert replacement could be done due to the fact that the manufacturer no longer produced the required insert. Also, the follow-up time was relatively short in the pre- sent case so recurrence may be possible in the future. Conclusions In this case we have shown a good clinical response to the combination of a DAIR procedure and pro- longed antibiotics for the treatment of a S. moniliformis periprosthetic joint infection. 16S rRNA gene sequenc- ing was vital for the diagnosis. Future cases of S. mon- iliformis periprosthetic joint infection could be treated by the combination of DAIR and prolonged antibiotics. In our case, clinical clues of PJI prompted us to per- form surgery on the TKA despite the fact that the cul- tures remained negative initially. We performed a DAIR procedure, based on steps agreed upon in our joint pro- tocol for treating periprosthetic joint infections [12]. The protocol consists of open arthrotomy, sampling at least five deep tissue cultures, followed by extensive debridement with excision of possible infected necrotic tissues and synovectomy. Exchangeable prosthesis PJI: Prosthetic joint infection; RBF: Rat bite fever; TKA: Total knee arthroplasty; DAIR: Debridement, antibiotics and implant retention; CRP: C-reactive protein; WBC: White blood cell count; MALDITOF: Matrix-assisted laser desorption/ioni- zation time-of-flight mass spectrometry; PCR: Polymerase chain reaction. Abbreviations PJI P h i j i PJI: Prosthetic joint infection; RBF: Rat bite fever; TKA: Total knee arthroplasty; DAIR: Debridement, antibiotics and implant retention; CRP: C-reactive protein; WBC: White blood cell count; MALDITOF: Matrix-assisted laser desorption/ioni- zation time-of-flight mass spectrometry; PCR: Polymerase chain reaction. Acknowledgements Not applicable Availability of data and materials Availability of data and materials Relevant data are available on reasonable request. Relevant data are available on reasonable request. Relevant data are available on reason Ethics approval and consent to participate The authors state that the treatment of the patient was not influenced because of this report. Written informed consent was obtained from the patient. Consent for publication Written informed consent was obtained from the patient for publication of this case report. Acknowledgements Not applicable Acknowledgements Not applicable Page 4 of 4 Fokkema et al. Arthroplasty (2022) 4:13 antimicrobial therapy. Experiences from the Northern Infection Network for Joint Arthroplasty (NINJA) in the Netherlands. Zijlstra WP, Ploegmakers JJW, Kampinga G, Toren-Wielema ML, Ettema HB, Knobben B, Jutte PC, Wouthuyzen-Bakker M, on behalf of the Northern Infection Network for Joint Arthroplasty (NINJA) Competing interests The authors declare that they have no competing interests and they were not involved in the journal’s review of or decisions related to this manuscript. 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Antimicrobial therapy of ratbite fever: a review. Arch Intern Med. 1965;116(1):39–54. https://doi.org/10.1001/archinte.1965. 03870010041007. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 9. Publisher’s Note S i N i Funding No funding was received for this case report. No funding was received for this case report. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Availability of data and materials Author details 1 Department of Orthopedic Surgery, Medical Center Leeuwarden, Henri Dunantweg 2, Leeuwarden, The Netherlands 8934 AD. 2 Department of Inter- nal Medicine, Medical Center Leeuwarden, Leeuwarden, The Netherlands. 3 Certe Center For Infectious Diseases Friesland, Leeuwarden, The Netherlands. Received: 18 October 2021 Accepted: 6 January 2022 Received: 18 October 2021 Accepted: 6 January 2022 • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: References Kimura M, Tanikawa T, Suzuki M, Koizumi N, Kamiyama T, Imaoka K, et al. Detection of streptobacillus spp. in feral rats by specific polymerase chain reaction. Microbiol Immunol. 2008;52(1):9–15. 10. Stehle P, Dubuis O, So A, Dudler J. Rat bite fever without fever. Ann Rheum Dis. 2003;62(9):894–6. 11. Smallbones M, Monem M, Baganeanu M, Okocha M, Sofat R. Near-fatal periprosthetic infection with streptobacillus moniliformis: case and review. J Bone Jt Infect. 2020;5(1):50–3. 12. (NINJA) NINJA. Protocol gewrichtsprothese infecties (PJI) bij volwassenen [Internet]. 2021. p. 1–21. Available from: https://www.ninja-noord.nl/ wp-content/uploads/2021/05/NINJA-protocol-PJI-2021.pdf. Accessed 15 October 2021. Also submitted to Arthroplasty, as: A protocol for peripros- thetic joint infections: clinical aspects, diagnosis, surgical treatment, and
https://openalex.org/W2161003704	http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889760/pdf/zdb1616.pdf	English	null	Early Hepatic Insulin Resistance Precedes the Onset of Diabetes in Obese C57BLKS-<i>db/db</i> Mice	Diabetes	2,010	cc-by	9,414	Early Hepatic Insulin Resistance Precedes the Onset of Diabetes in Obese C57BLKS-db/db Mice Richard C. Davis,1 Lawrence W. Castellani,1 Maryam Hosseini,1,2,3 Osnat Ben-Zeev,1,2 Hui Z. Mao,2,3 Michael M. Weinstein,1 Dae Young Jung,4,5 John Y. Jun,5 Jason K. Kim,4,5,6 Aldons J. Lusis,1 and Miklo´s Pe´terfy1,2,3 are normoglycemic throughout their life, obese leptin- deﬁcient (BKS-ob) or leptin receptor–deﬁcient (BKS-db) mice develop severe hyperglycemia. Second, the natural history of diabetes in BKS-ob or BKS-db is reminiscent of the human disease. These mice initially compensate for obesity-associated insulin resistance by increasing plasma insulin levels, but exhibit -cell failure and insulin deﬁ- ciency later in life. Finally, similarly to humans, diabetes in BKS-db is determined by multiple genetic factors (4,5). Despite extensive genetic analysis, the genes responsible for diabetes susceptibility in the BKS strain remain to be identiﬁed (6–8). OBJECTIVE—To identify metabolic derangements contributing to diabetes susceptibility in the leptin receptor–deﬁcient obese C57BLKS/J-db/db (BKS-db) mouse strain. RESEARCH DESIGN AND METHODS—Young BKS-db mice were used to identify metabolic pathways contributing to the development of diabetes. Using the diabetes-resistant B6-db strain as a comparison, in vivo and in vitro approaches were applied to identify metabolic and molecular differences between the two strains. RESULTS—Despite higher plasma insulin levels, BKS-db mice exhibit lower lipogenic gene expression, rate of lipogenesis, hepatic triglyceride and glycogen content, and impaired insulin suppression of gluconeogenic genes. Hepatic insulin receptor substrate (IRS)-1 and IRS-2 expression and insulin-stimulated Akt-phosphorylation are decreased in BKS-db primary hepato- cytes. Hyperinsulinemic-euglycemic clamp studies indicate that in contrast to hepatic insulin resistance, skeletal muscle is more insulin sensitive in BKS-db than in B6-db mice. We also demon- strate that elevated plasma triglyceride levels in BKS-db mice are associated with reduced triglyceride clearance due to lower lipase activities. ( ) Early studies on BKS-db mice indicated that the devel- opment of diabetes is associated with progressive -cell degranulation and a precipitous decrease in -cell mass and plasma insulin levels (2). In vivo radio-labeling studies revealed that after an initial phase of hyperproliferation at 4–6 weeks of age, the replication of -cells gradually decreases despite increasing glucose levels (9). In contrast to BKS, introduction of the db mutation into the C57BL/6J (B6) genetic background produces a dramatically different -cell phenotype (2,4). Although similarly obese as BKS- db, B6-db mice compensate for insulin resistance by -cell hyperplasia, increased islet mass, and hyperinsulinemia and maintain only mildly elevated blood glucose levels throughout their life. From the 1Department of Medicine, University of California, Los Angeles, Los Angeles, California; the 2Lipid Research Laboratory, VA Greater Los Angeles Healthcare System, Los Angeles, California; the 3Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, California; the 4Program in Molecular Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Massachusetts Medical School, Worcester, Massachusetts; the 5Department of Cellular and Molecular Physiology, Pennsylvania State University School of Medicine, Hershey, Pennsylvania; and the 6Department of Medicine, Division of Endocrinology, Metabolism and Diabetes, Univer- sity of Massachusetts Medical School, Worcester, Massachusetts. Corresponding author: Miklos Peterfy, mpeterfy@ucla.edu. Received 11 June 2009 and accepted 5 April 2010. Published ahead of print at http://diabetes.diabetesjournals.org on 14 April 2010. DOI: 10.2337/db09-0878. R.C.D. and L.W.C. contributed equally to this work. © 2010 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for proﬁt, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ORIGINAL ARTICLE ORIGINAL ARTICLE Early Hepatic Insulin Resistance Precedes the Onset of Diabetes in Obese C57BLKS-db/db Mice The markedly different -cell re- sponses to obesity in BKS-db and B6-db mice suggest that genetically determined variation in -cell viability/survival in the face of chronic glycemic stress is responsible for differences in diabetes susceptibility between the two strains. Consistent with this hypothesis, BKS -cells are more sensitive than B6 to cell death triggered by -cell toxins, such as alloxan and streptozotocin (10–12), and glucose-stimulated islet cell replication is diminished in BKS (13). In conclusion, previous studies suggest that variant -cell functions underlie the differences in diabetes susceptibility between BKS-db and B6-db mice. CONCLUSIONS—Our study demonstrates the presence of met- abolic derangements in BKS-db before the onset of -cell failure and identiﬁes early hepatic insulin resistance as a component of the BKS-db phenotype. We propose that defects in hepatic insulin signaling contribute to the development of diabetes in the BKS-db mouse strain. Diabetes 59:1616–1625, 2010 E E stablished in the 1940s, the C57BLKS (BKS) inbred mouse strain represents one of the ﬁrst animal models of type 2 diabetes (1). Develop- ment of diabetes in these mice captures several aspects of the human disease (2,3). First, diabetes in this model is associated with obesity. Whereas lean BKS mice In the current study, we reﬁne the current -cell–centric model of diabetes susceptibility in BKS-db by demonstrat- ing metabolic defects preceding the onset of -cell failure. In particular, BKS-db mice exhibit elevated hepatic insulin resistance associated with altered lipogenic and gluconeo- genic pathways relative to B6-db. We propose that early hepatic insulin resistance contributes to the development of diabetes in the BKS-db strain. diabetes.diabetesjournals.org From the 1Department of Medicine, University of California, Los Angeles, Los Angeles, California; the 2Lipid Research Laboratory, VA Greater Los Angeles Healthcare System, Los Angeles, California; the 3Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, California; the 4Program in Molecular Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Massachusetts Medical School, Worcester, Massachusetts; the 5Department of Cellular and Molecular Physiology, Pennsylvania State University School of Medicine, Hershey, Pennsylvania; and the 6Department of Medicine, Division of Endocrinology, Metabolism and Diabetes, Univer- sity of Massachusetts Medical School, Worcester, Massachusetts. RESEARCH DESIGN AND METHODS Animals. C57BL/6J-db/db (B6-db) and C57BLKS/J-db/db (BKS-db) mice were purchased from The Jackson Laboratory. Mice were maintained in the animal care facilities of the University of California and VA Greater Los Angeles Healthcare System on a 12-h light/dark cycle. The mice were maintained on a standard diet (Harlan Teklad LM-485). Before all experiments, mice were fasted for 4 h (9:00 A.M. to 1:00 P.M.) unless indicated otherwise. Protocols were 1616 DIABETES, VOL. 59, JULY 2010 R.C. DAVIS AND ASSOCIATES approved by the Institutional Animal Care and Use Committees of the respective institutions. approved by the Institutional Animal Care and Use Committees of the respective institutions. Manassas, VA) and fungizone (Invitrogen, Carlsbad, CA) and allowed to adhere for 4 h. The media were then removed, the cells washed once with PBS, and fresh media added. The following day, the media were changed and replaced with the same media except without the fungizone. After 24 h in culture, cells were serum-starved overnight in 199 medium supplemented with glutamine, NaHCO3, and pen/strep. After incubation in fresh serum-free media for 2 h, cells were stimulated with 100 nmol/l of porcine insulin for 10 min in triplicate wells. Cells were solubilized in lysis buffer (50 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl, 1% NP-40) containing inhibitors of proteases (Complete Mini; Roche, Indianapolis, IN) and phosphatases (PhosStop; Roche). Protein concentration was determined by the BCA assay (Pierce, Rockford, IL). Manassas, VA) and fungizone (Invitrogen, Carlsbad, CA) and allowed to adhere for 4 h. The media were then removed, the cells washed once with PBS, and fresh media added. The following day, the media were changed and replaced with the same media except without the fungizone. After 24 h in culture, cells were serum-starved overnight in 199 medium supplemented with glutamine, NaHCO3, and pen/strep. After incubation in fresh serum-free media for 2 h, cells were stimulated with 100 nmol/l of porcine insulin for 10 min in triplicate wells. Cells were solubilized in lysis buffer (50 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl, 1% NP-40) containing inhibitors of proteases (Complete Mini; Roche, Indianapolis, IN) and phosphatases (PhosStop; Roche). Protein concentration was determined by the BCA assay (Pierce, Rockford, IL). approved by the Institutional Animal Care and Use Committees of the respective institutions. Phenotypic characterization of mice. Plasma lipids, glucose, and insulin were determined as described previously (14). RESULTS Clearance of TG (10 l peanut oil/g body weight delivered by gavage) was determined by following plasma TG levels at hourly intervals after the administration of lipid. Post-heparin plasma. Ten units of heparin was administered by tail-vein injection followed by retro-orbital blood collection 10 min later as described (20). Plasma lipase activities were determined as described below. Post-heparin plasma. Ten units of heparin was administered by tail-vein injection followed by retro-orbital blood collection 10 min later as described (20). Plasma lipase activities were determined as described below. Gene expression. Total RNA was isolated with Trizol (Invitrogen) and reverse transcribed using the Omniscript RT kit (Qiagen). Quantitative real-time RT-PCR was performed as described previously (21) using primers shown in supplementary Table 1, available in an online appendix at http:// diabetes.diabetesjournals.org/cgi/content/full/db09-0878/DC1. Each sample was measured in triplicates. Expression of the TATA-box binding protein (Tbp) and hypoxanthine guanine phosphorybosyl transferase (Hprt) genes were used for normalization. Gene expression. Total RNA was isolated with Trizol (Invitrogen) and reverse transcribed using the Omniscript RT kit (Qiagen). Quantitative real-time RT-PCR was performed as described previously (21) using primers shown in supplementary Table 1, available in an online appendix at http:// diabetes.diabetesjournals.org/cgi/content/full/db09-0878/DC1. Each sample was measured in triplicates. Expression of the TATA-box binding protein (Tbp) and hypoxanthine guanine phosphorybosyl transferase (Hprt) genes were used for normalization. Hyperinsulinemic-euglycemic clamp. Euglycemic clamps were performed in conscious 1-month-old mice using a 10 mU/kg/min insulin dose as described (22). Insulin signaling in primary hepatocytes. Hepatocytes were isolated by a two-step perfusion protocol that is based on minor modiﬁcations of the method described by Seglen (23). Brieﬂy, mice were anesthetized by intra- peritoneal injection of pentobarbital (50 mg/kg). The abdominal cavity was opened and the liver perfused via the portal vein ﬁrst with a Ca2 Mg free EDTA chelating solution, followed by perfusion with a Ca2 Mg replete buffer containing type I collagenase (Worthington Biochemical, Lakewood, NJ). The hepatocytes were then dissociated by cutting the liver capsule and gently shaking in the collagenase solution. A series of ﬁve low-speed (50g) centrifugations were performed to wash and differentially sediment hepato- cytes from other cell types, particularly Kupffer and endothelial cells. The resulting cell pellet was re-suspended, and an aliquot was taken to determine cell number and viability by trypan blue exclusion. RESEARCH DESIGN AND METHODS Fatty acid (FA) synthesis and oxidation were measured in liver slice cultures (15). FA oxidation. After an overnight fast, mice were anesthetized with isoﬂurane and killed by cervical dislocation. The left caudal lobe of the liver was rapidly excised, and fresh liver sections of uniform thickness (1 mm, averaging 60 mg) were obtained using a Stadie-Riggs microtome. The liver sections were weighed and immediately incubated at 37°C for 40 min in Krebs-Henseleit buffer under 95% O2:5% CO2 that contained 5.5 mmol/l glucose, 3% BSA, 1 mmol/l oleate, and [14C-U]-palmitic acid (2.5 Ci/ml). The average time from euthanizing the animals to getting the liver section into the buffer was 2 min. Rates of FA oxidation were assessed by determining 14CO2 trapped in hyamine hydroxide saturated ﬁlter paper as described previously (16). Statistical analysis. Data are presented as mean SE. Differences between two groups were considered statistically signiﬁcant at P 0.05 using Student’s t test. ANOVA was used for multiple group comparisons followed by Bonfer- roni’s post hoc test, as implemented in the SigmaPlot software package (Systat Software, San Jose, CA). Non-normally distributed trait values were transformed before analysis as indicated. Hepatic lipogenesis. Liver sections were obtained and incubated essentially as described above, except that 3H2O (0.5 mCi/vessel) was used as the radioactive tracer and incubations were carried out for 1 h (17). Media and tissue were then collected and total lipids extracted as described above. Total lipid isolates were then hydrolyzed in an acid/acetonitrile solution as de- scribed previously (18), and the lipids were re-extracted and dried under nitrogen and radioactivity in the fatty acid fraction determined by liquid scintillation spectrometry. RESEARCH DESIGN AND METHODS The Luminex 100 system was used to measure plasma leptin, resistin, tumor necrosis factor-, and inter- leukin-6 levels. Adiponectin, corticosterone (R&D Systems, Minneapolis, MN), and glucagon (ALPCO Diagnostics, Salem, NH) were determined by enzyme- linked immunosorbent assays. Hepatic glycogen content was assayed at the Mouse Metabolic Phenotyping Center at Yale University. Hepatic triglyceride (TG) content was assayed using the L-type TG H kit (Wako Diagnostics, Richmond, VA) with modiﬁcations. Brieﬂy, liver tissue was homogenized in ice-cold PBS with a Polytron homogenizer. After Folch extraction, samples were centrifuged and the lower (organic) phase was transferred to new tubes. The organic volume was dried in vacuum and re-dissolved in 90 l of 10% Triton X-100 in isopropanol by vortexing. After the addition of 1.5 ml Wako Reagent A, samples were vortexed and incubated at 37° C for 5 min. Samples were vortexed until clear and 0.75 ml Wako Reagent B was added followed by incubation at 37°C for 30 min. Samples were vortexed again until clear and then put on ice until measurement of optical density at 600 nm. A calibration curve was established using the Wako Lipid Calibrator. Immunoblot analysis. Tissue and cell lysates were analyzed with Western blotting. Primary antibodies against insulin receptor substrate (IRS)-1 (1:500 dilution, Upstate Biotechnology, Lake Placid, NY), IRS-2 (1:2,000 dilution, Upstate), Akt (1:5,000 dilution, Cell Signaling Technologies, Danvers, MA), and phospho-Akt (1:1,000 dilution, CST) were used followed by goat anti- rabbit–horseradish peroxidase (1:10,000 dilution; Jackson ImmunoResearch, West Grove, PA) secondary antibody. Blots were visualized using the ECL chemiluminescent detection system (Roche). Lipase assays. The activity of lipoprotein lipase (LPL) and hepatic lipase was measured using the respective triolein substrates prepared by sonication (24). To discriminate between LPL and hepatic lipase activities further, we took advantage of the difference in salt sensitivity between these lipases. Lipase activities were measured using the LPL substrate in the presence and absence of 1 mol/l NaCl; LPL activity was calculated as the salt-inhibited fraction. Tissues were homogenized in 25 mmol/l Tris-HCl, pH 7.5, containing 10% glycerol, 0.2% deoxycholic acid (sodium salt), and 10 /ml heparin. Homoge- nization (100 mg/ml) was carried out for 30 s on ice, using a Tekmar tissue homogenizer, followed by centrifugation for 10 min at 10,000g to remove insoluble debris and, in the case of adipose tissue, to separate the ﬂoating lipid layer from the lysate. Assays of lipid metabolism. diabetes.diabetesjournals.org RESULTS Metabolic differences between young BKS-db and B6-db mice. To characterize the development of diabetes in the BKS-db strain, we performed a metabolic time- course study (Fig. 1, line graphs, and supplementary Table 3). B6-db mice were used as controls, because they are diabetes resistant despite their genetic similarity to BKS- db. Consistent with previous reports (2,4), BKS-db and B6-db mice exhibited similar body weights and adiposity, with the exception of male BKS-db, which started losing weight at 3 months because of severe diabetes (Fig. 1A and B). Despite similar degrees of obesity, BKS-db and B6-db mice displayed dramatic differences in glucose homeosta- sis. B6-db mice compensated for insulin resistance associ- ated with obesity by sharply increasing plasma insulin levels (Fig. 1D). In contrast, insulin levels in BKS-db gradually declined and were associated with progressive hyperglycemia (Fig. 1C). These data are consistent with previous studies implicating impaired -cell viability in BKS-db as the primary determinant of diabetes suscepti- bility (10,11,13). However, our time-course study also revealed metabolic differences between the strains at 1 month of age, before the onset of diabetes in BKS-db (Fig. 1, right panels). At this age, BKS-db mice maintained similar glucose levels, but over twofold higher insulin concentrations in comparison to B6-db mice (Fig. 1C and D). Moreover, these mice exhibited elevated plasma TG (Fig. 1E), but lower free fatty acid (Fig. 1F) and total and HDL cholesterol levels (data not shown) relative to their B6-db counterparts. To assess the effects of strain back- ground and genetic interaction with the db mutation, we also phenotyped lean B6 and BKS mice (supplementary Table 2). Among the traits analyzed, only HDL cholesterol Hepatic TG secretion. TG secretion was determined by comparing plasma TG levels at 0 and 30 min after intravenous injection of Triton WR1339 (Tyloxapol, Sigma Chemical) as described (19). Calculations assumed plasma volume of 3.5% of body weight. Hepatic TG secretion. TG secretion was determined by comparing plasma TG levels at 0 and 30 min after intravenous injection of Triton WR1339 (Tyloxapol, Sigma Chemical) as described (19). Calculations assumed plasma volume of 3.5% of body weight. Triglyceride clearance. Clearance of TG (10 l peanut oil/g body weight delivered by gavage) was determined by following plasma TG levels at hourly intervals after the administration of lipid. Triglyceride clearance. RESULTS Body weight (A), adiposity (B), blood glucose (C), plasma insulin (D), TG (E), and free fatty acids (FFA) (F) are shown at 1, 3, and 7 months of age (left panels). F, B6-db males (n 10); f, B6-db females (n 11); E, BKS-db males (n 10); , BKS-db females (n 7). Labels of statistical signiﬁcance have been omitted from the charts for clarity. Bar graphs (right panels) show data at 1 month of age. P < 0.05 for differences between strains. IMPAIRED HEPATIC METABOLISM IN C57BLKS-db MICE 0 10 20 30 Male Female 0 10 20 30 40 50 60 70 80 1 3 7 gram A Body Weight Adiposity Age (month) Age (month) 0 10 20 30 40 50 Male Female 0 10 20 30 40 50 60 70 1 3 7 % B 0 10 20 30 Male Female 0 10 20 30 40 50 60 70 80 1 3 7 gram A Body Weight Age (month) Adiposity Age (month) 0 10 20 30 40 50 Male Female 0 10 20 30 40 50 60 70 1 3 7 % B Body Weight B A Glucose Age (month) 0 100 200 300 400 Male Female 0 100 200 300 400 500 600 700 800 900 1000 1 3 7 mg/dL C Insulin Age (month) D 0 2 4 6 8 10 12 Male Female * 0 2 4 6 8 10 12 14 1 3 7 ng/mL D D C Female FFA Age (month) 0 20 40 60 Male Female * * 0 10 20 30 40 50 60 1 3 7 mg/dL F Plasma TG Age (month) E 0 20 40 60 Male Female * * 0 20 40 60 80 100 120 140 1 3 7 mg/dL F E FIG. 1. Phenotype of B6-db and BKS-db mice. Body weight (A), adiposity (B), blood glucose (C), plasma insulin (D), TG (E), and free fatty acids (FFA) (F) are shown at 1, 3, and 7 months of age (left panels). F, B6-db males (n 10); f, B6-db females (n 11); E, BKS-db males (n 10); , BKS-db females (n 7). Labels of statistical signiﬁcance have been omitted from the charts for clarity. Bar graphs (right panels) show data at 1 month of age. P < 0.05 for differences between strains. cemic clamps were performed. RESULTS The cells were plated at a density of 2.0 106 cells on 60-mm collagen-coated plates (BD Biosciences, San Jose, CA) in hepto-stim media (BD Biosciences, San Jose, CA) containing 10% FBS (ATCC, Manassas, VA) and supplemented with pen/strep (ATCC, Insulin signaling in primary hepatocytes. Hepatocytes were isolated by a two-step perfusion protocol that is based on minor modiﬁcations of the method described by Seglen (23). Brieﬂy, mice were anesthetized by intra- peritoneal injection of pentobarbital (50 mg/kg). The abdominal cavity was opened and the liver perfused via the portal vein ﬁrst with a Ca2 Mg free EDTA chelating solution, followed by perfusion with a Ca2 Mg replete buffer containing type I collagenase (Worthington Biochemical, Lakewood, NJ). The hepatocytes were then dissociated by cutting the liver capsule and gently shaking in the collagenase solution. A series of ﬁve low-speed (50g) centrifugations were performed to wash and differentially sediment hepato- cytes from other cell types, particularly Kupffer and endothelial cells. The resulting cell pellet was re-suspended, and an aliquot was taken to determine cell number and viability by trypan blue exclusion. The cells were plated at a density of 2.0 106 cells on 60-mm collagen-coated plates (BD Biosciences, San Jose, CA) in hepto-stim media (BD Biosciences, San Jose, CA) containing 10% FBS (ATCC, Manassas, VA) and supplemented with pen/strep (ATCC, DIABETES, VOL. 59, JULY 2010 1617 diabetes.diabetesjournals.org IMPAIRED HEPATIC METABOLISM IN C57BLKS-db MICE 0 10 20 30 Male Female 0 10 20 30 40 50 60 70 80 1 3 7 gram A Body Weight Glucose Plasma TG FFA Insulin Adiposity Age (month) Age (month) Age (month) Age (month) Age (month) Age (month) 0 10 20 30 40 50 Male Female 0 10 20 30 40 50 60 70 1 3 7 % 0 100 200 300 400 Male Female 0 100 200 300 400 500 600 700 800 900 1000 1 3 7 mg/dL C E D 0 2 4 6 8 10 12 Male Female * 0 2 4 6 8 10 12 14 1 3 7 ng/mL 0 20 40 60 Male Female * * 0 20 40 60 80 100 120 140 1 3 7 mg/dL 0 20 40 60 Male Female * * 0 10 20 30 40 50 60 1 3 7 mg/dL B F FIG. 1. Phenotype of B6-db and BKS-db mice. diabetes.diabetesjournals.org RESULTS Hepatic FA oxidation was not signiﬁcantly different between the two strains, although there was a trend toward increased oxidation in BKS-db (Fig. 3D). We also tested the potential involvement of circulating factors in the observed metabolic differences by measuring plasma levels of various adipokines and hormones implicated in hepatic metabolism (25). Plasma concentrations of adiponectin, leptin, resistin, corticoste- rone, and glucagon were similar in B6-db and BKS-db mice (supplementary Table 3). Taken together, our results indicate altered lipid and carbohydrate metabolism con- sistent with elevated hepatic insulin resistance in BKS-db mice. Altered hepatic gene expression in young BKS-db mice. To determine if altered gene expression underlies the metabolic differences observed between BKS-db and B6-db mice, we measured mRNA levels of genes involved in hepatic lipid and carbohydrate metabolism. Consistent with reduced lipogenesis in BKS-db mice, expression levels of all lipogenic genes tested (liver X receptor- [LXR], sterol regulatory element binding protein-1c [Srebp1c], peroxisome proliferator-activated receptor- [Ppar], acetyl-CoA carboxylase-1 [AccI], fatty acid syn- thase [Fas], ATP citrate lyase [Acl], stearoyl-CoA desatu- rase-1 [Scd1], and cytosolic malic enzyme [Me1]) were signiﬁcantly lower in this strain (Fig. 4A). The expression of enzymes involved in TG synthesis, including glycerol-3- phosphate acyltransferase (Gpat) and lipin (Lpin1), was also lower in BKS-db. As Srebp1c is a direct transcriptional activator of lipogenic genes and Gpat (26), suppressed Srebp1c expression is likely to be responsible for dimin- ished hepatic lipid synthesis in BKS-db mice. Other targets of Srebp1c, such as glucokinase (Gk) and the FA trans- porter Cd36, also exhibited reduced expression in this strain. In contrast, mRNA levels of acyl-CoA:diacylglycerol acyltransferase-2 (Dgat2), a TG biosynthetic enzyme not known to be regulated by Srebp1c, were similar in the two strains. Data are means SE. P 0.0005; †P 0.05. failed to achieve any suppression of hepatic glucose production in either strain. In conclusion, these data indicate relative muscle insulin sensitivity in BKS-db, but severe hepatic insulin resistance in both strains. Reduced insulin signaling in BKS-db hepatocytes. As insulin sensitivity in muscle is expected to protect against, rather than contribute to, the diabetic phenotype of BKS- db, we focused on liver metabolism in subsequent exper- iments. To investigate hepatic insulin resistance, we compared insulin signaling in the two strains. Immunoblot analysis revealed lower levels of both IRS-1 and IRS-2 proteins in BKS-db liver, suggesting reduced proximal insulin signaling in this strain (Fig. 2A). RESULTS To conﬁrm this prediction, we assessed downstream signaling in primary hepatocytes treated with insulin. Consistent with reduced IRS expression, insulin-stimulated Akt phosphorylation was reduced by 50% in BKS-db hepatocytes (Fig. 2B). These results indicate a higher degree of hepatic insulin resistance in BKS-db mice. We also analyzed gluconeogenic gene expression (Fig. 4B). After a 4-h fast, which results in more than twofold higher insulin levels in BKS-db than in B6-db mice (Fig. 4B, left panel), the mRNA levels of phosphoenopyruvate car- boxykinase (Pepck), fructose-1,6-bisphosphatase (Fbp), and glucose-6-phosphatase (G6p) were similar in the two strains (Fig. 4B, right panel). Conversely, similar plasma insulin levels after an overnight fast were associated with elevated expression of Pepck and Fbp, but not G6p, in BKS-db mice (Fig. 4B). In conclusion, our gene expression results indicate diminished hepatic insulin action in BKS-db mice. Consistent with a proximal defect in insulin signaling, hepatic insulin resistance affects both the lipogenic and anti-gluconeogenic actions of insulin in this strain. Altered hepatic metabolism in young BKS-db mice. Next, we investigated the metabolic consequences of elevated hepatic insulin resistance in BKS-db. Upon visual examination of internal organs, BKS-db livers appeared darker than their B6-db counterparts (Fig. 3A), suggesting reduced steatosis in the former. Indeed, despite higher plasma insulin levels in BKS-db mice, hepatic TG content was over threefold lower in this strain (Fig. 3B). Similarly, hepatic glycogen content was also signiﬁcantly reduced in BKS-db mice (Fig. 3E). Because plasma free fatty acid levels are lower in BKS-db mice, reduced hepatic TG content may reﬂect reduced substrate delivery for TG biosynthesis. Alterna- tively, metabolic differences intrinsic to liver may also contribute to reduced steatosis in BKS-db mice. To dis- criminate between these possibilities, we used short-term in vitro liver slice cultures, which allow examination of metabolic pathways under conditions of controlled sub- strate delivery (15). We ﬁrst examined hepatic lipogenesis by measuring the incorporation of radioactivity into de novo synthesized FAs from 3H2O. Despite elevated plasma Reduced TG clearance in young BKS-db mice. BKS-db mice exhibit twofold higher plasma TG levels than B6-db mice at 1 month of age (Fig. 1E, right panel). Considering the lower hepatic TG content in BKS-db mice (Fig. 3B), this result was initially surprising. To investigate the mechanisms underlying these differences, we measured hepatic TG secretion in 1-month-old mice after inhibiting clearance with Triton WR1339. RESULTS Upon infusion with 2.5 mU/kg/min insulin, the two strains exhibited similar met- abolic parameters (data not shown). However, administra- tion of increased doses of insulin (10 mU/kg/min) unexpectedly revealed elevated glucose infusion rate and insulin-stimulated whole-body glucose turnover in BKS-db, indicating better systemic insulin sensitivity in this strain (Table 1). Consistent with these results, insulin-stimulated glucose uptake in gastrocnemius muscle was also higher in BKS-db mice, suggesting that muscle tissue is likely responsible for their relative insulin sensitivity. In con- trast, the clamp showed severe hepatic insulin resistance in both strains. In fact, even high-dose insulin infusion and insulin levels were different between wild-type B6 and BKS mice, whereas total cholesterol, free fatty acid, and glucose traits revealed signiﬁcant strain db interactions. In conclusion, our results indicate -cell–independent metabolic derangements in BKS-db mice. To investigate the role of different tissues in the early metabolic differ- ences observed between B6-db and BKS-db, we further characterized 1-month-old mice in subsequent studies. q Relative muscle insulin sensitivity in young BKS-db mice. Differences in plasma insulin and lipid levels are frequently associated with different insulin response in different tissues. To directly investigate insulin sensitivity in young BKS-db and B6-db mice, hyperinsulinemic-eugly- DIABETES, VOL. 59, JULY 2010 1618 diabetes.diabetesjournals.org R.C. DAVIS AND ASSOCIATES TABLE 1 Hyperinsulinemic-euglycemic clamp measurements B6-db BKS-db n 8 6 Body weight (g) 22.1 0.5 22.5 0.7 Fat mass (g) 8.0 0.4 7.9 0.5 Lean mass (g) 12.6 0.4 12.5 0.5 Basal glucose (mg/dl) 125 10 116 19 Basal insulin (pmol/l) 328 63 324 53 Clamp glucose (mg/dl) 125 7 135 9 Clamp insulin (pmol/l) 1,882 136 1,829 258 Glucose infusion rate (mg/kg/min) 7.2 0.9 21.3 2.8 Whole-body glucose turnover (mg/kg/min) 30.8 1.4 40.6 3.2† Whole-body glycolysis (mg/kg/min) 24.2 2.1 28.9 1.0 Whole-body glycogen synthesis (mg/kg/min) 6.6 2.0 11.7 3.3 Muscle glucose uptake (nmol/g/min) 162 20 314 46† Suppression of hepatic glucose production (%) 0 0 Data are means SE. P 0.0005; †P 0.05. TABLE 1 Hyperinsulinemic-euglycemic clamp measurements insulin levels in vivo, the rate of hepatic lipogenesis was signiﬁcantly lower in BKS-db liver slices (Fig. 3C), suggest- ing intrinsic metabolic differences in hepatic lipid synthe- sis between the strains. diabetes.diabetesjournals.org DIABETES, VOL. 59, JULY 2010 1619 RESULTS Western blots show phosphorylated Akt (upper panel) and total Akt (lower panel). Graphs on the right represent densitometric analysis of band intensities. P < 0.05. 0.5 1.0 1.5 2.0 arbitrary units Insulin: - + pAkt Akt - - - + + + B6-db B6-db BKS-db BKS-db B IRS 1 0.5 1.0 1.5 2.0 arbitrary units Insulin: - + pAkt Akt - - - + + + B6-db B6-db BKS-db BKS-db * pAkt/Akt B 0.0 0.5 1.0 1.5 2.0 arbitrary units Insulin: - + - + B6-db BKS-db Insulin: - + pAkt Akt - - - + + + B6-db B6-db BKS-db BKS-db * pAkt/Akt B 0.0 0.5 1.0 1.5 2.0 arbitrary units Insulin: - + - + B6-db BKS-db * pAkt/Akt B B FIG. 2. A: Western blots showing hepatic IRS-1 and IRS-2 expression in B6-db and BKS-db mice. Each lane represents equal total protein loading from individual animals. B: Insulin signaling in primary hepatocytes. Hepatocyte cultures were stimulated with 100 nmol/l insulin for 10 min. Western blots show phosphorylated Akt (upper panel) and total Akt (lower panel). Graphs on the right represent densitometric analysis of band intensities. P < 0.05. mice result from reduced clearance primarily due to low LPL activity in adipose. Further studies will be required to explore the underlying mechanisms in this tissue. sured plasma TG clearance after an oral lipid load. In B6-db mice, lipid ingestion resulted in a modest (less than twofold) rise in plasma TG levels peaking at 2 h after gavage and returning to baseline levels after 4 h (Fig. 5B). In contrast, BKS-db mice exhibited an over fourfold in- crease in TG at 2 h, and TG levels remained elevated threefold after 4 h. These data indicate signiﬁcantly re- duced clearance of circulating TG in BKS-db mice. diabetes.diabetesjournals.org RESULTS This analysis indicated similar rates of hepatic TG secretion in B6-db and BKS-db mice (Fig. 5A). As plasma TG levels are determined by the relative rates of secretion and clearance, we also mea- DIABETES, VOL. 59, JULY 2010 1619 IMPAIRED HEPATIC METABOLISM IN C57BLKS-db MICE plasma TG clearance after an oral lipid load In mice result from reduced clearance primarily due to lo 0.0 0.5 1.0 1.5 2.0 arbitrary units Insulin: - + - + B6-db BKS-db Insulin: - + pAkt Akt - - - + + + B6-db B6-db BKS-db BKS-db pAkt/Akt A B IRS-1 IRS-2 0 20 40 60 80 100 120 arbitrary units B6-db IRS-1 BKS-db B6-db BKS-db * * B6-db IRS-2 BKS-db IRS expression A: Western blots showing hepatic IRS-1 and IRS-2 expression in B6-db and BKS-db mice. Each lane represents equal total protein loadi dividual animals. B: Insulin signaling in primary hepatocytes. Hepatocyte cultures were stimulated with 100 nmol/l insulin for 10 m n blots show phosphorylated Akt (upper panel) and total Akt (lower panel). Graphs on the right represent densitometric analysis of ba ties. P < 0.05. A IRS-1 IRS-2 0 20 40 60 80 100 120 arbitrary units B6-db IRS-1 BKS-db B6-db BKS-db * B6-db IRS-2 BKS-db IRS expression A A 0.0 0.5 1.0 1.5 2.0 arbitrary units Insulin: - + - + B6-db BKS-db Insulin: - + pAkt Akt - - - + + + B6-db B6-db BKS-db BKS-db * pAkt/Akt B 0 20 B6-db IRS-1 BKS-db B6-db IRS-2 BKS-db FIG. 2. A: Western blots showing hepatic IRS-1 and IRS-2 expression in B6-db and BKS-db mice. Each lane represents equal total protein loading from individual animals. B: Insulin signaling in primary hepatocytes. Hepatocyte cultures were stimulated with 100 nmol/l insulin for 10 min. Western blots show phosphorylated Akt (upper panel) and total Akt (lower panel). Graphs on the right represent densitometric analysis of band intensities. P < 0.05. 0.0 0.5 1.0 1.5 2.0 arbitrary units Insulin: - + - + B6-db BKS-db Insulin: - + pAkt Akt - - - + + + B6-db B6-db BKS-db BKS-db pAkt/Akt B IRS 1 IRS 2 FIG. 2. A: Western blots showing hepatic IRS-1 and IRS-2 expression in B6-db and BKS-db mice. Each lane represents equal total protein loading from individual animals. B: Insulin signaling in primary hepatocytes. Hepatocyte cultures were stimulated with 100 nmol/l insulin for 10 min. DISCUSSION Mutations in the leptin receptor gene cause severe obesity and insulin resistance in rodents and humans (27–29). In leptin receptor–deﬁcient (db mutation) mouse strains, obesity is variably associated with hyperglycemia depend- ing on genetic background (30). Diabetes-resistant strains, such as B6-db, exhibit islet hypertrophy and insulin hyper- secretion and are able to maintain mildly elevated plasma glucose levels. In contrast, islets in the diabetes-suscepti- ble BKS-db strain undergo atrophy leading to insulin deﬁciency and hyperglycemia. The genes underlying these phenotypic differences are largely unknown. Based on reduced viability and proliferative capacity of BKS -cells both in vivo and in vitro, diabetes susceptibility of this To test the potential role of lipases in reduced TG clearance in BKS-db mice, we measured the activities of these enzymes in postheparin plasma. Consistent with reduced TG clearance, total lipase, LPL, and hepatic lipase activities are signiﬁcantly lower in BKS-db plasma (Fig. 5C). To identify the tissue(s) responsible for differences in LPL activity, we assayed lipase activity in isolated tissues (Fig. 5D). Adipose and, to a smaller extent, heart from BKS-db mice exhibit lower LPL activities, whereas skeletal muscle shows the opposite pattern. In conclusion, these data suggest that elevated plasma TG levels in BKS-db 620 DIABETES, VOL. 59, JULY 2010 diabetes.diabetesjournals.org 1620 R.C. DAVIS AND ASSOCIATES 0 20 40 60 80 µg TG/mg liver wet weight A C E B B6-db Glycogen Hepatic TG FA oxidation FA synthesis BKS-db * * * 0 10 20 30 40 50 DPM in FA/g liver/1000 0 10 20 30 DPM in CO2/g liver/1000 0 50 100 150 200 250 NS 300 mmol/kg liver D FIG. 3. Hepatic fatty acid metabolism in 1-month-old male B6-db (f) and BKS-db () mice. A: Representative images of B6-db and BKS-db livers. B: Hepatic TG content in B6-db (n 10) and BKS-db (n 10) mice. Rates of in vitro FA synthesis (C) (n 4 4) and oxidation (n 7 7) in liver slice cultures (D) are shown. E: Hepatic glycogen content (n 10 10). DPM, disintegration per minute. P < 0.05. (A high-quality color representation of this ﬁgure is available in the online issue.) R.C. diabetes.diabetesjournals.org DISCUSSION DAVIS AND ASSOCIATES 0 20 40 60 80 µg TG/mg liver wet weight B Hepatic TG BKS-db B A B6-db B6-db 0 E Glyco FA oxidation 0 10 20 30 DPM in CO2/g liver/1000 0 50 100 150 200 250 NS 300 mmol/kg liver D 0 E Glycogen FA oxidation synthesis * * 0 10 20 30 DPM in CO2/g liver/1000 0 50 100 150 200 250 NS 300 mmol/kg liver D id metabolism in 1-month-old male B6-db (f) and BKS-db () mice. A: Representative images of B6-db and BKS-db livers. in B6-db (n 10) and BKS-db (n 10) mice. Rates of in vitro FA synthesis (C) (n 4 4) and oxidation (n 7 7) FA oxidation 0 10 20 30 DPM in CO2/g liver/1000 NS D C FA synthesis * 0 10 20 30 40 50 DPM in FA/g liver/1000 D E C DPM in FA/g liver/1000 FIG. 3. Hepatic fatty acid metabolism in 1-month-old male B6-db (f) and BKS-db () mice. A: Representative images of B6-db and BKS-db livers. B: Hepatic TG content in B6-db (n 10) and BKS-db (n 10) mice. Rates of in vitro FA synthesis (C) (n 4 4) and oxidation (n 7 7) in liver slice cultures (D) are shown. E: Hepatic glycogen content (n 10 10). DPM, disintegration per minute. P < 0.05. (A high-quality color representation of this ﬁgure is available in the online issue.) strain has been ascribed to genetic determinants acting in islets (10–13). However, it is conceivable that genetic differences manifested in other tissues may also contribute to -cell failure through mechanisms that increase meta- bolic stress on these cells (e.g., insulin resistance). Indeed, in the current study, we demonstrate increased hepatic insulin resistance in young BKS-db mice compared with B6-db mice and propose that this metabolic abnormality may lead to elevated -cell stress and the diabetic pheno- type in BKS-db. However, in contrast to muscle, hepatic insulin resistance is more severe in BKS-db than B6-db mice. Despite ele- vated plasma insulin levels, BKS-db mice exhibit signs of reduced hepatic insulin action including diminished lipo- genesis, glycogen accumulation, insulin-stimulated gene expression and TG secretion, and suppression of glu- coneogenic gene expression. Furthermore, direct analysis of hepatic insulin signaling demonstrated lower IRS-1 and IRS-2 expression as well as insulin-stimulated Akt phos- phorylation in BKS-db hepatocytes. DISCUSSION Diminished IRS-1/2 expression has been linked to reduced hepatic insulin signaling in various obese animal models in comparison to their lean counterparts (31–34). Thus, our data suggest that lower IRS-1/2 expression in BKS-db mice results in relative hepatic insulin resistance in this strain. However, in contrast to muscle, hepatic insulin resistance is more severe in BKS-db than B6-db mice. Despite ele- vated plasma insulin levels, BKS-db mice exhibit signs of reduced hepatic insulin action including diminished lipo- genesis, glycogen accumulation, insulin-stimulated gene expression and TG secretion, and suppression of glu- coneogenic gene expression. Furthermore, direct analysis of hepatic insulin signaling demonstrated lower IRS-1 and IRS-2 expression as well as insulin-stimulated Akt phos- phorylation in BKS-db hepatocytes. Diminished IRS-1/2 expression has been linked to reduced hepatic insulin signaling in various obese animal models in comparison to their lean counterparts (31–34). Thus, our data suggest that lower IRS-1/2 expression in BKS-db mice results in relative hepatic insulin resistance in this strain. Side-by-side phenotypic characterization of BKS-db and B6-db mice revealed differences in plasma lipid and insulin levels as early as 1 month of age, even before the onset of -cell dysfunction in BKS-db mice. These observations indicated metabolic differences in tissues other than -cells and prompted us to investigate the early BKS-db phenotype further. Unexpectedly, hyperinsulinemic-eugly- cemic clamp studies demonstrated higher whole-body and muscle insulin sensitivity in BKS-db, indicating that this tissue does not contribute to, but rather counteracts, the diabetes-sensitive phenotype of this strain. However, the clamp also revealed profound hepatic insulin resistance, which completely abolished the effect of high doses of insulin on hepatic glucose production in both strains. Hepatic insulin resistance is a principal component of type 2 diabetes (35). Decreased insulin sensitivity in liver leads to elevated hepatic glucose production, hyperinsu- linemia, increased -cell stress, and hyperglycemia (36). Animal models indicate that hepatic insulin resistance can play a primary role in the development of diabetes (37). For example, diminished insulin signaling due to hepatic insulin receptor (38) or IRS-1/2 deﬁciency (34,39) causes hyperglycemia. In line with these studies, we propose that DIABETES, VOL. diabetes.diabetesjournals.org DISCUSSION 59, JULY 2010 1621 IMPAIRED HEPATIC METABOLISM IN C57BLKS-db MICE early hepatic insulin resistance in BKS db mice contributes hepatic glycogen content observed in this strain Another 0 0.2 0.4 0.6 0.8 1 1.2 1.4 LXRa Srebp1c PPARg Acc1 Fas Acl Scd1 Me1 Gpat Lpin1 Dgat2 Gk CD36 mRNA amount (relative units) A B Lipogenesis TG Synthesis 0 0.5 1 1.5 2 2.5 Pepck 16h 16h 4h 4h G6p Fbp Pepck G6p Fbp mRNA amount (relative units) 0 2 4 6 8 10 12 1 2 Insulin (ng/ml) Gene expression Insulin Gene expression * * * * * * * * * * * * * * FIG. 4. A: Hepatic mRNA levels for enzymes involved in lipogenesis and TG synthesis in 1-month-old male B6-db (f, n 5) and BKS-db (, n 5) mice fasted for 4 h. Relative mRNA levels are normalized to B6-db values. B: Left panel: Plasma insulin levels after 4 h (n 10 10) and 16 h (n 5–5) of fasting. Right panel: Gluconeogenic gene expression after 4 h (n 5 5) and 16 h of fasting (n 5 5). P < 0.05 for differences between strains. IMPAIRED HEPATIC METABOLISM IN C57BLKS-db MICE 0 0.2 0.4 0.6 0.8 1 1.2 1.4 LXRa Srebp1c PPARg Acc1 Fas Acl Scd1 Me1 Gpat Lpin1 Dgat2 Gk CD36 mRNA amount (relative units) A Lipogenesis TG Synthesis Gene expression * * * * * * * * * * * A Gene expression TG Synthesis B 0 0.5 1 1.5 2 2.5 Pepck 16h 16h 4h 4h G6p Fbp Pepck G6p Fbp mRNA amount (relative units) 0 2 4 6 8 10 12 1 2 Insulin (ng/ml) Insulin Gene expression * * * FIG. 4. A: Hepatic mRNA levels for enzymes involved in lipogenesis and TG synthesis in 1-month-old male B6-db (f, n 5) and BKS-db (, n 5) mice fasted for 4 h. Relative mRNA levels are normalized to B6-db values. B: Left panel: Plasma insulin levels after 4 h (n 10 10) and 16 (n 5–5) of fasting. Right panel: Gluconeogenic gene expression after 4 h (n 5 5) and 16 h of fasting (n 5 5). diabetes.diabetesjournals.org DISCUSSION P < 0.05 for differenc 0 0.5 1 1.5 2 2.5 Pepck 16h 4h G6p Fbp Pepck G6p Fbp mRNA amount (relative units) Gene expression * Gene expression B FIG. 4. A: Hepatic mRNA levels for enzymes involved in lipogenesis and TG synthesis in 1-month-old male B6-db (f, n 5) and BKS-db (, n 5) mice fasted for 4 h. Relative mRNA levels are normalized to B6-db values. B: Left panel: Plasma insulin levels after 4 h (n 10 10) and 16 h (n 5–5) of fasting. Right panel: Gluconeogenic gene expression after 4 h (n 5 5) and 16 h of fasting (n 5 5). P < 0.05 for differences between strains. early hepatic insulin resistance in BKS-db mice contributes to the diabetes-susceptible phenotype of this strain. hepatic glycogen content observed in this strain. Another relevant ﬁnding is markedly reduced lipogenic capacity in the BKS-db liver. It has been proposed that active lipogen- esis reduces fuel partitioning into the gluconeogenic path- way, thereby reducing the rate of glucose production at the substrate level (40). Thus, reduced lipogenesis result- ing from proximal defects in insulin signaling in BKS-db mice may contribute to elevated gluconeogenesis, in- creased -cell stress, and the development of diabetes in What are the metabolic consequences of hepatic insulin resistance in BKS-db mice? Although we did not directly demonstrate it in our study, hepatic glucose output is expected to be higher in BKS-db mice than in B6-db mice. Diminished insulin signaling in BKS-db liver results in impaired suppression of gluconeogenic gene expression and likely elevated glycogenolysis, consistent with lower 1622 DIABETES, VOL. 59, JULY 2010 1622 diabetes.diabetesjournals.org R.C. DAVIS AND ASSOCIATES A C D B 0 500 1000 1500 2000 2500 Triglyceride secretion (mg/kg body weight/h) 0 10 20 30 40 50 60 0 1 2 3 4 Time (h) Plasma triglyceride (mg/dL) TG secretion TG clearance Post-heparin plasma Adipose Heart Sk. DISCUSSION muscle Total LPL HL 0 500 1000 1500 2000 2500 3000 Lipase activity (mu/mL) 0 500 1000 1500 0 100 200 300 400 500 * * * * * 0 100 200 300 Lipase activity (mU/mg) 0 20 40 60 80 100 0 2 4 6 8 10 12 14 A C B 0 500 1000 1500 2000 2500 Triglyceride secretion (mg/kg body weight/h) 0 10 20 30 40 50 60 0 1 2 3 4 Time (h) Plasma triglyceride (mg/dL) TG secretion TG clearance Post-heparin plasma A 0 500 1000 1500 2000 2500 Triglyceride secretion (mg/kg body weight/h) TG secretion B 0 10 20 30 40 50 60 0 1 2 3 4 Time (h) Plasma triglyceride (mg/dL) TG clearance Post-heparin plasma B A TG clearance C Post-heparin plasma D Total LPL HL 0 500 1000 1500 2000 2500 3000 Lipase activity (mu/mL) 0 500 1000 1500 0 100 200 300 400 500 * * * D Adipose Heart Sk. muscle 0 0 * * * 0 100 200 300 Lipase activity (mU/mg) 0 20 40 60 80 100 0 2 4 6 8 10 12 14 FIG. 5. A: Hepatic TG secretion in 1-month-old male B6-db (f, n 4) and BKS-db (, n 6) mice as measured after the intravenous injection D D Sk. muscle FIG. 5. A: Hepatic TG secretion in 1-month-old male B6-db (f, n 4) and BKS-db (, n 6) mice as measured after the intravenous injection of Triton. B: TG clearance after an oral lipid load (n 5–5). C: Postheparin plasma levels of total lipase activity, LPL, and hepatic lipase (HL). D: Lipase activities in adipose, heart, and skeletal (Sk.) muscle tissues. A total of 4–6 animals of each strain were averaged in lipase activity measurements. *P > 0.05 for differences between strains. this strain. Consistent with this hypothesis, decreased hepatic lipogenesis and steatosis are also associated with diabetes susceptibility in the obese BTBR-ob (40), lipoatro- phic FVB-AZIP (41), and liver-speciﬁc Ppar knockout mouse models (42). Moreover, increased hepatic lipogen- esis and steatosis achieved by overexpressing Srebp1c DIABETES, VOL. 59, JULY 2010 1623 phic FVB-AZIP (41), and liver-speciﬁc Ppar knockout mouse models (42). Moreover, increased hepatic lipogen- esis and steatosis achieved by overexpressing Srebp1c phic FVB-AZIP (41), and liver-speciﬁc Ppar knockout mouse models (42). Moreover, increased hepatic lipogen- esis and steatosis achieved by overexpressing Srebp1c DIABETES, VOL. REFERENCES 25. Guerre-Millo M. Adipose tissue and adipokines: for better or worse. Diabete Metab 2004;30:13–19 1. Hummel KP, Dickie MM, Coleman DL. Diabetes, a new mutation in the mouse. Science 1966;153:1127–1128 26. McPherson R, Gauthier A. Molecular regulation of SREBP function: the Insig-SCAP connection and isoform-speciﬁc modulation of lipid synthesis. Biochem Cell Biol 2004;82:201–211 2. Hummel KP, Coleman DL, Lane PW. The inﬂuence of genetic background on expression of mutations at the diabetes locus in the mouse. I. 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https://openalex.org/W2438644162	https://scipost.org/10.21468/SciPostPhys.2.1.002/pdf	English	null	Conformal field theory for inhomogeneous one-dimensional quantum systems: the example of non-interacting Fermi gases	SciPost physics	2,017	cc-by	11,368	Abstract Conformal ﬁeld theory (CFT) has been extremely successful in describing large-scale uni- versal effects in one-dimensional (1D) systems at quantum critical points. Unfortunately, its applicability in condensed matter physics has been limited to situations in which the bulk is uniform because CFT describes low-energy excitations around some energy scale, taken to be constant throughout the system. However, in many experimental contexts, such as quantum gases in trapping potentials and in several out-of-equilibrium situa- tions, systems are strongly inhomogeneous. We show here that the powerful CFT meth- ods can be extended to deal with such 1D situations, providing a few concrete examples for non-interacting Fermi gases. The system’s inhomogeneity enters the ﬁeld theory ac- tion through parameters that vary with position; in particular, the metric itself varies, resulting in a CFT in curved space. This approach allows us to derive exact formulas for entanglement entropies which were not known by other means. Copyright J. Dubail et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. Received 15-11-2016 Accepted 26-01-2017 Published 13-02-2017 Check for updates doi:10.21468/SciPostPhys.2.1.002 Contents 1 Introduction 2 2 The one-dimensional Fermi gas 3 2.1 Translation-invariant Fermi gas and the euclidean 2D Dirac action 3 2.2 Harmonic trap and the euclidean Dirac action in curved 2d space 4 3 Application to the entanglement entropy 5 3.1 Entanglement entropy of the Fermi gas in a harmonic trap 5 3.2 Fermi gas in an arbitrary external potential 6 4 A non-equilibrium situation 8 1 Copyright J. Dubail et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. Received 15-11-2016 Accepted 26-01-2017 Published 13-02-2017 Check for updates doi:10.21468/SciPostPhys.2.1.002 Copyright J. Dubail et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. SciPost Phys. 2, 002 (2017) Conformal ﬁeld theory for inhomogeneous one-dimensional quantum systems: the example of non-interacting Fermi gases Conformal ﬁeld theory for inhomogeneous one-dimensional quantum systems: the example of non-interacting Fermi gases Jérôme Dubail1, Jean-Marie Stéphan2,3, Jacopo Viti4 and Pasquale Calabrese5 1 CNRS & IJL-UMR 7198, Université de Lorraine, F-54506 Vandoeuvre-les-Nancy, France 2 Max Planck Institut für Physik komplexer Systeme, Nöthnitzer Str. 38 D01187 Dresden, Germany 3 CNRS & Institut Camille Jordan-UMR 5208, Université Lyon 1, 69622 Villeurbanne, France 4 ECT & Instituto Internacional de Fisica, UFRN, Lagoa Nova 59078-970 Natal, Brazil 5 SISSA and INFN, Via Bonomea 265, 34136 Trieste, Italy Germany 3 CNRS & Institut Camille Jordan-UMR 5208, Université Lyon 1, 69622 Villeurbanne, France 4 ECT & Instituto Internacional de Fisica, UFRN, Lagoa Nova 59078-970 Natal, Brazil 5 SISSA and INFN, Via Bonomea 265, 34136 Trieste, Italy stephan@math.univ-lyon1.fr * stephan@math.univ-lyon1.fr 4 A non-equilibrium situation 1 Introduction Low-dimensional quantum systems are a formidable arena for the study of many-body physics: in one or two spatial dimensions (1D or 2D), the effects of strong correlations and interac- tions are enhanced and lead to dramatic effects. Celebrated examples from condensed mat- ter physics include such diverse cases as the fractionalization of charge and emergence of topological order in the quantum Hall effect, high-Tc superconductivity, or the breakdown of Landau’s Fermi liquid theory in 1D, replaced by the Luttinger liquid paradigm [1]. In the past decade, breakthroughs in the ﬁeld of optically trapped ultra-cold atomic gases [2] have lead to a new generation of quantum experiments that allow to directly observe fundamental phenomena such as quantum phase transitions [3] and coherent quantum dynamics [4,5] in low-dimensional systems, including 1D gases [6–11]. These revolutionary experiments are an ideal playground for the interplay with theory, as they allow to directly realize, in the labora- tory, ideal setups that were previously regarded only as oversimpliﬁed thought experiments. On the theory side, many exact results in 1D can be obtained by a blend of methods that comprises lattice integrability [12, 13] and non-perturbative ﬁeld theory approaches, in par- ticular 2D (1+1D) conformal ﬁeld theory (CFT) [14,15] and integrable ﬁeld theory [16]. CFT has been incredibly successful at making exact universal predictions for 1D condensed mat- ter systems at a quantum critical point; these include the Kondo effect and other quantum impurity problems [17], and the many insights on quantum quenches [18] as well as univer- sal characterization of entanglement at quantum criticality [19–21]. Entanglement entropies, in particular, are currently in the limelight, as they have become experimentally measurable both in- [22] and out-of-equilibrium [23], opening the route to a direct comparison between experimental data and many exact analytical results obtained by CFT. Indeed, entanglement entropies are usually difﬁcult to compute in microscopic models [24, 25], but their scaling limit is obtained within the powerful CFT approach by solving elementary exercises on Rie- mann surfaces [21]. There is a caveat in the CFT approach to 1D physics though: since it describes low-energy excitations around some ﬁxed energy scale (e.g. the Fermi energy), CFT does not accommodate strong variations of that scale throughout the system. This rules out a priori the possibility of tackling inhomogeneous systems, in which the relevant energy scale varies. Contents 1 Introduction 2 2 The one-dimensional Fermi gas 3 2.1 Translation-invariant Fermi gas and the euclidean 2D Dirac action 3 2.2 Harmonic trap and the euclidean Dirac action in curved 2d space 4 3 Application to the entanglement entropy 5 3.1 Entanglement entropy of the Fermi gas in a harmonic trap 5 3.2 Fermi gas in an arbitrary external potential 6 4 A non-equilibrium situation 8 1 4 A non-equilibrium situation 1 SciPost Phys. 2, 002 (2017) 5 Conclusion 10 A The Dirac action in curved space-time 11 B The entanglement entropies of a ﬁnite interval [x1, x2] 12 B.1 The harmonically trapped Fermi gas 12 B.2 The domain-wall quench 13 C The domain wall quench for a more general dispersion relation 13 C.1 Imaginary time treatment 13 C.2 An explicit example 15 References 16 16 1 Introduction This caveat is important, as inhomogeneous systems are the rule rather than the exception in the realm of quantum experiments: quantum gases at equilibrium always lie in trapping potentials (often 2 SciPost Phys. 2, 002 (2017) harmonic) and therefore usually come with a non-uniform density; this is also the case of many out-of-equilibrium situations, such as trap releases. harmonic) and therefore usually come with a non-uniform density; this is also the case of many out-of-equilibrium situations, such as trap releases. In this paper, we make one step forward. We focus on the example of the free Fermi gas, in a few illustrative in- and out-of-equilibrium inhomogeneous situations. The free Fermi gas is technically simpler than interacting models, and yet it allows to draw interesting lessons that will hold more generally. We ﬁnd that the varying energy scale is taken into account rather naturally in the effective ﬁeld theory, in the form of varying parameters in the action. Interestingly, the metric is one such parameter, so one generically ends up with a CFT in curved 2D space. These conclusions are very general and hold under the reasonable assumption of separation of scales: there must exist an intermediate scale ℓwhich is simultaneously large compared to the microscopic scale (the inter-particle distance ∼ρ−1, where ρ is the density), but small compared to the scale on which physical quantities vary macroscopically (of order ρ\|∂xρ\|−1). Indeed, at the intermediate scale ℓ, the system is well described by continuous ﬁelds because ρ−1 ≪ℓ, and is locally homogeneous because ℓ\|∂xρ\|/ρ ≪1, so one knows that it corresponds to a standard (i.e. ﬂat-space, translation-invariant) ﬁeld theory. From there, it is clear that unravelling the global theory for the inhomogeneous system is a problem of geometric nature: it is about understanding the global geometric data (e.g. metric tensor, coupling constants, gauge ﬁelds, etc.) that enter the action. This is the program we illustrate with the few simple examples below. We demonstrate the power of this formalism by providing new exact asymptotic formulas for entanglement entropies. 2.1 Translation-invariant Fermi gas and the euclidean 2D Dirac action Let us start by considering a free Fermi gas in 1D H = Z ∞ −∞ d x c†(x) −ℏ2 2m∂2 x −µ + V(x) c(x), (1) (1) in the absence of an external potential, i.e. V(x) = 0. For the reader’s convenience, we brieﬂy review the well known relation between this (homogeneous) 1D system and the CFT of massless Dirac fermions in 2D euclidean space-time. The question is: what is the proper ﬁeld theory framework that captures the behavior of long-range correlations of arbitrary local observables 〈φ1(x1, y1)...φn(xn, yn)〉? Here and below, the y-coordinate is imaginary time. The starting point to answer this question is the ground-state propagator c†(x, y)c(0,0) = Z kF −kF dk 2πe−i[kx+iϵ(k) y ℏ], (2) (2) where ϵ(k) = ℏ2k2 2m −µ is the dispersion relation and kF = 1 ℏ p 2mµ is the Fermi momentum. Its large distance behavior is obtained by linearizing the dispersion relation around the two Fermi points k± F = ±kF, ϵ(k) ≃±vFℏ(k ∓kF) with Fermi velocity vF = 1 ℏ dϵ dk\|k=kF . One ﬁnds straightforwardly, for x, vF y ≫1/kF, c†(x, y)c(0,0) ≃ Z kF −∞ dk 2πe−i[kx+i(k−kF)vF y] + Z ∞ −kF dk 2πe−i[kx−i(k+kF)vF y] = i 2π e−ikFx x + ivF y − eikFx x −ivF y . (3) (3) 3 3 SciPost Phys. 2, 002 (2017) These two terms coincide with the right(R)-/left(L)-components of a massless Dirac fermion in 2D euclidean spacetime, 〈ψ† R,L(x, y)ψR,L(0,0)〉= 1 x±ivF y , that derive from the action (with z = x + ivF y, ¯z = x −ivF y): S = 1 π Z dzd¯z ψ† R∂¯zψR + ψ† L∂zψL . (4) (4) The action (4) is invariant under conformal transformations z7→f (z), ψR(z)7→( d f dz ) 1 2 ψR(f (z) ¯ 1 The action (4) is invariant under conformal transformations z7→f (z), ψR(z)7→( d f dz ) 2 ψR(f (z)), ψL(¯z) 7→( d ¯f d¯z ) 1 2 ψR( ¯f (¯z)), where f (z) is a holomorphic function of z. The phase factors in (3) may be incorporated into (4) with a chiral gauge transformation ψR,L →e±iα(x)ψR,L. Here we do not keep track of these phase factors, as they are unimportant for our purposes, and simply discard them; these aspects are discussed in appendix A. 2.2 Harmonic trap and the euclidean Dirac action in curved 2d space Consider the Fermi gas (1) in a harmonic potential V(x) = mω2x2/2. We ask: what is the underlying Dirac action? The system is now inhomogeneous, so there should be parameters in the effective action that vary with position. But what are these parameters in (4)? In order to understand this, one needs to ﬁnd the density proﬁle ﬁrst. The latter is obtained from the exact solution of the microscopic problem in the thermodynamic limit, which in this case is extremely simple. The single-particle eigenstates are just those of the harmonic oscillator, and the many- body ground state is obtained by simply ﬁlling up all eigenstates with negative energies. In the thermodynamic limit (µ ≫ℏω), the density proﬁle ρ(x) = c†(x)c(x) follows the Wigner semicircle law 1 ρ(x) = 1 π Æ 2(µ −x2/2), (5) (5) (5) which is non-vanishing in the interval [−L, L] with L = p 2µ. Here and in the following, we set ℏ= m = ω = 1. The total number of particles is N = R ρ(x)d x = µ ≫1. Away from the edges x = ±L, there is an intermediate scale ℓsuch that N −1 2 ∼ρ−1 ≪ℓ≪L ∼N 1 2 . At this scale, the system can be viewed as homogeneous, with a local Fermi momentum kF(x) = πρ(x): in a window of width ∼ℓaround the position x, the system consists of all states ﬁlled in the interval [−kF(x),+kF(x)]. Thus, around a point (x, y) ∈[−L, L] × R in spacetime, the behaviour of the propagator must be the same as in the translation-invariant gas with kF = kF(x): c†(x + δx, y + δy)c(x, y) ≃ i 2π e−iδϕ(x,y) δx + ivF(x)δy − eiδϕ(x,y) δx −ivF(x)δy , (6) (6) where vF(x) = ϵ′(kF(x)), δϕ = kF(x)δx + ivF(kF(x))δy, and δϕ is its complex conjugate. Like in (3), we would like to view the terms (δx ± ivF(x)δy)−1 as the R-/L-components of a massless Dirac ﬁeld. Of course, in the neighborhood of (x, y), one can always do that. But the real question is: is there a consistent Dirac theory deﬁned globally on the entire domain (x, y) ∈[−L, L] × R, such that its propagator has the required local behavior everywhere? If the reader is familiar with quantum ﬁeld theories in curved background, they will proba- bly have guessed that Eq. 2.1 Translation-invariant Fermi gas and the euclidean 2D Dirac action Let us start by considering a free Fermi gas in 1D From Wick’s theorem, it then follows that the large-scale behavior of arbitrary multi-point correlations of the original fermions can be obtained from correlators in the massless Dirac theory. 2.2 Harmonic trap and the euclidean Dirac action in curved 2d space (6) is, in fact, related to the propagator of the massless Dirac fermion in curved space. The action of the latter theory in 2D is S = 1 2π Z dzd¯z eσ(x,y) ψ† R ↔ ∂¯zψR + ψ† L ↔ ∂zψL , (7) (7) 4 SciPost Phys. 2, 002 (2017) written in isothermal coordinates (z, ¯z) and in a ﬁxed frame (see appendix A for all details). The underlying Riemannian metric is ds2 = e2σdzd¯z . (8) (8) o connect this theory to Eq. (6), notice that, in the coordinate z, the propagator behaves cally as 〈ψ† R(z + δz)ψR(z)〉= 1 eσδz . (9) (9) hus, to prove that Eq. (6) is the propagator of a Dirac fermion in a curved metric, it is fﬁcient to exhibit a map (x, y) 7→z(x, y) such that eσ(x,y)δz(x, y) = δx + ivF(x)δy, (10) (10) for some real-valued function σ(x, y). This equation can be solved easily. First, notice that it is equivalent to eσ∂xz = 1, eσ∂yz = ivF. Writing that ∂x∂yz = ∂y∂xz, we ﬁnd a constraint on σ: (ivF ∂x −∂y)σ = i∂x vF. Looking for a solution that is independent of y, we can set eσ(x) = vF(x), which implies ∂xz = 1/vF(x) and ∂yz = 1. The solution is straightforward: up to an additive constant, we ﬁnd the complex coordinate system (z, ¯z), which lives on the inﬁnite strip [−π 2 , π 2 ] × R: z(x, y) = arcsin x L + i y , eσ = vF = p L2 −x2 . (11) (11) This ﬁxes the underlying geometry of the problem. 3 Application to the entanglement entropy To illustrate the power of this approach, we exhibit new exact results for the entanglement entropies of the Fermi gas in external trapping potentials. Such calculations are, in general, difﬁcult. But within the framework we just developed, they boil down to elementary manipu- lations of complex analytic functions. The Renyi entanglement entropies of a subsystem A are deﬁned as 1 Sn = 1 1 −n lntr(ρn A), (12) (12) where ρA is the reduced density matrix of A and n is an arbitrary real number. For n →1, Sn reduces to the von Neumann entropy of the subsystem which is usually referred to as entan- glement entropy. where ρA is the reduced density matrix of A and n is an arbitrary real number. For n →1, Sn reduces to the von Neumann entropy of the subsystem which is usually referred to as entan- glement entropy. The main property we will use in the following is that the Renyi entanglement entropies are related to the expectation values of the twist ﬁelds Tn [21,26,27] which under conformal mapping share the same transformation properties of primaries with dimension ∆n = c 12 n −1 n , (13) (13) with c the central charge of the CFT (c = 1 for the free Fermi gas). 3.1 Entanglement entropy of the Fermi gas in a harmonic trap Let us now apply Eqs. (8)-(11) to the problem of calculating the entanglement entropy in a harmonic trap. 5 SciPost Phys. 2, 002 (2017) We start with the case of a bipartition A ∪B consisting of two semi-inﬁnite systems, A = [−∞, x], B = [x,+∞]. In a homogeneous system, the Renyi entanglement entropy is [21] Sn(x) ≃ 1 1 −n lnε∆n 〈Tn(x, y = 0)〉. (14) (14) ε is a UV cutoff, sometimes dismissed in homogeneous systems, because it simply appears in the form of a non-universal constant offset. In inhomogeneous situations, however, it is crucial to have a closer look at this cutoff: since the energy scale changes throughout the system, why shouldn’t ε vary as well? And, if ε(x) varies, then it must obviously affect the dependence of Sn on x. And indeed, there is a good reason why ε should vary with position: the continuous Fermi gas is locally galilean invariant, and the only relevant microscopic scale is the inverse density 1/ρ(x), or equivalently k−1 F (x). So the UV cutoff must simply be proportional to that scale: ε(x) = ε0/kF(x), for some dimensionless constant ε0. Coming back to the harmonic potential, we now make use of the coordinate system (z, ¯z), with z(x, y) = arcsin(x/L) + i y. To evaluate 〈Tn〉, we ﬁrst perform a Weyl transformation e2σdzd¯z→dzd¯z, which changes 〈Tn〉into eσ∆n 〈Tn〉. Next, we notice that under the z 7→ g(z) mapping deﬁned above,〈Tn(z, ¯z)〉becomes dg(z) dz ∆n 〈Tn(g(z), g(z))〉uhp. The latter factor, which is the one-point function in the upper half-plane, is equal to (Im g(z))−∆n. Putting everything together, 〈Tn(z,z)〉= eσ(z,¯z) d g(z) dz −1 Im g(z) −∆n . (15) (15) 4) with ε(x) = ε0/kF(x), we ﬁnally have for the entanglement entropy Sn = n + 1 12 n ln kF(x) eσ(z,¯z) d g(z) dz −1 Im g(z) , (16) (16) up to an additive constant and subleading corrections, which we systematically drop from now on. This gives 1 Sn(x) = n + 1 12 n ln L2 1 −(x/L)23/2 . (17) (17) A more complicated bipartition A∪B that can be considered in our framework is A = [x1, x2] and B = [−∞, x1] ∪[x2,+∞], where −L < x1 < x2 < L. The calculation is straightforward, but rather cumbersome and so we report it in appendix B.1. 3.1 Entanglement entropy of the Fermi gas in a harmonic trap The ﬁnal result, setting ζi = xi/L, can be written as Sn = n + 1 6 n ln L2 + ln (1 −ζ2 1)3/4(1 −ζ2 2)3/4 2(ζ2 −ζ1) + ln 1 −ζ1ζ2 − q (1 −ζ2 1)(1 −ζ2 2) , (18) (18) which is a highly non-trivial generalization of recent results (for x2 = −x1) obtained by means of random matrix theory [28]. We checked the validity of this formula against exact ﬁnite size computations for lattice and continuous Fermi gases using the approaches of Refs. [29,30]. 3.2 Fermi gas in an arbitrary external potential The generalization to arbitrary V(x) is very simple, even though the single particle problem is not always exactly solvable for a general potential. Indeed, we are always interested in the thermodynamic limit, where the single particle states that matter are those up in the spec- trum, and for which the semi-classical approximation becomes exact. Thus, focusing on the thermodynamic limit (µ ≫ℏω), we proceed as follows: semi-classically, the single-particle 6 SciPost Phys. 2, 002 (2017) 1.0 0.5 0.0 0.5 1.0 x / system width 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Entanglement entropy S1 N =16 N =32 N =64 analytic x k ε(k) + V (x) > 0 ε(k) + V (x) < 0 +kF(x) −kF(x) Figure 1: An example: a Fermi gas trapped in a double-well potential. To obtain non trivial results, we scaled the potential with the number N of particles so as to have a ﬁnite density inside each well. Our analytic prediction for the entanglement entropy is checked against numerical data for n = 1: the agreement is excellent, and improves further when increasing N. Inset: in phase space, the quasi-classical orbitals are equipotentials enclosing an area that is an integer multiple of 2π. 1.0 0.5 0.0 0.5 1.0 x / system width 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Entanglement entropy S1 N =16 N =32 N =64 analytic x k ε(k) + V (x) > 0 ε(k) + V (x) < 0 +kF(x) −kF(x) Figure 1: An example: a Fermi gas trapped in a double-well potential. To obtain non trivial results, we scaled the potential with the number N of particles so as to have a ﬁnite density inside each well. Our analytic prediction for the entanglement entropy is checked against numerical data for n = 1: the agreement is excellent, and improves further when increasing N. Inset: in phase space, the quasi-classical orbitals are equipotentials enclosing an area that is an integer multiple of 2π. eigenstates correspond to the equipotentials (x, k) that satisfy the Bohr-Sommerfeld quantiza- tion rule. To get the many-body ground state, one simply ﬁlls up all eigenstates with negative energies. The equipotentials can be locally parametrized as (x,±kF(x)), where eigenstates correspond to the equipotentials (x, k) that satisfy the Bohr-Sommerfeld quantiza- tion rule. To get the many-body ground state, one simply ﬁlls up all eigenstates with negative energies. 3.2 Fermi gas in an arbitrary external potential The equipotentials can be locally parametrized as (x,±kF(x)), where kF(x) = Æ 2(µ −V(x)). (19) (19) The position-dependent Fermi velocity is vF(x) = kF(x). The metric that underlies the problem is ds2 = d x2 + vF(x)2d y2, one can obtain a complex coordinate system on the worldsheet just like before; the result for a general potential V(x) reads The position-dependent Fermi velocity is vF(x) = kF(x). The metric that underlies the problem is ds2 = d x2 + vF(x)2d y2, one can obtain a complex coordinate system on the worldsheet just like before; the result for a general potential V(x) reads z(x, y) = Z x d x′ vF(x′) + i y, eσ = vF(x). (20) (20) Notice that the real part of z(x, y) is the time that a massless excitation takes to arrive at point x, starting from some reference point x0 with Rez(x0, y) = 0. The coordinate z always lives on an inﬁnite strip [τ1,τ2] × R, whose width τ2 −τ1 depends on V(x) and µ, and which can be conformally mapped onto the upper half-plane by z 7→g(z) = eiπ z−τ1 τ2−τ1 . Correlation functions are then evaluated exactly as in the harmonic case above. Formula (16) holds, and can be used to derive new exact results for the entanglement entropy. For instance, considering V(x) ∝\|x\|p one recovers the results from the so-called trap size scaling [31]. Notice that the real part of z(x, y) is the time that a massless excitation takes to arrive at point x, starting from some reference point x0 with Rez(x0, y) = 0. The coordinate z always lives on an inﬁnite strip [τ1,τ2] × R, whose width τ2 −τ1 depends on V(x) and µ, and which can be conformally mapped onto the upper half-plane by z 7→g(z) = eiπ z−τ1 τ2−τ1 . Correlation functions are then evaluated exactly as in the harmonic case above. Formula (16) holds, and can be used to derive new exact results for the entanglement entropy. For instance, considering V(x) ∝\|x\|p one recovers the results from the so-called trap size scaling [31]. One example that, to the best of our knowledge, cannot be solved with other means is that of a double well potential V(x) = α4x4 −α2x2 , α2,α4 > 0. (21) (21) 7 7 SciPost Phys. 3.2 Fermi gas in an arbitrary external potential 2, 002 (2017) 0 0.2 0.4 0.6 0.8 1.0 x 0 y = −R y = +R × (x, y) Figure 2: Imaginary time density proﬁle corresponding to the quench from a semi-inﬁnite box packed with fermions (ﬁlled black circles on the picture, the holes are shown with empty circles). The density at coordinate (x, y) is deﬁned as (25). Green colors correspond to a density close to one, blue to a density close to zero. In both cases the system is said to be “frozen”: observables do not ﬂuctuate at all. Intermediate colors correspond to ﬁnite densities, and have non trivial ﬂuctuations. The ﬂuctuating region is a disk of radius R, with density given by (25). 0 0.2 0.4 0.6 0.8 1.0 x 0 y = −R y = +R × (x, y) Figure 2: Imaginary time density proﬁle corresponding to the quench from a semi-inﬁnite box packed with fermions (ﬁlled black circles on the picture, the holes are shown with empty circles). The density at coordinate (x, y) is deﬁned as (25). Green colors correspond to a density close to one, blue to a density close to zero. In both cases the system is said to be “frozen”: observables do not ﬂuctuate at all. Intermediate colors correspond to ﬁnite densities, and have non trivial ﬂuctuations. The ﬂuctuating region is a disk of radius R, with density given by (25). In Fig. 1 we compare some exact ﬁnite size calculations for this potential with our novel prediction. The ﬁgure shows that (apart from well-known ﬁnite size oscillations [32]) exact numerical results match perfectly our CFT prediction. time t is [36] ρ(x, t) = 1 πarccos x t . (24) (24) Again, the question we want to answer is: what is the effective theory that captures long-range correlators 〈φ1(x1, t1)...φn(xn, tn)〉in this inhomogeneous system? We expect that it should be a (lorentzian) Dirac theory in curved 1+1D spacetime. There are technical issues, however, associated with the lorentzian formulation of the problem—for instance, the metric would be degenerate, ds2 = (d x−x t dt)2, and there would be no clear distinction between right- and left- movers—, so we chose to look at the problem in imaginary time, as routinely done in quench problems in CFT [18]. In this imaginary time approach to quantum quenches, the initial state becomes a boundary condition on the two sides of an inﬁnite strip of width 2R in imaginary time direction y [18, 42, 43]. Real-time correlators are recovered by ﬁrst performing a Wick rotation y →it, and then sending R →0. We focus on correlators 〈φ1(x1, y1)...φn(xn, yn)〉R, where yj ∈[−R,R] (see Fig. 2 for the application to the domain wall quench). For example, the imaginary-time density proﬁle is [44] 〈ρ(x, y)〉R = 〈ψ\|e−(R−y)Hc† xcxe−(R+y)H\|ψ〉 〈ψ\|e−2RH\|ψ〉 = 1 πarccos x p R2 −y2 , (25) (25) which gives back the real-time proﬁle (24) after performing the Wick rotation y →it and tak- ing the limit R →0. The density is different from zero or one only inside the disc x2 + y2 < R2; thus, there is a phase separation phenomenon, known as arctic circle [45]. This is shown in Fig. 2. In Ref. [44], the ﬁeld theory that describes long-range correlations inside the disc was unraveled: it is a Dirac theory in curved space, with euclidean metric ds2 = d x2 + 2 x y R2 −y2 d xd y + R2 −x2 R2 −y2 d y2 . (26) (26) Once we know this, it is again a straightforward exercise in CFT to compute correlation func- tions. As an illustration, we calculate again the entanglement entropy, for the bipartition A∪B with A = [−∞, x], B = [x,+∞] at times t > x (without loss of generality we assume x > 0). This can be done by performing elementary manipulations similar to those of Sec. 3.1 The main difference with the previous calculation is that, due to the presence of a ﬁnite lattice spacing, Eq. (16) needs to be modiﬁed. 4 A non-equilibrium situation We now show how the above framework can be adapted to deal with out-of-equilibrium sit- uations. The most natural problem to attack is the Fermi gas (1) released from a harmonic trap. However, this problem may be solved by other methods, and it is known, for instance, that various observables obey a dynamical symmetry [33], including the entanglement en- tropy [30,34,35]. This symmetry relates time-dependent quantities to their time-independent, ground-state, counterpart, simply by rescaling the coordinate x to x/ p 1 + ω2t2. Since the dynamical symmetry allows to deal with this problem in an efﬁcient way, it is not the most illustrative example for our purposes. Instead, we turn to a lattice gas, released from a semi-inﬁnite box: the initial state is such that all sites x < 0 are ﬁlled, and all sites x ≥0 are empty (this is also known as a quench from a domain wall initial state [36–41]). At t > 0, one lets the system evolve with the Hamiltonian H = −1 2 X x∈Z c† xcx+1 + c† x+1cx . (22) (22) In Fourier space the Hamiltonian is (up to an additive constant) In Fourier space the Hamiltonian is (up to an additive constant) H = Z π −π dk 2πϵ(k)c† kck , (23) (23) with dispersion relation ϵ(k) = −cos k. with dispersion relation ϵ(k) = −cos k. The relevant regime for an effective ﬁeld theory description is that of large distances and late times, in a way such that the ratio x/t is kept ﬁnite. In this limit the density proﬁle at 8 SciPost Phys. 2, 002 (2017) time t is [36] ρ(x, t) = 1 πarccos x t . (24) SciPost Phys. 2, 002 (2017) time t is [36] Finally, recalling that [44] to the upper half plane by the conformal map g(z) = eiz. Finally, recalling that [44] kF(x, y) = Re[z(x, y)] = arccos x p R2 −y2 , (29) (29) elementary algebraic manipulations and the use of (27) lead to the following expression for the Renyi entropies in imaginary time Sn(x, y) = n + 1 12n ln (R2 −x2 −y2)3/2 R2 −y2 . (30) (30) The analytic continuation to real time is obtained by ﬁrst substituting y →it, and then sending R →0. This gives + 1 Sn(x, t) = n + 1 12 n ln t(1 −(x/t)2)3/2 , (31) (31) a formula which was guessed from numerics in Ref. [48], and was calling for an analytic derivation. We just provided this derivation, which crucially relies on the metric (26) that underlies the whole problem. In Figure 3, we report a comparison of this prediction with nu- merical data and the agreement is perfect, up to the usual ﬁnite-size effects. The entanglement entropy for other bipartitions can be calculated as well, but the resulting formulas are more complicated and are therefore deferred to the appendix B.2. We mention that it is also possible to study different dispersion relations, but the results are rather technical; they are reported in appendix C. time t is [36] Namely, the position-dependent cut-off is no longer simply proportional to kF. In the homogeneous problem, it is known from the exact lattice so- lution [46] that the cut-off enters the formula for the Renyi entropies as sin(kF), instead of kF in the continuous gas. As a consequence of separation of scales, in the inhomogeneous case, we thus need to replace the local cut-off kF(x, y) in Eq. (16) by sin(kF(x, y)). This leads to the following formula for the Renyi entropies in imaginary time, Sn = n + 1 12n ln sin(kF(x, y)) eσ(z,¯z) d g(z) dz −1 Im g(z) . (27) (27) Here the complex coordinate z = z(x, y) must be compatible with the conformal structure of the metric (26), i.e. one must have ds2 = e2σdzd¯z, for some function eσ. Both z and eσ are given in Ref. [44], z(x, y) = arccos x p R2 −y2 −iarcth y R , eσ = Æ R2 −x2 −y2. (28) (28) The function (x, y) 7→z maps the interior of the disc x2 + y2 < R2, namely the interior of the critical region in the (x, y) plane, onto the inﬁnite strip [0,π] × R. The strip itself can be sent 9 SciPost Phys. 2, 002 (2017) 0 0.5 1 1.5 −1.5 −1 −0.5 0 0.5 1 1.5 0 0.5 1 S(x, t) x/t numerics CFT ρ(x, t) Figure 3: von Neumann entanglement entropy as a function of x/t after a quench from the domain wall initial state. The numerical simulations are performed using a ﬁnite system with 4096 sites. Data for t = 512 are reported (black circles) and compared to our analytical prediction (red line): the agreement is nearly perfect. Inset: corresponding density proﬁle ρ(x, t) after the quench. 0 0.5 1 1.5 −1.5 −1 −0.5 0 0.5 1 1.5 0 0.5 1 S(x, t) x/t numerics CFT ρ(x, t) Figure 3: von Neumann entanglement entropy as a function of x/t after a quench from the domain wall initial state. The numerical simulations are performed using a ﬁnite system with 4096 sites. Data for t = 512 are reported (black circles) and compared to our analytical prediction (red line): the agreement is nearly perfect. Inset: corresponding density proﬁle ρ(x, t) after the quench. to the upper half plane by the conformal map g(z) = eiz. 5 Conclusion Inhomogeneous 1D quantum systems are difﬁcult to tackle and this is motivating an enor- mous activity in order to provide exact results in some regimes, as for example the recently developed integrable hydrodynamics [49,50] (anticipated in [47]) which may have important ramiﬁcations into transport in 1D systems [51,52] such as quantum wires. To shed some light on these timely problems, we have shown here, with a few free fermion examples, that CFT 10 SciPost Phys. 2, 002 (2017) methods may be extended to attack this class of problems. The key assumption of our work is separation of scales: the system is locally homogeneous (but only locally), which is also the working limit of all approaches constructed so far [47, 49, 50]. In this regime, one can write a ﬁeld theory action with varying parameters. In the examples tackled in this paper, we found that the metric tensor should vary, leading to CFT in curved space. In particular, this new approach allows us to compute in a simple manner the entanglement entropies of these inhomogeneous systems (both in- and out-of-equilibrium) which are otherwise very difﬁcult (in most cases impossible) to obtain with other methods. It is important to stress that the background metric and the inhomogeneous cut-off are non-universal functions and must be viewed as inputs of the formalism. They should be obtained a priori by a proper microscopic computation. We believe anyway that the results of this paper should open the door to several new devel- opments. For example, by following the CFT approach of Ref. [53], our method could be used not only to determine the entanglement entropy, but also the entanglement negativity which is a proper measure of entanglement in mixed states [54] and it is not yet known how to compute it exactly for free fermionic systems. Another interesting development would be to recover by random matrix techniques [28, 55] the results we obtained for the entanglement entropy in a harmonic potential, possibly unveiling new structures of the random matrices. Having ex- act results also for arbitrary trapping potential could also help understanding whether these general cases could be tackled by random matrix techniques. From a physical point of view, the main open problem is how to describe inhomogeneous interacting 1D systems, most importantly Luttinger liquids such as Heisenberg spin chains and Lieb-Liniger gases. Acknowledgements We thank Nicolas Allegra and Masud Haque for collaboration on closely related topics [41,44], and Viktor Eisler for useful discussions. Funding information This work was supported partially by the ERC under Starting Grant 279391 EDEQS (PC), and by the CNRS Interdisciplinary Mission and Région Lorraine (JD). JD, JMS, and JV thank SISSA for hospitality. 5 Conclusion In these cases, one expects also the coupling constant (or Luttinger parameter K) to vary with position in spacetime. Fixing such parameters is a challenging problem. We hope that this paper will stimulate activity in that direction. A The Dirac action in curved space-time Here we explain the form of the Dirac action in a curved space-time, that was used in the main text. The most generic form of the Dirac action in a curved two-dimensional eulidean space-time is given by S = 1 2π Z d2xpgeµ a ¯Ψγa 1 2 ↔ ∂µ +iA(v) µ + iA(a) µ γ5 Ψ , (32) (32) where we chose γ1 = −σ2, γ2 = σ1, γ5 = σ3 and ¯Ψ = Ψ†γ2. The σα are the usual Pauli matrices. In this representation the two components of the spinor Ψ are the chiral components ψR and ψL; the function A(v) µ and A(a) µ are the vector and axial gauge ﬁeld associated with the U(1) gauge symmetry and the (anomalous) chiral symmetry. The matrix eµ a is called a tetrad: it 11 SciPost Phys. 2, 002 (2017) maps the tangent space of the manifold into R2 while preserving the inner product. It satisﬁes gµνeµ a eν b = δab, where gµν is the metric tensor and δab the ﬂat metric. In two dimensions, it is convenient to use complex coordinates z = x1+ix2 and ¯z = x1−ix2. The metric is conformally ﬂat and off-diagonal, the line element thus reads ds2 = e2σdz d¯z. The tetrad is diagonal if one chooses complex coordinates both in the tangent space and in R2. Its only non-vanishing components are complex conjugated and have modulus e−σ. Using ﬁnally pg = e2σ we can then rewrite the Dirac action in complex coordinates as maps the tangent space of the manifold into R2 while preserving the inner product. It satisﬁes gµνeµ a eν b = δab, where gµν is the metric tensor and δab the ﬂat metric. In two dimensions, it is convenient to use complex coordinates z = x1+ix2 and ¯z = x1−ix2. The metric is conformally ﬂat and off-diagonal, the line element thus reads ds2 = e2σdz d¯z. The tetrad is diagonal if one chooses complex coordinates both in the tangent space and in R2. Its only non-vanishing components are complex conjugated and have modulus e−σ. A The Dirac action in curved space-time Using ﬁnally pg = e2σ we can then rewrite the Dirac action in complex coordinates as S = 1 2π Z d2z eσ+iθ[ψ† R( ↔ ∂¯z +iA(v) + iA(a))ψR] + 1 2π Z d2z eσ−iθ[ψ† L( ↔ ∂z +i ¯A(v) −i ¯A(a))ψL], (33) (33) ( ) where A(v/a) and ¯A(v/a) are the complex z, ¯z components of the vector and axial potential. The rotation of angle θ in (33) between the tangent space of the manifold and the ﬂat euclidean space does not alter any of the conformal maps discussed in the main text. This holds because the twist ﬁelds needed to compute the entropy are spinless [27]. The extra phase factors in the fermionic propagators may be restored by performing a U(1) gauge transformation and a chiral transformation. The former acts on the chiral fermions as ψR/L →eiϕψR/L, and on the gauge ﬁeld as A(v) →A(v) −∂¯zϕ, ¯A(v) →¯A(v) −∂zϕ; the chiral transformation instead acts as ψR/L →e±iϕψR/L, and the gauge ﬁelds get modiﬁed as A(a) →A(a)−∂¯zϕ and ¯A(a) →¯A(a)−∂zϕ. where A(v/a) and ¯A(v/a) are the complex z, ¯z components of the vector and axial potential. The rotation of angle θ in (33) between the tangent space of the manifold and the ﬂat euclidean space does not alter any of the conformal maps discussed in the main text. This holds because the twist ﬁelds needed to compute the entropy are spinless [27]. The extra phase factors in the fermionic propagators may be restored by performing a U(1) gauge transformation and a chiral transformation. The former acts on the chiral fermions as ψR/L →eiϕψR/L, and on the gauge ﬁeld as A(v) →A(v) −∂¯zϕ, ¯A(v) →¯A(v) −∂zϕ; the chiral transformation instead acts as ψR/L →e±iϕψR/L, and the gauge ﬁelds get modiﬁed as A(a) →A(a)−∂¯zϕ and ¯A(a) →¯A(a)−∂zϕ. B.1 The harmonically trapped Fermi gas This is the result reported in the main text. B.2 The domain-wall quench For the case of a ﬁnite interval, the Renyi entanglement entropy are easily obtained by using Eq. (35) and the conformal mapping from the strip [0,π] × R to the upper half-plane g(z) = eiz. Setting y1 = y2 ≡y (i.e. equal imaginary times) and using (28), we get Sn(x1, x2, y) = Sn(x1, y) + Sn(x2, y) + n + 1 12n ln q R2 −x2 1 −y2 − q R2 −x2 2 −y2 −i(x1 −x2) q R2 −x2 1 −y2 + q R2 −x2 2 −y2 −i(x1 −x2) 2 = n + 1 6n ln (R2 −x2 1 −y2)3/4(R2 −x2 2 −y2)3/4 (R2 −y2)3/2 + + ln R2 −x1x2 −y2 − q (R2 −x2 1 −y2)(R2 −x2 2 −y2) (x1 −x2) . (37) (37) Analytically continuing the result (37) to real time and using the rescaled variables ζi = xi/t we obtain Analytically continuing the result (37) to real time and using the rescaled variables ζi = xi/ we obtain Sn(x1, x2, t) = n + 1 6n ln t(1 −ζ2 1)3/4(1 −ζ2 2)3/4(1 −ζ1ζ2 − q (1 −ζ2 1)(1 −ζ2 2)) ζ1 −ζ2 . (38) (38) This formula is valid in the regime t > \|x1\|,\|x2\|. Notice that this is very similar to Eq. (36), and indeed it has the same dependence of the rescaled variables ζi. However, the leading dimensional term in one case scales like ln t while in the other as ln L2 (times (n + 1)/(6n) in both cases). This is somehow reminiscent of a similar anomalous scaling found in local quenches [59] and it is unclear whether there is a connection between the two. This formula is valid in the regime t > \|x1\|,\|x2\|. Notice that this is very similar to Eq. (36), and indeed it has the same dependence of the rescaled variables ζi. However, the leading dimensional term in one case scales like ln t while in the other as ln L2 (times (n + 1)/(6n) in both cases). This is somehow reminiscent of a similar anomalous scaling found in local quenches [59] and it is unclear whether there is a connection between the two. B.1 The harmonically trapped Fermi gas The entropies of a ﬁnite interval [x1, x2] are slightly more complicated to calculate than the case of the semi-inﬁnite line (cf. Eq. (17)). Following Ref. [21], the Renyi entanglement entropy of order n is related to the two-point function of the twist ﬁeld Tn on the desired geometry. Here we work out the generalization to inhomogeneous systems and in particular for the case of fermions in a harmonic trap. In this case, the two-point function can be related to the one of the upper half plane by the combined action of a Weil transformation and the conformal mapping g(z) = ei(z+π/2) from the strip [−π 2 , π 2 ] × R to the upper half-plane. This allows us to write such two-point function in the z coordinate as 〈Tn(z1) ˜ Tn(z2)〉= e−σ(z1) d g(z1) dz1 ∆n e−σ(z2) d g(z2) dz2 ∆n 〈Tn(g(z1)) ˜ Tn(g(z2))〉uhp. (34) (34) The ﬁeld ˜ Tn is the conjugated twist ﬁeld [21, 27]. The calculation of two-point functions of twist ﬁelds in the upper half plane is, in general, a very challenging problem (indeed by images trick, it can be turned into a four point function in the complex plane which have been considered e.g. in Refs. [56, 57]). However, in the case of the massless free fermion ﬁeld- theory, this two-point function in the half plane simpliﬁes considerably and our desired object boils down to [30,58] (up to unimportant multiplicative constants) 〈Tn(z1) ˜ Tn(z2)〉= e−σ(z1) d g(z1) dz1 e−σ(z2) d g(z2) dz2 \|g∗(z1) −g(z2)\|2 Im(g(z1))Im(g(z2))\|g(z1) −g(z2)\|2 ∆n . (35) (35) 12 SciPost Phys. 2, 002 (2017) In our setup z1 = arcsin(x1/L) and z2 = arcsin(x1/L). Using again g(z) = ei(z+π/2) and (11), we obtain Sn(x1, x2) = Sn(x1) + Sn(x2) + n + 1 12n ln r 1 − x2 1 L2 − r 1 − x2 2 L2 + i (x1−x2) L r 1 − x2 1 L2 + r 1 − x2 2 L2 + i (x1−x2) L 2 =n + 1 6n ln   2L3 1 −( x1 L )23/4 1 −( x2 L )23/4 1 −x1x2 L2 − s 1 − x2 1 L2 1 − x2 1 L2 (x1 −x2)  , (36) where Sn(xi) is given in Eq. (17). This is the result reported in the main text. where Sn(xi) is given in Eq. (17). C.1 Imaginary time treatment Here we come back to the entropy of a single interval (−∞; x] following a domain wall quench, but with a more general dispersion relation. We consider Hamiltonian (23) with a dispersion relation of the form ϵ(k) = − X n≥1 a2n+1 cos(2n + 1)k, (39) (39) 13 SciPost Phys. 2, 002 (2017) which contains only odd Fourier modes. The reason for choosing this special form is mainly technical. As we shall see it makes explicit computations much simpler due to the symmetries ϵ(−k) = ϵ(k) and ϵ(π −k) = −ϵ(k). In real time the stationary phase equation governing the long time dynamics is [41] v(k) = dϵ(k) dk = x t , (40) (40) and may be used to compute systematically all correlation functions. Now, due to the form (39), if ks(x, t) is a solution, then so is π −ks(x, t). In the following we will assume that there are at most two solutions ks(x, t) and π−ks(x, t) to the previous equation. Under these assumptions, the density proﬁle is and may be used to compute systematically all correlation functions. Now, due to the form (39), if ks(x, t) is a solution, then so is π −ks(x, t). In the following we will assume that there are at most two solutions ks(x, t) and π−ks(x, t) to the previous equation. Under these assumptions, the density proﬁle is ρ(x, t) = kF(x, t) π = π −2ks(x, t) 2π = 1/2 −ks(x, t) π (41) (41) As is emphasized in the main text and above, it is convenient to think of this problem in imaginary time. The stationary phase equation for the imaginary time problem reads [44] dΦx,y,R dk = 0, Φx,y,R(k) = kx + i yϵ(k) + R˜ϵ(k), (42) (42) with with ˜ϵ(k) = − X n≥1 a2n+1 sin(2n + 1)k, (43) (43) the Hilbert transform of ϵ(k). In case there is only a ﬁnite number of Fourier modes in the dispersion, solving (42) amounts to solving algebraic equations in eik, which can be done in principle systematically. Now we make the extra assumption that there are only two solutions k = z(x, y) and k = −z∗(x, y). The new variable z lives in an inﬁnite strip 0 ≤Rez ≤π. The metric then reads ds2 = eσ(x,y)dzd¯z, eσ(x,y) = d2Φx,y,R(k) dk2 k=z(x,y) . 1We are grateful to Viktor Eisler for pointing that out to us. C.1 Imaginary time treatment Obtaining the position-dependent cutoff is more tricky, but can nevertheless be done using exact equilib- rium results obtained for several Fermi seas1. We refer to Ref. [60] for the details. The lattice cutoff takes now the more complicated form Let us brieﬂy comment on the case with more than two solutions to the stationary phase equa- tion (40). With the form (39) of the dispersion they always come in pairs. We label them as k(i) s and π−k(i) s for i = 1,..., p. p may be interpreted as a number of Fermi seas. As before we compute the entanglement entropy using ﬁeld theory, and add to this the contribution coming from the position-dependent UV cutoff. The generalization of our ﬁeld-theoretical framework to account for several Fermi seas is straightforward: we simply introduce p different species of Dirac Fermions, one for each Fermi sea (pair of stationary modes). Being non-interacting par- ticles, all the Dirac species contribute independently to the entanglement entropy. Obtaining the position-dependent cutoff is more tricky, but can nevertheless be done using exact equilib- rium results obtained for several Fermi seas1. We refer to Ref. [60] for the details. The lattice cutoff takes now the more complicated form f (k(1) s ,..., k(p) s ) = p Y i=1 cos k(i) s Y 1≤i<j≤p sin k(i) s −k(j) s 2 cos k(i) s +k(j) s 2 (−2)i−j+1 . (52) (52) The ﬁnal result for the entropy reads The ﬁnal result for the entropy reads Sn(x, t) = 1 12 1 + 1 n    p X i=1 ln t v′(k(i) s )cos2 k(i) s −2 X 1≤i<j≤p (−1)i−j log sin k(i) s −k(j) s 2 cos k(i) s +k(j) s 2   . (53) (53) C.1 Imaginary time treatment (44) (44) The conformal distance to the boundary may be obtained by mapping the inﬁnite strip to the upper half plane. Such a mapping is once again provided by g(z) = eiz, so we obtain d(x, y) = eσ(x,y) d g dz −1 Img(z) = eσ(x,y) sin(Rez(x, y)). (45) (45) Separating the real and imaginary parts z(x, y) = κ(x, y)+iQ(x, y) = κ+iQ, the above reads d(x, y) = d2Φx,y,R(k) dk2 k=κ+iQ ! sinκ, (46) (46) where κ and Q satisfy the system of equations where κ and Q satisfy the system of equations X n (2n + 1)a2n+1 cos(2n + 1)κ[y sinh(2n + 1)Q + Rcosh(2n + 1)Q] = x, (47) X n (2n + 1)a2n+1 sin(2n + 1)κ[y cosh(2n + 1)Q + Rsinh(2n + 1)Q] = 0. (48) (47) (48) he reasoning to get the entanglement entropy should be clear at this point: ﬁrst we get κ and from the above set of equations, and then we use them to compute the conformal distance The reasoning to get the entanglement entropy should be clear at this point: ﬁrst we get κ and Q from the above set of equations, and then we use them to compute the conformal distance The reasoning to get the entanglement entropy should be clear at this point: ﬁrst we get κ an Q f h b f i d h h h f l di The reasoning to get the entanglement entropy should be clear at this point: ﬁrst we get κ and Q from the above set of equations, and then we use them to compute the conformal distance 14 SciPost Phys. 2, 002 (2017) to be plugged in the appropriate equation for the entropy. For example in the case of the standard cosine dispersion relation, using this procedure, we ﬁnd the already known result d(x, y) = (R2 −x2 −y2)/ p R2 −y2, which can then be continued to real time y = it,R →0. It is important to stress that the concept of conformal distance only makes sense in imaginary time: in principle only after computing d(x, y) in imaginary time we are allowed to make the analytic continuation to real time. to be plugged in the appropriate equation for the entropy. C.1 Imaginary time treatment For example in the case of the standard cosine dispersion relation, using this procedure, we ﬁnd the already known result d(x, y) = (R2 −x2 −y2)/ p R2 −y2, which can then be continued to real time y = it,R →0. It is important to stress that the concept of conformal distance only makes sense in imaginary time: in principle only after computing d(x, y) in imaginary time we are allowed to make the analytic continuation to real time. In practice, one can avoid ﬁnding the general solution of the above system of equations. Indeed, because of their analytic structure, we can perform the analytic continuation at the level of Eqs. (47) and (48), relaxing the requirement that κ and Q are real. Plugging y = it and taking the limit R →0, we ﬁnd that Q = iπ/2 is a trivial solution to (48), due to the absence of even Fourier modes. The variable κ is then the solution of x t = X n≥1 (−1)n+1 cos[(2n + 1)κ] = v(κ + π/2), (49) (49) and so we get κ = ks −π/2. The analytic continuation of the conformal distance becomes −π/2. The analytic continuation of the conformal distance becomes lim R→0 d(x, it) = t dv dk k=ks cos ks = tv′(ks)cos ks. (50) (50) Putting back the contribution from the UV cutoff, sin kF = cos ks, we ﬁnally obtain Putting back the contribution from the UV cutoff, sin kF = cos ks, we ﬁnally obtain Sn(x, t) = 1 12 1 + 1 n ln t v′(ks)cos2 ks . (51) (51) Let us brieﬂy comment on the case with more than two solutions to the stationary phase equa- tion (40). With the form (39) of the dispersion they always come in pairs. We label them as k(i) s and π−k(i) s for i = 1,..., p. p may be interpreted as a number of Fermi seas. As before we compute the entanglement entropy using ﬁeld theory, and add to this the contribution coming from the position-dependent UV cutoff. The generalization of our ﬁeld-theoretical framework to account for several Fermi seas is straightforward: we simply introduce p different species of Dirac Fermions, one for each Fermi sea (pair of stationary modes). Being non-interacting par- ticles, all the Dirac species contribute independently to the entanglement entropy. C.2 An explicit example Let us now work out an example where the formula (51) may be computed explicitely. We consider a dispersion of the form ϵ(k) = −cos k + α 9 cos(3k), 0 < α < 1. (54) (54) 1We are grateful to Viktor Eisler for pointing that out to us. 1We are grateful to Viktor Eisler for pointing that out to us. 15 SciPost Phys. 2, 002 (2017) 0 0.5 1 1.5 −1.5 −1 −0.5 0 0.5 1 1.5 0 0.5 1 S(x, t) x/t α = 1/10 analytic ρ(x, t) Figure 4: von Neumann entanglement entropy as a function of x/t after a quench from the domain wall initial state, for dispersion relation ϵ(k) = −cos k + αcos3k and α = 1/10. he numerical simulations are performed using a ﬁnite system with 4096 sites. Data for t = 512 are reported (black circles) and compared to our analytical prediction (red lines): the agreement is nearly perfect. Inset: corresponding density proﬁle ρ(x, t) after the quench. 0 0.5 1 1.5 −1.5 −1 −0.5 0 0.5 1 1.5 0 0.5 1 S(x, t) x/t α = 1/10 analytic ρ(x, t) Figure 4: von Neumann entanglement entropy as a function of x/t after a quench from the domain wall initial state, for dispersion relation ϵ(k) = −cos k + αcos3k and α = 1/10. he numerical simulations are performed using a ﬁnite system with 4096 sites. Data for t = 512 are reported (black circles) and compared to our analytical prediction (red lines): the agreement is nearly perfect. Inset: corresponding density proﬁle ρ(x, t) after the quench. The restriction on α ensures that there are at most two solutions to the stationary phase equa- tion, ks(x, t) and π −ks(x, t). ks may be obtained by solving a cubic equation: The restriction on α ensures that there are at most two solutions to the stationary phase equa- tion, ks(x, t) and π −ks(x, t). ks may be obtained by solving a cubic equation: ks(x, t) = arcsin 1 −α −B(x, t)2/3 2pα B(x, t)1/3 , B(x, t) = Æ 9α(x/t)2 + (1 −α)3 −3pα(x/t). (5 (55) The density proﬁle in the inhomogeneous region \|x/t\| ≤1 + α/3 is then given by ρ(x, t) = 1 π arccos 1 −α −B(x, t)2/3 2pα B(x, t)1/3 , (56) (56) and the entropy is obtained by plugging (55) in (51). C.2 An explicit example In Fig. 4 we report the numerical data for the von Neumann entanglement entropy for the dispersion relation with α = 0.1: this shows perfect agreement with our prediction. References [1] T. Giamarchi, Quantum physics in one dimension, Oxford Univ. Press (2004), doi:10.1093/acprof:oso/9780198525004.001.0001. [1] T. 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https://openalex.org/W1511817061	https://www.intechopen.com/citation-pdf-url/38060	English	null	Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment	InTech eBooks	2,012	cc-by	13,937	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 11 © 2012 Pokethitiyook and Poolpak, licensee InTech. This is an open access chapter 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. Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment Prayad Pokethitiyook and Toemthip Poolpak Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48741 1.1. Organochlorine pesticide and river delta The contamination of soil and sediments with persistent OCPs may be related to point sources such as industrial discharges and waste plant effluents, but more frequently, it is attributed to diffuse sources, precipitation, agricultural runoff and particle transport. Because of hydrophobic characteristics, OCPs are least soluble in water but show a high affinity for different surface area, and those with organic content show the highest adsorption capacity. The organochlorines bound with particulate matter ultimately sink, thereby enhancing the concentration of the pollutant in the bottom sediment. Therefore, sediment acts as one of the main sinks and potential sources for OCPs in the environment [3]. Various reports have already confirmed the contamination with OCPs from different parts of the world. Chang and Doong [4], reported the contamination of BHCs, aldrin, α- and β-endosulfan, dieldrin, endrin and DDTs in estuaries surface sediments of Taiwan. In the same year, Pandit et al. [5] also reported the distribution of BHCs and DDTs in coastal marine of Mumbai. In 2005, Wure and Obbard [6] studied the distribution of organochlorine compounds, BHCs and DDTs, in the sea surface microlayer, water column and sediment of Singapore’s coastal environment. Additionally, Cheevaporn and Menasveta [7] reviewed and reported the water pollution in the Gulf of Thailand, which included some OCPs such as BHCs, DDTs, aldrin, dieldrin, α-endosulfan, heptachlor and heptachlor epoxide. Although the major sink of organochlorine compounds is the sediment, such compounds can bioaccumulate in the biota, especially for the benthic organisms and then enter the food chain. Menone et al. [8] reported that the burrowing crabs, Chasmagnathus granulatus, showed bioaccumulation of OCPs from sediments. They suggested that the pesticide levels are more related to the physico-chemical characteristics (e.g. total organic carbon and particle size distribution) of the sediment where the pesticides remain. Boonyatumanond et al. [9] reported the concentration of OCPs compounds in green mussel, Cara viridis, the bioindicator for water assessment, in coastal waters along the Gulf of Thailand. Agricultural pesticides most often are applied as liquids sprayed on the crop or the soil or as a seed treatment. An application of pesticide, depending on crop stage, formulation, intended target, application technique, and weather conditions is distributed between soil, plant foliage or crop residues, and losses due to drift. Chemicals used in the formulation of the pesticide can change the agronomic effects such as effectiveness and phytotoxicity of the formulated product. 1.1. Organochlorine pesticide and river delta Chemical pesticides are substances that kill or control an uninvited organism, which all share the common property of blocking a vital metabolic process of the target to which they are toxic [1]. Practically pesticides have been a concern since their introduction, because of the potential impact on human health through chemical contamination and their accumulation in food chain and environment. Organic pesticides were developed during and after World War II to displaced inorganic and metal organic substances such as sulfur, arsenic, sodium fluoride and boric acid, which used by society for thousands of year. Some of these pesticides were OCPs which are organic compounds that contain chlorine. These OCPs were preferred because of their effective against the target pests even if only small amount was applied to the environment. Neither small amounts of chemicals should enter the environment; however, the organic substances are generally much less toxic to human than the inorganic and metal organic compounds. Many OCPs share several remarkable properties together with stability against decomposition or degradation in the environment and their very low solubility properties in water, unless oxygen or nitrogen is also present in the molecules. In addition, their high ability to dissolve in hydrocarbon-like environments, fatty material in living matter for example. And the important one is OCPs is relatively high toxicity to insects, but low toxicity to humans [1]. OCPs can enter the environment from direct application and runoff, disposal of contaminated wastes into landfills, emissions from waste incinerators, and releases from manufacturing plants that produce these chemicals. Some OCPs are volatile or can adhere to soil or particles in the air. In aquatic systems, OCPs are adsorbed onto sediments in water that can then bioaccumulate in fish and other marine mammals. 218 Pesticides – Recent Trends in Pesticide Residue Assay Massive volumes of OCPs have been widely used for more than two decades as the backbone of a pest control strategy. Their lipophilic nature and stability contribute to their high bioaccumulation potential and long persistence. Organochlorine compounds have become an interesting object of study all over the world and are monitored continuously because of their proven toxicity to human beings, animal and plant life, and also food chain. Residues of OCPs were detected in almost all environmental compartments including water bodies, food chain, as well as in humans. Soil is the main repository of these OCPs [2]. 1.1. Organochlorine pesticide and river delta Chemicals also affect the environmental impact, as dispersion patterns may be altered and the functional activity period of the active ingredient may be lengthened or its degradation delayed. The pesticides that reach the soil or plant material in the target area begin to disappear by degradation or dispersion. Pesticides may volatilize into the air, Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 219 219 runoff or reach into surface water and groundwater, accumulate by plant or soil organisms or stay in the soil [10]. Human and ecological system exposure to micro-organic contaminants is related to their distribution between the various compartments of the environment (atmosphere, soils, surface fresh waters, ground waters, and oceans), to their transport between compartments, and to their availability for uptake and bioaccumulation by organisms. All the compartments have their importance but each has its own significance. The most obvious concern relating to the pollutants in the freshwater compartment is the possibility of adverse effects upon the freshwater ecosystem. In addition, micro-organics in rivers can be washed out to the seashore and so freshwater can also act as a major source of the pollutants to the oceans, where they may result in adverse ecological effects, particularly in the coastal zone. Human exposure can then occur through eating both freshwater and saltwater fish and other seafood. Within the freshwater compartment, it is particularly important to consider the sediment phase, because the largely hydrophobic nature of many micro-organic pollutants, which are known to associate strongly with natural sediments and dissolved organic matter. Sediments therefore have the strong potential to influence micro-organic pollutants fate in bed sediment, both freshwater and coastal marine. 2. Sources and transport of pesticides in freshwater sedimentary i The sources and transport of OCPs to and within freshwater sedimentary environments are obviously a crucial factor in determining the concentrations and the distribution of contaminants in freshwaters [11]. After applied to the soil zone, many pesticides transport to sedimentary systems, through leaching, surface run-off, spray drift, soil erosion and volatilization. A complex range of factors determines the fate of pesticides applied to agricultural land. These include the method of application of the active ingredient, and the weather conditions such as temperature, wind direction and speed, and rainfall following the application. The antecedent conditions, topography, soil type, farming practice and crop grown on the land, also affect water movement and dispersal of pesticides. These factors are reported to be more important than the physico-chemical parameters of the compound [12]. Other major routes of these organics compounds into freshwater sedimentary environments include direct discharges from industry, treated effluent discharge from both domestic and industrial sources, storm-water discharge from storm drain and aerial deposition. When wastewaters are treated before discharge to rivers and lakes, the concentration of a range of pesticide in the effluents is likely to be lower than in direct discharges because of possible sorption to solids and degradation during waste treatment. A huge range of the compounds is used and produced in industrial processes and may be discharged either from treatment plants or direct into freshwater. In some situations, industrial effluents are a very complex mixture of the OCPs and it is impossible to know the entire chemical composition of even the commercial products used 220 Pesticides – Recent Trends in Pesticide Residue Assay in manufacturing processes. This is because many commercial formulations are not pure chemicals but contain by-products of the production process. This, together with the metabolites formed during sewage treatment, leads to a wide range of compounds delivered to freshwater from a single commercial-product source. Hence any investigation of a point- source discharge containing micro-organic compounds must consider not only compounds that have been designed to be toxic, such as pesticide, but a wide range of commercial and natural products (possibly containing impurities not essential for the product application) that may have partially degraded in the pipeline or in the wastewater treatment process. Moreover, storm-water discharges, overflow from roadside and other urban drains during heavy rainfall events, can be sources of a range of micro-organic compounds to freshwaters. 3. The association of pesticides with sedimentary material and dissolved organic matter The sorption of pesticide on soil reduces its mobility, with the extent of reduction dependent upon the physical and chemical properties of the soil as well as the molecular characteristics of the pesticide. Since soil organic matter is the primary soil constituent responsible for sorption of non-ionic pesticides, a sorption constant based on only the organic carbon present (Koc, dm3 kg-1) can be used to assess pesticide mobility, Koc = Kd/oc. Where Kd is a measure of the extent of pesticide sorption by the soil, and oc is the fraction of the organic C present in the soil [14]. A sorption constant expressed in this manner is dependent only on the pesticide and is essentially independent of soil type. However, in soils with extremely low or high organic C and for ionizable or ionic pesticides the use of Koc to estimate Kd may be subject to large error. A significant proportion, typically ranging from 20 to 70%, of a pesticide or its degradation products may remain in the soil as a persistent residue bound to the soil colloids [15]. In this bound state, these compounds are difficult to extract and characterize, and tend to lose their biological activities. Many pesticides formerly believed to be readily degraded and lost from the soil were later shown to form these bound residues. Although no firm evidence is available, concern has been expressed that these bound residues may be released and be taken up by crop plants or leach into groundwater. In any water body there are areas of bed-sediment erosion and of sediment accumulation, largely determined by flow and wave patterns. Fine suspended sediment particles, weather from external influxes to the water body or from resuspended of bed sediments will be deposited, in the long term, in accumulation zones. In general, fine silty sediments will also have a higher organic C content than coarse sandy sediments. Because of these and because of their higher surface area to volume ratio, fine sediments are likely to contain higher concentrations of micro-organic compounds, on a weight for weight basis. The overall effect is that away from direct sources, bed-sediments in accumulation zones are likely to have a much higher loading of micro-organic compounds than bed sediments in other areas [11]. The effects of flow rate can also be very important in the transport of micro-organics in freshwaters. 3. The association of pesticides with sedimentary material and dissolved organic matter In freshwater, micro-organic contaminants from OCPs can exist in a variety of forms: as a freely dissolved phase, as a colloidal phase or associated with sedimentary material. In terms of the fate and effects of micro-organics between these various phases is a central issue. It strongly influences bed and suspended-sediment concentrations, freely dissolved concentrations (both in pore-waters and in the overlying water) and colloidally bound concentrations, and therefore also have a strong influence on sediment and water quality [11]. Natural sediments are complex mixtures of inorganic mineral offer environments, which may be favorable for micro-organics to become associated with. The association of micro-organics with sediments and soils is known as sorption, a general term, which covers two types of process, adsorption and absorption. Outstanding to the compositional complexity of sediments, sorption is not, however, the result of a single process, but may result from both adsorption and adsorption in/on a range of matrices. Behavior of pesticides in soil, suspended sediment and bed sediment were determined by several processes including degradation by soil microorganism, chemical degradation (e.g. hydrolysis), sorption and binding by organic and mineral soil components, uptake by plant roots, volatilization and the diluting effects of water flow process. Losses of pesticide in the soil via microbiological and chemical pathways are collectively termed degradation. Rate of degradation in soil usually increases with temperature and with soil water content [13]. Half-lives can be very long in dry soil. Degradation is often described satisfactorily by simple exponential decay, which assumes that the amount degraded per unit time is directly proportional to the amount present. The rate of degradation can be characterized by a half-life (DT50). As pesticide degradation products (metabolites) may have environmentally undesirable characteristics, the evaluation of the environmental fate of a pesticide should also take the fate of its major metabolites into consideration. However, the pesticide properties of the chemicals remain unaffected. Field-measured half-lives generally are shorter than those measured under controlled laboratory conditions because of first, multiple degradation pathways operating under field 221 Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment conditions, resulting in more rapid degradation. The last reason is losses by volatilization and photodecomposition. Both processes are occurring in the field and not in the laboratory field-measured soil; half-lives may also include dissipation losses by runoff, leaching and uptake by plants, which are not degradation pathway. 4. Degradation of organochlorine pesticides in freshwater sedimentary environment Degradation of OCPs contaminants in freshwater environments can occur either in the water column, or in the sediment bed following deposition. For pesticides in particular, compound half-lives and stabilities under various abiotic conditions are available from manufactures’ information. However, the situation is complicated in natural waters by the presence of varied microbial populations, suspended sediments, dissolved ions, dissolved organic matter and the sediment bed. Sediment, in particular, has the potential to strongly influence degradation, because many micro-organic compounds are known to associate strongly with sediments. In addition, microbial populations are also known to be largely associated with surfaces in the environment rather than living free in solution. Those OCPs contaminants can undergo degradation, either abiotically, through purely chemical or photochemical pathways, or biologically, with the microorganism assistant. Degradation can be complete, with compounds being mineralized to form CO2 and other simple chemical species, or partial, with the resultant degradation products more closely resembling the parent species. Different degradation pathways can occur simultaneously, with the relative importance of the various pathways being dependent upon the compound and the environmental conditions. Even if it is difficult to generalize because of the multitude of compound and types of environment, biotic degradation pathways are often found to be more important in the degradation of micro-organic contaminants. In particular, microbial pathways usually lead to complete mineralization of microorganism compounds. 3. The association of pesticides with sedimentary material and dissolved organic matter In relatively slow-flowing rivers such as the Rivers Aire and Calder in Yorkshire, bed and suspended sediments have been found to contain relatively high concentrations of a range of pesticides, notably including the synthetic pyrethroids [16]. In contrast, some fast-flowing rivers have high self-purification ability against pollution, with high water-discharge and sediment loads. This is the case for a number of rivers in some catchments in western China, where in spite of heavy industrial discharges of HCHs, DDT and PCBs, relatively low concentrations have been found in surficial river sediment (depth <3 cm) [17] compared with the concentrations found in other sediment surveys [18]. Where contaminated river have high purification ability because of rapid flushing and bed- scouring, compounds that are not found in river sediments may accumulate in coastal zones, estuaries and lakes. 222 Pesticides – Recent Trends in Pesticide Residue Assay Spatial variations in suspended-sediment-bound concentrations, as well as in bed sediment concentrations, can occur because of different sedimentation and flow regimes. Spatial variations in suspended-sediment-bound concentrations can also reflect source differences [16]. There is also likely to be a dilution of suspended sediments carrying contaminants by clean suspended material as the distance away from the source increases [19]. Temporal variations in non-tidal stretches of rivers usually result from changes in flow levels into and through the river, often varying with season [16]. Moreover, temporal variations can be complex if the source of the micro-organic compound is flow-related as in the case of resuspension of contaminated bed sediments, runoff from storm drains and erosion of contaminated soils, for example due to flooding. Otherwise, high flow rates giving rise to increased sediment transport will act to dilute suspended-sediment micro-organic compound concentrations. 6. Biotic degradation Microbial populations are responsible for the vast majority of biotic transformations of micro-organics in the environment. Microorganisms involved in five basic types of transformation including, microbial metabolism, in which the compound serves as a growth substrate, which leads to mineralization and co-metabolism, in which the compound is transformed by metabolic processes, but does not serve as an energy source. The third transformation type is polymerization or conjugation, in which microbial reactions result in the polymerization of the compound with natural organic matter. The last two transformation types are accumulation, in which the compound is incorporated into the tissues of the organism itself, but not for growth, and secondary effect transformations, whereby transformation results from changes in redox potential or pH as a result of microbial reactions. 5. Abiotic degradation pathways The two most important types of abiotic chemical degradation reactions are hydrolysis, usually either acid or based-catalyzed and redox reactions. Hydrolysis can be a significant 223 Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 223 degradation pathway for compounds containing ester, ether or amide functional groups, s- triazine and carbamate for example, and for substituted haloalkanes. In terms of chemical oxidation, the major oxidants present in the environment are O2, and Fe (III) and Mn (III) and (IV) oxides, with susceptible compounds including anilines and polyalkylated phenols. Whilst reduction is known to be a degradation route for a wide range of micro-organic compounds under appropriate environmental conditions, it is often difficult to identify the chemical reducing agents responsible. It is generally thought that low abundance but highly reactive species such as quinoid-type compounds, Fe porphyrines and some transition metal ions may be involved [11]. In terms of abiotic photochemical degradation or photolysis, there are few micro-organic contaminants including highly conjugated species such as polyene, nitroaromatics and some polyaromatic compounds, that will degrade directly following absorption of natural light. Indirect photolysis, in which dissolved natural organic matter becomes electronically excited after absorbing natural light, resulting in the production of a wide range of highly reactive species such as the hydroxyl radical (OH), which then may react with micro-organic pollutants. It is thought to be the more significant photolysis pathway for micro-organic compounds, with alkyl-substituted phenols, anilines, mercaptans, furans and other dienes being particularly susceptible [11]. 7. The study area The study area is Mae Klong River basin, which is located in the central plain of Thailand. The climate in Thailand can be divided into 3 seasons including winter (October-February), summer (February-May) and rainy season (May-October). Approximate annual rainfall levels are up to 1,100 mm in the rainy season (85% in May to September). The temperatures are between 25-40ºC. The humidity is around 56-79% [20]. The Mae Klong River basin is one of three major river basins in the Chao Phraya Delta, which are an important agricultural area in rice production and other agronomic in 224 Pesticides – Recent Trends in Pesticide Residue Assay Thailand for some centuries. The other two major river basins are the Chao Phraya and Tha Chin River basins. The Mae Klong River basin covers a total area of 30,800 km2 in the west central of Thailand with the very flat delta. It is periodically flooded in rainy season (mostly during August to November) [21]. The most important farming activities in this region are rice paddies and orchards including coconut, lime, lychee and mango. The main waterway in this basin is Mae Klong River which ends at the Gulf of Thailand in Samut Songkhram Province, and has the length of 132 km. The river receives water from both the irrigation canal systems and natural tributaries, which flow through cultivated areas where OCPs are intensively used to improve crop yields [7,22]. The discharge of untreated waste from agriculture is a major source of OCPs contamination in this area, and has been a major concern to citizens and officials for more than two decades [7,20,22]. Some organochlorine pesticides accumulation and their degradation along Mae Klong River basin and some of its tributaries will be discussed in this chapter. Figure 1. Map of Thailand showing the five tributaries of the Mae Klong River in the study area (Poolpak et al., 2008) Figure 1. Map of Thailand showing the five tributaries of the Mae Klong River in the study area Figure 1. Map of Thailand showing the five tributaries of the Mae Klong River in the study area (Poolpak et al., 2008) Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 225 Figure 2. The land use in Mae Klong Basin in the area of Samut Songkram Province with the major land use is agricultural area (Poolpak et al., 2008) Figure 2. 7. The study area The land use in Mae Klong Basin in the area of Samut Songkram Province with the major land use is agricultural area (Poolpak et al., 2008) The five sampling sites were on the Mae Klong River tributaries at Tumbon Prag Nam Dang, Amphawa, Samut Songkram Province (Figure 1). These tributaries are also major sources for household and industrial water supply in Samut Songkhram Province. As showed in Figure 2, the areas surrounding the sampling sites are a traditional rice- cultivation area in which farmers cultivate rice twice per year. The first crop (rainy-season rice) cultivation typically starts in August and harvested in November or December. An irrigated dry-season crop is planted in April or May and ends in July or August. Five sites were chosen in such a way as to include hot spots of pollution discharges along the Mae Klong River such as industrial regions, paddy fields and orchards for the first year. For the second year of monitoring, sediment samples were collected from Rang Tub Tab canal (site R) and Phi Lok canal (site P), which is an important crossing point of several tributaries in study area. Site R is also the tributary connected to the Gulf of Thailand. Sediment samples were collected monthly for a period of two years, during August 2003 to February 2005. The study areas and sampling locations along the selected tributaries were illustrated in figure 3. Furthermore, the basic physico-chemical properties of natural surface sediment samples within the area are shown in Table 1. Pesticides – Recent Trends in Pesticide Residue Assay 226 igure 3. The study areas and sampling locations along the selected tributaries during August 2003 to uly 2004 (A) and March 2004 to February 2005 (B) (Poolpak et al., 2008) Figure 3. 7. The study area The study areas and sampling locations along the selected tributaries during August 2003 to July 2004 (A) and March 2004 to February 2005 (B) (Poolpak et al., 2008) Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 227 Sampling year Sampling sites Geographical coordinate Water content (%) pH Sand (%) Silt (%) Clay (%) 2003 Kud Kamnan Somboon (K) (598863N, 1475923E) 50.79 5.9 20 24 56 Saphan Han (SP) (598450N, 1475000E) 64.15 5.8 30 21 49 Phi Lok (P) (595992N, 1475052E) 56.88 5.7 25 23 52 Sam-nga (SN) (595235N, 1472125E) 64.41 5.6 32 20 48 Rang Tub Tab (R) (593496N, 1472125E) 61.79 5.8 29 30 41 2004 Phi Lok (P) (595986N, 1472125E; 59.34 5.7 24 23 53 594718N, 1471496E; 595133N, 1475347E) Rang Tub Tab (R) (588357N, 1474380E; 42.44 5.8 29 30 41 589628N, 1474503E; 592581N, 1473464E; 593310N, 1474090E; 593059N, 1472441E; 593899N, 1476626E) Table 1. Sampling areas and codes of the tributaries of Mae Klong River, their coordinates and the basic physicochemical properties of natural surface sediment samples within the area Table 1. Sampling areas and codes of the tributaries of Mae Klong River, their coordinates and the basic physicochemical properties of natural surface sediment samples within the area Table 1. Sampling areas and codes of the tributaries of Mae Klong River, their coordinates and the basic physicochemical properties of natural surface sediment samples within the area 8. OCPs accumulation and their distribution in study area The organichlorine compounds in this investigation were hexachlorocyclohexane (technical HCH including -HCH, -HCH, -HCH, -HCH), heptachlor and its epoxide, -, - endosulfan and endosulfan sulfate, DDT, DDE, and DDD. They were chosen in the subject of their persistence and their importance in agricultural activities. The chemical structures and some properties of the selected OCPs were presented in table 2 and 3. Name Molecular formula Molecular weight Chemical structure α-HCH C6H6Cl6 290.83 β-HCH C6H6Cl6 290.83 Pesticides – Recent Trends in Pesticide Residue Assay 228 Name Molecular formula Molecular weight Chemical structure γ-HCH C6H6Cl6 290.83 δ-HCH C6H6Cl6 290.83 DDT C14H9Cl5 354.49 DDD C14H10Cl4 320.05 DDE C14H12Cl2 318.03 α-Endosulfan C9H6Cl6O3S 406.93 β-Endosulfan C9H6Cl6O3S 406.93 Heptachlor C10H5Cl7 373.32 Heptachlor epoxide C10H5Cl7O 389.32 Table 2. Organochlorine compound structures and their molecular properties Table 2. Organochlorine compound structures and their molecular properties Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 229 Substance Melting Pta Boiling Pta Vapor pressureb Water solubilityc Log P Henry’s law law constantd Atmospheric OH rate constantd α-HCH 159.5 228 4.50E-05 2 3.80 1.22E-05 1.40E-13 β-HCH 314.5 60 at 5.80E-01 mmHg 3.60E-06 0.24 3.78 4.40E-07 5.73E-13 γ-HCH 112.5 323.4 4.20E-05 7.3 3.72 5.14E-06 1.90E-13 δ-HCH 141.5 60 at 3.40E-01 mmHg 3.52E-05 10 4.14 4.29E-07 5.73E-07 DDT 108.5 NA 1.60E-07 0.0055 6.91 8.32E-06 3.44E-12 DDD 109.5 350 1.35E-06 0.09 6.02 6.60E-06 4.34E-12 DDE 56 320 6.00E-06 0.04 6.51 4.16E-05 7.43E-12 α- Endosulfan NA NA 3.00E-06 0.51 3.83 7.09E-06 8.17E-12 β - Endosulfan NA NA 6.00E-07 0.45 3.83 3.91E07 8.17E-12 Endosulfan sulfate 181-182 NA 2.80E-07 0.48 3.66 3.25E-07 8.17E-12 Heptachlor 95.5 310 4.00E-04 0.18 6.10 2.94E-04 6.11E-11 Heptachlor epoxide 160 NA 1.95E-05 0.2 4.98 2.10E-05 5.17E-12 NA indicates that the property is not applicable a at 1 atmosphere, ºC b at 25ºC, mmHg c at 25ºC, mg/L d at 25ºC NA indicates that the property is not applicable a at 1 atmosphere, ºC b at 25ºC, mmHg c at 25ºC, mg/L d at 25ºC Table 3. Physical and physico-chemical properties of some organochlorine pesticides Table 3. Physical and physico-chemical properties of some organochlorine pesticides The variations of the OCPs levels for different rice-cropping seasons in surface sediments from the five tributaries during August 2003 and July 2004 are summarized in Table 4, whereas Tables 5 show the mean concentration of OCPs residues in the sediment samples taken along site P and site R during March 2004–February 2005. 8. OCPs accumulation and their distribution in study area Despite the official ban and restriction on the usage of some OCPs, pesticide residues were detected in all sampling sites [20]. The range of total OCPs concentration in the sediments from five tributaries were 0.09-96.48 μg/g dw at site K, 0.01-110.92 μg/g dw at site SP, 0.06-102.05 μg/g dw at site P, 0.11-84.00 μg/g dw at site SN and 0.02-274.16 μg/g dw at site R. In the second year of observation, sites P and R were then chosen for further monitoring. Total residue concentrations of OCPs ranged from 0.15 to 99.60 at site P and 0.14 to 129.82 at site R. Comparing with the others, site R was found to be the most OCPs contaminated site in this study. 230 Pesticides – Recent Trends in Pesticide Residue Assay The higher concentration of OCPs at five selected tributaries indicated the high irrigation of these compounds in and around the basin. These OCPs are believed to have originated from various pesticide-rich sources, mainly agricultural areas and household. The natural processes of leaching and runoff and irrigation are likely to enhance their transfer to the main course of these tributaries, especially during the rice cultivation season [20]. The relatively high pesticide levels at site R can be explained from the input of pollutants in discharge from agricultural fields. This tributary was connected to and received water from other tributaries so it may contain pesticides from extraneous areas as well as from the immediate surroundings. OCPs residues in dry-season rice cultivation periods were higher than during the wet season in both years. This meant that some OCPs could be released from the surrounding areas to the tributaries in the irrigation from paddy fields. Moreover, the still water in dry season is one of the major basis. Decreased OCPs concentrations in the rainy-season cultivation can be explained in term of lower upstream discharge and dilution effects. Where non-agricultural activities in cool season infer to the low levels of the chemicals found in this sampling season [20]. 8. OCPs accumulation and their distribution in study area Pesticides Rainy –season cultivationa Non-rice cultivationb Dry-season rice cutivationc K SP P SN R K SP P SN R K SP P SN R -HCH 0.05 (0.06) 0.25 (0.18) 0.12 (0.10) 0.25 (0.18) 0.33 (0.08) 0.28 (0.05) 0.28 (0.20) 0.26 (0.11) 0.25 (0.14) 0.30 (0.13) 0.26 (0.09) 0.39 (0.07) 0.35 (0.00) 0.41 (0.04) 0.33 (0.08) -HCH 0.11 (0.02) 0.18 (0.02) 0.13 (0.04) 0.17 (0.02) 0.21 (0.08) 0.17 (0.06) 0.02 (0.02) 0.17 (0.03) 0.18 (0.03) 0.23 (0.07) 0.21 (0.10) 0.18 (0.02) 0.15 (0.02) 0.19 (0.03) 0.33 (0.19) -HCH 0.21 (0.05) 0.35 (0.00) 0.26 (0.05) 0.32 (0.06) 0.30 (0.05) 0.23 (0.04) 0.35 (0.00) 0.29 (0.00) 0.28 (0.09) 0.33 (0.00) 0.23 (0.04) 0.35 (0.00) 0.29 (0.00) 0.32 (0.06) 0.31 (0.06) -HCH 0.54 (0.24) 0.91 (0.41) 0.76 (0.72) 0.68 (0.22) 3.31 (1.32) 0.86 (0.81) 1.28 (1.51) 1.15 (1.13) 1.67 (2.49) 1.99 (1.01) 2.64 (1.08) 2.89 (2.13) 1.96 (1.35) 1.85 (1.22) 2.95 (0.95) Heptachlor 0.06 (0.07) 0.15 (0.10) 0.21 (0.19) 0.13 (0.10) 0.19 (0.14) 0.04 (0.04) n/a 0.09 (0.07) n/a 0.25 (0.19) 0.10 (0.04) 0.13 (0.05) 0.15 (0.11) 0.21 (0.12) 0.61 (0.91) Heptachlor epoxide 11.61 (4.39) 13.99 (5.96) 16.52 (20.78) 9.23 (7.87) 63.22 (24.72) 20.41 (25.67) 20.82 (27.51) 29.79 (38.96) 33.01 (56.12) 58.77 (49.73) 64.46 (29.63) 76.11 (44.67) 55.74 (19.57) 41.76 (26.84) 152.17 (55.50) Endosulfan I 0.12 (0.00) 0.16 (0.00) 0.13 (0.00) 0.16 (0.00) 0.15 (0.00) 0.11 (0.02) 0.15 (0.03) 0.13 (0.00) 0.16 (0.00) 0.16 (0.02) 0.10 (0.02) 0.17 (0.02) 0.11 (0.03) 0.16 (0.00) 0.12 (0.05) Endosulfan II 0.03 (0.02) n/a 0.05 (0.04) 0.01 (0.03) 0.05 (0.04) 0.03 (0.03) 0.01 (0.03) 0.04 (0.02) 0.01 (0.03) 0.14 (0.25) 0.03 (0.07) n/a 0.05 (0.04) 0.09 (0.06) 0.07 (0.05) Endosulfan sulfate 0.07 (0.06) 0.13 (0.03) 0.08 (0.05) 0.15 (0.04) 0.15 (0.03) 0.11 (0.05) 0.08 (0.08) 0.17 (0.08) 0.07 (0.10) 0.38 (0.46) 0.15 (0.01) 0.21 (0.07) 0.19 (0.02) 0.19 (0.02) 0.58 (0.51) p,p-DDE 0.08 (0.03) 0.04 (0.08) 0.09 (0.06) 0.06 (0.08) 0.11 (0.04) 0.09 (0.08) 0.17 (0.10) 0.13 (0.09) 0.12 (0.14) 0.16 (0.07) 0.10 (0.03) 0.21 (0.04) 0.12 (0.06) 0.18 (0.08) 0.16 (0.07) p,p-DDD 0.01 (0.02) 0.06 (0.12) 0.03 (0.03) 0.02 (0.03) 0.02 (0.03) 0.13 (0.05) 0.03 (0.04) 0.03 (0.03) 0.03 (0.06) n/a 0.14 (0.10) 0.13 (0.27) n/a 0.11 (0.18) n/a p,p-DDT 0.13 (0.08) 0.17 (0.12) 0.14 (0.13) 0.26 (0.11) 0.08 (0.05) 0.26 (0.07) 0.31 (0.13) 0.25 (0.05) 0.38 (0.05) 0.20 (0.14) 0.37 (0.11) 0.35 (0.08) 0.22 (0.10) 0.37 (0.03) 0.63 (0.74) n/a means no available a August 2003-November 2003 b December 2003-March 2004 c April 2004-July 2004 Table 4. 8. OCPs accumulation and their distribution in study area Mean and standard deviation (S.D.) of OCPs (μg g-1 dw) detected in the surface sediment in five tributaries of the Mae Klong river during August 2003-July 2004 (n=60/cultivation period) p J y Table 4. Mean and standard deviation (S.D.) of OCPs (μg g-1 dw) detected in the surface sediment in five tributaries of the Mae Klong river during August 2003-July 2004 (n=60/cultivation period) Table 4. Mean and standard deviation (S.D.) of OCPs (μg g-1 dw) detected in the surface sediment in five tributaries of the Mae Klong river during August 2003-July 2004 (n=60/cultivation period) Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 231 Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 231 Pesticides Dry–season cultivationa Rainy-season rice cultivationb Non-rice cutivationc P R P R P R -HCH 0.21 (0.15) 0.28 (0.15) 0.32 (0.06) 0.02 (0.03) n/a 0.33 (0.08) -HCH 0.93 (0.83) 3.66 (3.29) 0.12 (0.02) 0.09 (0.02) 0.11 (0.02) 0.08 (0.19) -HCH 0.18 (0.08) 0.21 (0.12) 0.08 (0.13) 0.05 (0.08) 0.02 (0.02) 0.01 (0.06) -HCH 5.50 (3.32) 10.09 (6.31) 1.08 (1.28) 0.82 (0.94) 0.64 (0.23) 0.43 (0.95) Heptachlor 0.53 (0.59) 2.16 (2.61) 0.04 (0.09) 0.04 (0.04) 0.04 (0.03) 0.02 (0.02) Heptachlor epoxide 72.43 (30.90) 110.39 (56.79) 19.51 (28.43) 14.84 (21.82) 7.66 (3.41) 4.59 (1.50) Endosulfan I 0.07 (0.05) 0.18 (0.12) 0.10 (0.05) 0.06 (0.02) 0.02 (0.01) 0.01 (0.05) Endosulfan II 0.07 (0.04) 0.10 (0.03) 0.06 (0.04) 0.07 (0.03) 0.02 (0.01) n/a Endosulfan sulfate 0.17 (0.07) 4.62 (7.47) 0.04 (0.02) 0.05 (0.03) 0.01 (0.01) n/a p,p-DDE 0.66 (0.12) 0.11 (0.05) 0.03 (0.01) 0.03 (0.03) 0.03 (0.01) n/a p,p-DDD 1.03 (0.63) 0.17 (0.09) 0.02 (0.02) 0.02 (0.01) 0.01 (0.01) n/a p,p-DDT 1.05 (0.85) 2.99 (2.59) 0.05 (0.04) 0.05 (0.02) 0.02 (0.02) n/a n/a means no available a March 2004-June 2004 b July 2004-October 2004 c November 2004-Febuary 2005 Table 5. Mean and standard deviation (S.D.) of OCPs (μg g-1 dw) detected in the surface sediment at site P and R during March 2004-Febuary 2005 (n=36/cultivation period) No e be 00 ebua y 005 Table 5. Mean and standard deviation (S.D.) of OCPs (μg g-1 dw) detected in the surface sediment at site P and R during March 2004-Febuary 2005 (n=36/cultivation period) Table 5. 8. OCPs accumulation and their distribution in study area Mean and standard deviation (S.D.) of OCPs (μg g-1 dw) detected in the surface sediment at site P and R during March 2004-Febuary 2005 (n=36/cultivation period) Even though usage of some imported pesticides such as DDT was officially banned in 1983, there is the official report presented that these OCPs were parts of organochlorine insecticides active ingredients not included in the ban list. That is why they were still detected in sediment samples along with their metabolites [22-24]. Additionally, the appearance of some OCPs such as endosulfan and heptachlor in the sediment may indicate continued usage of these pesticides despite the official ban. Illegal smuggling of these pesticides from the neighborhood countries has been reported several times in local newspapers. Furthermore, some pesticides are still used in some developing countries around the tropical belt [25,26] which demonstrates the degradation persistence of these substances, even in tropical environment [27]. Heptachlor epoxide was found at the highest concentration in sediments from station R. The enzymatic epoxidation of heptachlor to heptachlor epoxide in soil and animals is most likely responsible for the significant greater incidence of heptachlor epoxide than heptachlor [28]. This chemical was also extensively used for termite control in Thailand in the past, and additionally, was used in seed treatments and control of soil insects such as ants. Although the usage of heptachlor was officially banned in 1988, the observed high residue concentrations of heptachlor epoxide at all sampling sites could come either from persistence in the environment or from continued use reflecting either illegal smuggling or use of old stocks [20]. Heptachlor and its epoxide were also detected in both water samples Pesticides – Recent Trends in Pesticide Residue Assay 232 [23] and agricultural soil samples [24] from the Chao Phraya Basin. Furthermore, the other unlikely sources of heptachlor and its metabolite may be chlordane which is widely used against termite infestation and could be released into the environment primarily from its application as an insecticide [9]. It has the main components including α- and γ-chlordane, γ-nonachlor and heptachlor and generally metabolized in the environment and in the human body to oxychlordane and may be heptachlor epoxide found in the absence of either oxychlordane or γ-nonachlor [29]. In the case of endosulfan, the concentration of its α-isomer (the main component of the commercial product) was higher than β-endosulfan (a secondary component) at all sampling sites during the two years. 8. OCPs accumulation and their distribution in study area Total endosulfan showed values in the range >0.001- 1.34 μg/g dw in 2003, with 0.05-0.20 μg/g dw for the α-isomer and only >0.001-0.51 μg/g dw for the β-isomer. Endosulfan sulfate, an endosulfan degradation by-product, was found in large concentration in the sediment samples (range >0.001 to 1.34 μg/g dw). The total endosulfan in 2004 was 22.60 μg/g dw with the highest proportion as endosulfan sulfate (>0.001-15.72 μg/g dw) [20]. Endosulfan was extensively used to control golden apple snails in rice fields in this area and was officially banned in 2004. Endosulfan and its by-products, endosulfan sulfate, were found in relatively high concentrations, both during the dry and rainy-rice cultivation seasons. These data reflects the wide use of endosulfan in the surrounding agricultural areas over a long period [20]. Moreover, during warehouse storage, β-endosulfan can be slowly converted to α-endosulfan, which has higher toxic insecticidal properties than the β-isomer [30]. For some OCPs, chemical transformation and microbial decomposition may occur, e.g. DDT is transformed to DDE and δ-HCH to α-HCH. However, complete mineralization of organochlorine compounds does usually either not takes place, or is an extremely slow process [3]. Composition of DDT metabolites and HCH isomers were examined in detail because their differing composition in the environment could indicate different sources of contamination [31]. 9. DDTs In the first year of monitoring, levels of DDTs were found elsewhere, for example, between 0.08 and 1.83 μg/g dw in sediment from site R. The ratios of (DDE+DDD)/∑DDT in surface sediments ranged from 0.21 to 0.61 μg/g dw in 2003 and from 0.10 to 1.00 μg/g dw during 2004 (Figure 4 and 5). Most values were less than 0.5 for the first year, whereas they were greater than 0.5 for the second year. As to the individual DDTs, the concentrations in surface sediment were generally found in the order of DDT>DDE>DDD in most sampling sites and sampling seasons (Figure 6). The same trend was also reported for the second year (Figure 7) except for sites P and R (non-rice cultivation areas), at which DDE showed the highest percentage. In addition, DDE was usually present at a higher concentration than DDD [20]. 9. DDTs In the first year of monitoring, levels of DDTs were found elsewhere, for example, between 0.08 and 1.83 μg/g dw in sediment from site R. The ratios of (DDE+DDD)/∑DDT in surface sediments ranged from 0.21 to 0.61 μg/g dw in 2003 and from 0.10 to 1.00 μg/g dw during 2004 (Figure 4 and 5). Most values were less than 0.5 for the first year, whereas they were greater than 0.5 for the second year. As to the individual DDTs, the concentrations in surface sediment were generally found in the order of DDT>DDE>DDD in most sampling sites and sampling seasons (Figure 6). The same trend was also reported for the second year (Figure 7) except for sites P and R (non-rice cultivation areas), at which DDE showed the highest percentage. In addition, DDE was usually present at a higher concentration than DDD [20]. Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 233 Heptachlor and Its Metabolite: Accumulation and Degradation in Sedim Figure 4. Relationship between (DDE+DDD)/∑DDT and DDD/DDE in surface sediments during August 2003 to July 2004 (Poolpak et al., 2008) Figure 5. Relationship between (DDE+DDD)/∑DDT and DDD/DDE in surface sediments during Mar 2004 to February 2005 (Poolpak et al., 2008) Figure 4. Relationship between (DDE+DDD)/∑DDT and DDD/DDE in surface sediments during August 2003 to July 2004 (Poolpak et al., 2008) Figure 4. Relationship between (DDE+DDD)/∑DDT and DDD/DDE in surface sediments during August 2003 to July 2004 (Poolpak et al., 2008) Figure 5. Relationship between (DDE+DDD)/∑DDT and DDD/DDE in surface sediments during March 2004 to February 2005 (Poolpak et al., 2008) Figure 5. Relationship between (DDE+DDD)/∑DDT and DDD/DDE in surface sediments during March 2004 to February 2005 (Poolpak et al., 2008) Figure 5. Relationship between (DDE+DDD)/∑DDT and DDD/DDE in surface sediments during March 2004 to February 2005 (Poolpak et al., 2008) Pesticides – Recent Trends in Pesticide Residue Assay 4 Figure 6. Composition of DDTs in surface sediments, during August 2003 to July 2004 (Poolpak et al., 2008) Figure 7. Composition of DDTs in surface sediments, during March 2004 to February 2005 (Poolpak et al., 2008) Pesticides – Recent Trends in Pesticide Residue Assay 234 Figure 6. Composition of DDTs in surface sediments, during August 2003 to July 2004 (Poolpak et al., 2008) Figure 6. Composition of DDTs in surface sediments, during August 2003 to July 2004 (Poolpak et al., 2008) Figure 7. 9. DDTs Composition of DDTs in surface sediments, during March 2004 to February 2005 (Poolpak et al., 2008) Figure 7. Composition of DDTs in surface sediments, during March 2004 to February 2005 (Poolpak et al., 2008) 235 Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 235 The result showed that DDE was usually present in a higher concentration than DDD, suggesting that the most DDT was transformed into DDE [32]. The higher occurrence of DDD and DDE are the results of DDT degradation and the higher stabilities of the metabolites [33-35]. The metabolite DDE is produced from DDT under aerobic conditions such as that occurs in upland soils. On the other hand, DDT is converted to DDD with a half-life of a few days under anaerobic conditions such as in the aquatic sediment [3]. Microbial degradation of DDTs is generally slow resulting in environmental persistence of these compounds [36]. The relative concentrations of the parent DDT and its metabolites can indicate whether DDTs input are from the former or present uses [26,34,37]. Data from the relative concentrations of the parent DDT and its metabolites (DDE and DDD) shows that most values were less than 0.5 for the first year (2003), whereas they were greater than 0.5 for the second year (2004) [20]. The ratios of (DDE+DDD)/∑DDT > 0.5 are reported to indicate long-term weathering [26,38-41]. This suggested that the DDT compounds in sediments collected in 2003 were from the current use whereas the 2004 values which were mostly > 0.5 indicated that degraded metabolites formed a significant proportion of total DDT compounds and the contamination of the area has started long time ago. Since the DDD/DDE ratios were less than the unity at both sampling sites, this indicates that the sediment samples were dominated by the products of aerobic degradation and the contamination of the area is fairly recent. It also indicates that the ongoing use of DDT and its metabolites were derived under aerobic conditions before transported by surface runoff to the waterways sediment [20]. This may be explicable by the relatively higher transportability of DDE than the other form in the atmosphere [18]. 10. HCHs The concentrations of HCH isomers detected in this study in 2003 were in the sequence β- HCH<α-HCH<γ-HCH<δ-HCH at most sampling sites, where the order in 2004 was α- HCH≤γ-HCH<β-HCH<δ-HCH. In addition, β-HCH represented the lowest proportion (7.61% of the total HCHs compared to 11.2% and 12.0% for α- and γ-isomers, respectively during 2003 (Figure 8). On the other hand, the decreased proportion of α-HCH and the increased proportion of β-HCH in 2004 were detected (Figure 9). Among HCH isomers analyzed, γ-HCH (lindane) contributed 12% and then 2.22% of the total HCH concentrations in sediment samples in 2003 and 2004, respectively [20]. HCH residues are among the most widely distributed and frequently detected organochlorine contaminants in the environment [36]. Technical HCH was formerly used as a broad-spectrum pesticide for agricultural purpose in Thailand until it was banned in 1980 [9] whereas the γ-isomer, commonly known as lindane, (the only isomer with insecticidal activity) was used for controlling agricultural and medical pests [43] and banned in 2001. Generally, the most common isomers of HCHs in the environment are α-HCH, β-HCH and γ-HCH [44]. Whereas there were high concentrations of δ-HCH in sediments at most sampling sites in the present study, the results are similar to the levels detected at Beijing Guanting reservoir, where the possible reasons of contamination are still unclear [44]. The Pesticides – Recent Trends in Pesticide Residue Assay 36 Figure 8. Composition of HCHs in surface sediments, during August 2003 to July 2004 (Poolpak et al., 2008) Figure 9. Composition of HCHs in surface sediments, during March 2004 to February 2005 (Poolpak et al., 2008) Pesticides – Recent Trends in Pesticide Residue Assay 236 Figure 8. Composition of HCHs in surface sediments, during August 2003 to July 2004 (Poolpak et al., 2008) Figure 8. Composition of HCHs in surface sediments, during August 2003 to July 2004 (Poolpak et al., 2008) Figure 9. Composition of HCHs in surface sediments, during March 2004 to February 2005 (Poolpak et al., 2008) Figure 9. Composition of HCHs in surface sediments, during March 2004 to February 2005 (Poolpak et al., 2008) Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 237 237 typical technical HCH generally contains all four isomers and their physico-chemical properties are different. The most stable β-HCH, relatively resistant to microbial degradation due to its lowest water solubility and vapor pressure, represents a good indicator of contamination [31,33,43,45]. 11. Degradation of heptachlor by soil microcosm According to Poolpak [53], the biodegradation of heptachlor and its oxidative metabolite, epoxide in the microcosm were selected in the mean of their highest contamination in the filed study. The effect of initial concentrations and kinetic study of heptachlor, biodegradation in the sediment, effect of additional carbon sources, soil:water ratio and temperature on degradation of heptachlor were studied to assess its degradation in the fresh water environment. Sediments in degradation study were collected from Rang Tub Tab canal to determine the biodegradation behavior of the indigenous microorganisms in studied site. 10. HCHs Many studies have reported that β-isomer was dominant in sediments from river or estuary environment after long-term migration and transformation [38,46,47]. In addition, α-isomer is typically predominant in ambient air as well as ocean waters and can be converted into the β-isomer in the environment [31,42]. The sequence of HCHs in this study can be explained by the resistance to chemical degradation of these isomers in this environmental condition and the recent use of technical HCH in this area [45]. Various technical HCH formulations have different amounts of the α- , β-, and γ-isomers; however, the long range atmospheric transport favors α-HCH, the most volatile isomer [48]. On the other hand, the decrease in proportion of α-HCH and the increase in proportion of β-HCH in 2004 implied that the HCHs contamination in the sediment samples might have originated from a relatively distant source or long-term accumulation. Additionally, this data indicates the use of technical HCHs rather than lindane (γ-HCH) and may be explained by the degradation of γ-HCH in this environmental condition because γ-HCH is degraded by microorganisms [49,50] and photochemically isomerized to the α-isomer [45,51]. Although, the predominance of α-isomer in some environmental samples reflects the recent use of technical HCH [52], the higher concentration of α-isomer than γ-isomer in this study may establish the evidence of lindane usage in the past. Since there is no historical data concerning HCHs contamination in this central plain, thus the data presented here can be used to establish a baseline for the future monitoring and management of pesticides in this area [20]. 12. The effects of initial concentrations of heptachlor on microcosm acti ities In addition, Chiu et al. [56] reported that 100 μg/mL heptachlor and DDT were completely inhibited anaerobic microorganisms and no metabolite was found. Growth of microorganism represented inhibition of higher heptachlor concentrations, which may decrease the degradation efficiencies of the chemical in this study. Similar results were presented in degradation of DDT by serratia marcescens [55] and biodegradation of heptachlor and DDT by sediment microbial [56]. In addition, Chiu et al. [56] reported that 100 μg/mL heptachlor and DDT were completely inhibited anaerobic microorganisms and no metabolite was found. pH and redox potential of different concentrations of heptachlor in liquid culture were measured to evaluate the relationship between growth and metabolic activities of the microcosms and pesticide concentrations. The culture pH was slightly decreased to an acidic range in early treatments before slightly increased to neutral at the end of the experiment in all treatments except for the liquid culture at 150 μg/mL heptachlor concentration where the pH was decreased to acidic range until the end of the experiment. This phenomenon may be due to the metabolic activities of the growing microorganisms. The decreased of pH in liquid culture in biodegradation experiment were also reported [56,58]. In addition, the redox potential values of the liquid culture were positive earlier then decreased to the negative value at the end of the experiment. The effects of abiotic factors such as temperature, pH and organic matter on degradation of heptachlor in sediment were also determined at 30°C for 28 days. Heptachlor was degraded in sediment only 19.62% in abiotic control compared to 51.17% in biotic control. Furthermore, its metabolite, heptachlor epoxide was not detected under abiotic condition, whereas its concentration under biotic condition was 21.13±1.62 μg/g dw or 25.58% of the parent heptachlor. Because of heptachlor epoxide can be degraded by oxidative reaction by organisms, as a result abiotic control presented no concentration of heptachlor epoxide. Additionally, heptachlor epoxide concentration in this studied was 25.58% of the parent heptachlor which higher than that reported by Bidleman et al. [59] who suggested that heptachlor epoxide accounted for about 20% of the original heptachlor. They also suggested heptachlor degradation in soil proceeded at by at least two routes, 1-hydroxy chlordane and the more persistent, epoxide which subsequently volatilized. Consequently, the oxidation product of indigenous microbial in this study was heptachlor epoxide. 12. The effects of initial concentrations of heptachlor on microcosm acti ities The effects of different initial concentrations of heptachlor on the degradation and rate of metabolism of soil microcosms were performed in the liquid culture at the heptachlor concentrations of 50, 100 and 150 μg/mL at temperature of 30°C, 150 rpm for 7 days. The decline of different concentrations of heptachlor and the transformation of heptachlor epoxide, the oxidative metabolite, were examined. The liquid cultures without heptachlor but with 1% yeast extract were used as control. Degradation of heptachlor and heptachlor epoxide formation at various initial concentrations was illustrated in figure 10. Pesticides – Recent Trends in Pesticide Residue Assay 238 Figure 10. Degradation of heptachlor and heptachlor epoxide formation at various initial concentrations (Heptachlor residue at  50 μg/mg heptachlor (abiotic);  100 μg/mg heptachlor (abiotic);  150 μg/mg heptachlor (abiotic); ---- 50 μg/mg heptachlor; ----100 μg/mg heptachlor; and ---- 150 μg/mg heptachlor. Heptachlor epoxide concentration at  50 μg/mg heptachlor;  100 μg/mg heptachlor; and  150 μg/mg heptachlor) (Modified from Poolpak, 2008) Figure 10. Degradation of heptachlor and heptachlor epoxide formation at various initial concentrations (Heptachlor residue at  50 μg/mg heptachlor (abiotic);  100 μg/mg heptachlor (abiotic);  150 μg/mg heptachlor (abiotic); ---- 50 μg/mg heptachlor; ----100 μg/mg heptachlor; and ---- 150 μg/mg heptachlor. Heptachlor epoxide concentration at  50 μg/mg heptachlor;  100 μg/mg heptachlor; and  150 μg/mg heptachlor) (Modified from Poolpak, 2008) The highest degradation efficiencies were observed in 100 μg/mL heptachlor as 88.36%, whereas at the concentration of 150 μg/mL heptachlor, the efficiencies were lower. Accordingly, 100 μg/mL heptachlor were conditioned in further experiments. Transformation of heptachlor to heptachlor epoxide in liquid culture was evaluated and showed that its metabolite was mostly found in 50 μg/mL heptachlor treatments or 27.85% of parent heptachlor with the positive values of redox potential suggesting the oxidative activities of microorganisms. Lu et al. [54] reported the transformation of heptachlor into 1- hydroxychlordene and 1-hydroxychlordene epoxide was relatively rapid but small proportion of heptachlor epoxide was formed which suggested that heptachlor epoxide formed environmentally largely in vivo by microsomal multifunction oxidase action. 239 Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 239 Growth of microorganism represented inhibition of higher heptachlor concentrations, which may decrease the degradation efficiencies of the chemical in this study. Similar results were presented in degradation of DDT by serratia marcescens [55] and biodegradation of heptachlor and DDT by sediment microbial [56]. 12. The effects of initial concentrations of heptachlor on microcosm acti ities This result is relatively similar to the field survey data which the major contaminant in the tributaries of the Mae Klong River was heptachlor epoxide [20]. The pH values were slightly acidic in biotic than abiotic condition. The redox potentials were positive in both conditions, except for later incubation, when those of biotic condition showed the negative value. Microorganisms were increased in number when the incubation time was increased. The positive redox potential was related to the microbial metabolism employing oxidative activities. DeLaune et al. [60] reported that soil generally had a redox range from -250 to +700 mV. In the flooded soil, oxygen is consumed and disappears at a redox potential of approximately +350 mV. Accordingly, aerobic soils represent a redox range of +350 to +700 mV, and anaerobic soil a range of -250 to +350 mV. 240 Pesticides – Recent Trends in Pesticide Residue Assay 13. Effects of additional carbon source on heptachlor degradation The additional carbon sources other than the target compounds may influence the degradation rates in the system, so the effects of additional carbon sources including sodium succinate, sodium acetate and glucose supplements at 1% concentration were performed and compared with abiotic control (no additional carbon source) to observe heptachlor degradation and heptachlor epoxide evolution. Higher heptachlor residue in the experiment amended with 1% glucose, without additional carbon sources and 1% succinate (40.33±2.06, 40.33±1.79 and 39.84±0.77 μg/g dw, respectively), where 1% sodium acetate showed the least heptachlor (37.13±0.39 μg/g dw) as showed in figure 11. The results illustrated that without carbon supplement, the degradation efficiency was at its highest, whereas 1% of sodium succinate and sodium acetate addition increased the efficiency of degradation. In contrast, many studied reported the inhibition of the degradation of pesticides by microbial when the presence of the more favorable carbon sources has been found [55,56,61]. Moreover, increasing organic contents by adding carbon sources made sediment microorganisms more vigorous in consuming oxygen and turned the incubating condition into anaerobic condition [62]. Figure 11. Degradation of heptachlor and heptachlor epoxide formation at various additional carbon sources (Heptachlor residue at  abiotic control; ---- w/o carbon source; ---- 1% glucose; ---- 1% sodium succinate; and ---- 1% sodium acetate. Heptachlor epoxide concentration at  w/o carbon source;  1% glucose;  1% sodium succinate; and  1% sodium acetate) (Modified from Poolpak, 2008) Figure 11. Degradation of heptachlor and heptachlor epoxide formation at various additional carbon sources (Heptachlor residue at  abiotic control; ---- w/o carbon source; ---- 1% glucose; ---- 1% sodium succinate; and ---- 1% sodium acetate. Heptachlor epoxide concentration at  w/o carbon source;  1% glucose;  1% sodium succinate; and  1% sodium acetate) (Modified from Poolpak, 2008) 241 Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment Heptachlor epoxide appeared in experiments with adding sodium succinate (21.73% of the parent heptachlor) and sodium acetate (22.02% of the parent heptachlor) was lower than the other two (25.87% and 25.58% for glucose added and without carbon source added, respectively). By means of heptachlor epoxide concentrations decreased when carbon sources were added, the degradation product of heptachlor was no longer heptachlor epoxide. The anaerobic degradation of heptachlor by study of Chiu et al. 13. Effects of additional carbon source on heptachlor degradation [56] indicated that sodium acetate increased the anaerobic degradation of heptachlor in river sediment better than glucose and chlordane was produced as the product of degradation. Accordingly, heptachlor could be metabolized to chlordane and 1-hydroxychlordene-2,3-epoychlordene under anaerobic condition [63]. Microorganisms grew well with 1% glucose as compared to other carbon sources. With sodium succinatqe added, the pH was increased with the increase in incubation time whereas the number of microorganisms and redox potential values were decreased. The same trend was observed in sodium acetate addition experiment, less microorganism and redox potential values were found. 14. Effect of soil:water ratio on the heptachlor degradation The effect of water volume on heptachlor degradation in sediment, the 1:1, 1:2 and 1:4 soil:water (w/v) ratio experiments were determined and the biodegradation effects were presented in figure 12. When the soil:water ratio was decreased, the degradation rate was decreased. Together with higher heptachlor residues in the 1:1 soil:water ratio (47.89±1.52 μg/g dw), heptachlor epoxide concentration was also decreased when soil:water ratio was increased to 1:1 w/v (18.81% of the parent heptachlor). The degradation efficiencies of heptachlor were also decreased when more sediment was added. Degradation of α- and γ- HCH in the soil slurry by Siddique et al. [57] showed that the soil:water ratio significantly decreased the rate of degradation at 4 weeks of chemical incubation. In addition, the study of Castro and Yoshida [64] found that heptachlor had degraded readily in flooded soil than under upland conditions. The fate of heptachlor in the environment is influenced by environmental factors including soil/water ratio. Decreasing the soil:water ratio decreased the degradation of heptachlor in the sediment, which might due to the adsorption of heptachlor to the soil particles, decreasing their bioavailability to the microorganisms. Furthermore, the conditions in sediments are principally controlled by the diffusion of dissolved oxygen from the overlying water into the sediment bed, which aerobic bacteria dominate, and oxidation reactions both abiotic and biotic relatively easy occurred [11]. The decrease in heptachlor epoxide productivity when soil:water ratio decreased, suggested that less oxidative metabolism by bacterial activities with the confirmation by negative redox potential. The redox profile is expected to have the greatest effect on OCPs degradation in bed sediments, since compounds will be degraded at different rates and may be degraded through different pathways under different redox conditions. In general, the degradation rates in soils and sediments have been observed to be significantly faster under Pesticides – Recent Trends in Pesticide Residue Assay 242 aerobic conditions, atrazine degradation in oxidizing conditions [60] and HCH degradation by microbial consortium [61,65]. This studied shows the same trend as previous studies as redox potential since 1:4 soil:water ratio were more positive than the others. Moreover the degradation efficiency of this soil:water ratio was better than other treatments. Figure 12. Degradation of heptachlor and heptachlor epoxide formation at various soil:water ratio (Heptachlor residue at  abiotic control; ---- 1:4 soil:water ratio; ---- 1:2 soil:water ratio; and ---- 1:1 soil:water ratio. 14. Effect of soil:water ratio on the heptachlor degradation Heptachlor epoxide concentration at  1:4 soil:water ratio;  1:2 soil:water ratio; and  1:1 soil:water ratio) (Modified from Poolpak, 2008). Figure 12. Degradation of heptachlor and heptachlor epoxide formation at various soil:water ratio (Heptachlor residue at  abiotic control; ---- 1:4 soil:water ratio; ---- 1:2 soil:water ratio; and ---- 1:1 soil:water ratio. Heptachlor epoxide concentration at  1:4 soil:water ratio;  1:2 soil:water ratio; and  1:1 soil:water ratio) (Modified from Poolpak, 2008). Even though degradation was slow under reducing or anaerobic condition, the anaerobic degradation of chlordane [66], atrazine [60] and endosulfan [67] were observed. Even though degradation was slow under reducing or anaerobic condition, the anaerobic degradation of chlordane [66], atrazine [60] and endosulfan [67] were observed. 15. Effect of incubation temperature on heptachlor degradation The influence of incubation temperatures on heptachlor degradation and heptachlor epoxide transformation was studied at 20°C and 30°C to evaluate the degradation efficiencies of the microcosm. A smaller amount heptachlor residue was presented at 30°C (40.33±2.06 μg/g dw) than that of 20°C (65.70±2.41 μg/g dw). Heptachlor epoxide concentration was decreased when the temperature was decreased, 25.58% and 14.75% of parent heptachlor at 30° C and 20°C, respectively (figure 13). Furthermore, the higher percentage of biodegradation at 30°C was also better than that of 20°C (51.17% and 26.30%, respectively). Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 243 Figure 13. Degradation of heptachlor and heptachlor epoxide formation at various temperature (Heptachlor residue at  abiotic control; ---- 20C; and ---- 30C. Heptachlor epoxide concentration at  20C; and  30C) (Modified from Poolpak, 2008). Figure 13. Degradation of heptachlor and heptachlor epoxide formation at various temperature (Heptachlor residue at  abiotic control; ---- 20C; and ---- 30C. Heptachlor epoxide concentration at  20C; and  30C) (Modified from Poolpak, 2008). Figure 13. Degradation of heptachlor and heptachlor epoxide formation at various temperature (Heptachlor residue at  abiotic control; ---- 20C; and ---- 30C. Heptachlor epoxide concentration at  20C; and  30C) (Modified from Poolpak, 2008). Many studies reported that the temperature of 30°C was suitable for chemical biodegradation including biodegradation of HCH isomer in soil slurry [57] and DDT and heptachlor degradation in river sediment of Taiwan [56]. Moreover, biodegradation of endosulfan by soil bacteria [68], abiotic degradation of endosulfan in a clay soil [70] and degradation of DDT by soil bacteria [54] yielded the maximum values at temperature of 30°C. However, microorganisms have the ability to degraded pesticides in a wide range of temperatures. For example, Serratia marcescens DT-1P can degrade DDT at the temperature from 4°C up to 50°C [55]. 16. Rate of heptachlor degradation In liquid culture and sediment, the degradation rate at the heptachlor concentration of 100 μg/g soil dw by indigenous microorganism fitted well with the second-order reaction and first order-reaction, respectively. The constant heptachlor degradation rate in liquid culture with the degradation rate constant, k = 0.0125 μg/mL·day. The degradation acted as the 244 Pesticides – Recent Trends in Pesticide Residue Assay concentrations of one second-order reactant, or two first-order reactants. Heptachlor degradation in sediment hold the degradation rate constant (k) of 3.106104 μg/g dw·day and acted as the first-order reaction. A first-order reaction depended on the concentration of only one reactant. Other reactants could be present but in zero-order reaction. The reaction rate of heptachlor biodegradation in both liquid culture and sediment was slightly decreased when the incubation time was increased. This may be due to the decrease in bacterial number when the incubation time was increased. Additionally, the conditions of the experiment were unfavorable for metabolic activities for microorganisms. Since the degradation rate in liquid culture fits to the second-order reaction, it indicates that not only bacterial activities affected heptachlor degradation but also other factors should be involved as well. Even though, degradation of sorbed pesticides in the soil is a complicated process affected by many factors, the degradation rate of heptachlor in sediment fitted well with first-order reaction. This suggests that microbial degradation of pesticide was the major factor in sediment degradation. Ghadiri et al. [70] reported the degradation rates of aldrin, dieldrin, endrin and chlordane were first-order, the same as rate of atrazine degradation in reactors studied by Ghosh and Philip [71]. 17. Dominant microorganism in heptachlor degradation experiment The second is microbial dechlorination of heptachlor produce chlordene, which undergoes microbial epoxidation to form the corresponding chlordene epoxide. The last route of biodegradation is oxidation of heptachlor by microorganism converts heptachlor to its epoxide by mixed-function oxidase system [63]. 17. Dominant microorganism in heptachlor degradation experiment In all experiment of heptachlor biodegradation by soil microorganisms collected from Rang Tub Tab canal. Three indigenous microorganisms, which dominated in the biodegradation experiment in both liquid culture and sediment, were identified by 16S rDNA sequencing as Bacillus subtilis RS-01, Bacillus cereus RS-02 and Pseudomonas putida RS-03. Although, the numbers of bacteria were low at earlier incubation, it was increased when the incubation time increased. Additionally, degradation rate increased when the number of bacteria was increased. Despite the fact these three bacteria were dominant in the experiment during heptachlor degradation; it is difficult to interpret the role that the bacteria play in the transformation process of heptachlor in view of the fact that the physiological actions of these isolates are still unknown. However, biodegradation of endosulfan contaminated soil and water in laboratory scale reactors by B. circulans-I and II enriched from contaminated soil of endosulfan processing industry has been reported by Kumar and Philip [71]. In addition, P. fluorescencens, B. cereus and Bacillus sp. were the consortium successful for ex-situ biodegradation of chlorobenzenes in soil [73]. Ambrosoli et al. [74] studied the degradation of carbofulan by bacterial population; the results showed that genera Pseudomonas and Bacillus rapidly degraded carbofuran in soil as carbon source. Furthermore, heptachlor epoxide concentrations in all biotic experiments represented oxidative metabolism of these bacterial population. The evidences suggest that these three bacteria might be responsible for the heptachlor degradation in sediment. The proposed pathways of heptachlor metabolism in this study were summarized in Figure 14. Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 245 Figure 14. Pathways of heptachlor metabolism by indigenous microorganisms (T. Poolpak, 2008; modified from Lal and Saxena, 1982). Figure 14. Pathways of heptachlor metabolism by indigenous microorganisms (T. Poolpak, 200 modified from Lal and Saxena, 1982). The first route of heptachlor degradation is the chemically hydrolysis of heptachlor to 1- hydroxychlordene in soil, and microbial attack on this product results in the production of 1-hydroxy-2,3-epoxychlordene. The second is microbial dechlorination of heptachlor produce chlordene, which undergoes microbial epoxidation to form the corresponding chlordene epoxide. The last route of biodegradation is oxidation of heptachlor by microorganism converts heptachlor to its epoxide by mixed-function oxidase system [63]. The first route of heptachlor degradation is the chemically hydrolysis of heptachlor to 1- hydroxychlordene in soil, and microbial attack on this product results in the production of 1-hydroxy-2,3-epoxychlordene. 18. Conclusion The surface sediment in the tributaries of Mae Klong River is polluted by the OCPs and relatively high levels of these compounds were observed in most study areas which summer was the season that highest OCPs residues were found in both sampling years. Heptachlor epoxide presented the highest concentration among detected OCPs. In addition, DDT and HCHs were found in slightly elevated levels representing a recent input of these two OCPs into the study area. For heptachlor biodegradation in liquid cultures, the highest degradation efficiency (88.36%) was observed in 100 μg/mL heptachlor where at 150 μg/mL heptachlor, the efficiency was lower. Moreover, growth of microorganism was inhibited at higher heptachlor concentration; as a result the degradation efficiency was decreased. In addition, heptachlor 246 Pesticides – Recent Trends in Pesticide Residue Assay epoxide, the oxidative product of heptachlor by microorganism was found at the highest concentration in the 50 μg/mL heptachlor treatments. This evidence enhanced the idea of inhibition of bacterial growth at high concentration of heptachlor. In abiotic condition of heptachlor biodegradation and the transformation to heptachlor epoxide in sediment, there was no detection of heptachlor epoxide. Confirms microorganisms play a major role in heptachlor oxidation to heptachlor epoxide. In contrast, heptachlor degradation was detected in biotic degradation; heptachlor epoxide concentration in this study was 25.58% of the parent heptachlor. A number of external parameters including additional carbon sources affected the degradation rates in this study. Without carbon supplement, the degradation efficiency was high. Moreover, when 1% of sodium succinate and sodium acetate was added, the efficiency of degradation was even higher. However, heptachlor epoxide concentrations decreased when these carbon sources were added and the degradation product of heptachlor was changed to chlordane instead of heptachlor epoxide. For soil:water ratio experiments, as the soil:water ratio was decreased, the degradation rate of heptachlor decreased. At 1:4 soil:water ratio, the redox potential values were more positive and higher degradation efficiency was observed. Also both degradation efficiency and transformation of heptachlor to epoxide at 20°C were lower than those at 30°C, these may be due to 30°C was the most favorable temperature for bacterial growth and heptachlor degradation. The degradation rate in liquid culture fitted to the second-order reaction where the degradation rate of heptachlor in sediment fitted well to the first-order reaction. Decrease of reaction rate as incubation time increased was seen in both liquid culture and sediment. 18. Conclusion This may be as a result of the decrease in bacterial number and the shortage of food source toward time. Three dominant bacteria were isolated and identified as B. subtilis RS-01, B. cereus RS-02 and P. putida RS-03. Degradation rate of heptachlor and heptachlor epoxide evolution increased when the number of bacteria was increased. Although these three bacteria were dominant in the experiment during heptachlor degradation, it is difficult to interpret the role that the bacteria play in the transformation process of heptachlor in view of the fact that the physiological actions of these isolates are still unknown. Nonetheless, heptachlor epoxide, oxidative products from microorganism metabolism, presented in biotic treatments, suggests that these bacteria might be responsible for the heptachlor degradation in sediment. Prayad Pokethitiyook* and Toemthip Poolpak Department of Biology, Faculty of Science, Mahidol University, Ratchathewi, Bangkok, Thailand * Corresponding Author Author details Prayad Pokethitiyook* and Toemthip Poolpak Department of Biology, Faculty of Science, Mahidol University, Ratchathewi, Bangkok, Thailand * Corresponding Author Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 247 247 Acknowledgement This work was supported by the Post-Graduate Education, Training and Research Program in Environmental Science, Technology and Management (ESTM) under Higher Education Development Project of the Ministry of Education and Mahidol University. It was also supported by and carried out in the framework of the Integrative Research Network of the Tha-chin and Mae Klong river basins. We gratefully acknowledge Department of Biology, Faculty of Science, Mahidol University. 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Heptachlor and Its Metabolite: Accumulation and Degradation in Sediment 251 251 [56] Chiu T, Yen J, Hsieh Y, Wang Y (2005) Reductive transformation of dieldrin under anaerobic sediment culture. Chemoshpere 60: 1182-1189. [56] Chiu T, Yen J, Hsieh Y, Wang Y (2005) Reductive transformation of dieldrin under anaerobic sediment culture. Chemoshpere 60: 1182-1189. [57] Siddique T, Okeke B C, Arshad M, Frankenberger Jr W T (2002) Temperature and pH effects on biodegradation of hexachlorocyclohexane isomers in water and a soil slurry. J Agric Food Chem 50: 5070-5076. [58] Okeke B C, Siddique T, Arbestain M C, Frankenberger W T (2002) Biodegradation of γ- hexachlorocyclohexane (lindane) and α-hexachlorocyclohexane in water and a soil slurry by a Pandoraea species. J Agric Food Chem 50: 2548-2555. [59] Bidleman, T F et al. (1998) Soil as a source of atmospheric heptachlor epoxide. Environ Sci Technol 32(10): 1546-1548. [60] DeLaune R D, Devai I, Mulbah C, Crozier C, Lindau C W (1997) The influence of soil redox conditions on atrazine degradation in wetlands. Agric Ecosys Environ 66: 41-46. [61] Manonmani H K, Chandrashekaraiah D H, Reddy N S, Elcey C D, Kunh A A M (2000) Isolation and acclimation of a microbial consortium for improved aerobic degradation of α-hexachlorocyclohexane. J Agric Food Chem 48: 4341-4351. [62] Chen I, Chang Y, Lin H (2004) Microbial dechlorination of hexachlorobenzene by untamed sediment microorganisms in Taiwan. Prac Period Hazard Toxic Radio Waste Manag 8(2): 73-78. [63] Lal R, Saxena D M (1982) Accumulation, metabolism, and effects of organochlorine insecticides on microorganisms. Microbiol rev 46: 95-127. [64] Castro T F, Yoshida T (1971) Degradation of organochlorine insecticides in flooded soil in the Philippines. J Agric Food Chem 19(6): 1168-1170. [65] Sahu S K, Patnaik K K, Sharmila M, Sethunathan N (1990) Degradation of α-, β- and γ- hexachlorocyclohexane by a soil bacterium under aerobic conditions. Appl Environ Microbiol 56: 3620-3622. [66] Hirano T, Ishida T, Oh K, Sudo R (2007) Biodegradation of chlordane and hexachlorobenzenes in river sediment. Chemosphere 67: 428-434. 19. References [67] Guerin T F (1999) The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments. Environ Pollut 106: 13-21. [68] Hussain S, Arshad M, Saleem M, Khalid A (2007) Biodegradation of α- and β- endosulfan by soil bacteria. Biodegrad 18: 731-740. [69] Ghadiri H, Rose C W (2001) Degradation of endosulfan in a clay soil from cotton farms of western Queensland. J Environ Manag 62: 155-169. [70] Ghadiri H, Rose C W, Connell D W (1995) Degradation of organochiorine pesticides in soils under controlled environment and outdoor conditions. J Environ Manag 43: 141- 151. [71] Ghosh P K, Philip L (2004) Atrazine degradation in anaerobic environment by a mixed microbial consortium. Water Res 38: 2277–2284. [72] Kumar M, Philip L (2006) Bioremediation of endosulfan contaminated soil and water- optimization of operating conditions in laboratory scale reactors. J Hazard Mater B 136: 354-364. Pesticides – Recent Trends in Pesticide Residue Assay [73] Guerin, T F (2008) Ex-situ bioremediation of chlorobenzenes in soil. J Hazard Mater 154: 9-20. [74] Ambrosoli R, Negre M, Gennari M (1996) Indications of the occurrence of enhanced biodegradation of carbofulan in some Italian soils. Soil Biol Biochem 28(12): 1749-1752. Pesticides – Recent Trends in Pesticide Residue Assay 252 [73] Guerin, T F (2008) Ex-situ bioremediation of chlorobenzenes in soil. J Hazard Mater 154: 9-20. [74] Ambrosoli R, Negre M, Gennari M (1996) Indications of the occurrence of enhanced biodegradation of carbofulan in some Italian soils. Soil Biol Biochem 28(12): 1749-1752.
https://openalex.org/W1548293091	https://journalhosting.ucalgary.ca/index.php/TLI/article/download/57390/43163	English	null	Weaving SoTL into Our Everyday Lives	Teaching & learning inquiry	2,015	cc-by	967	INTRODUCTION Weaving SoTL into Our Everyday Lives The third year of Teaching & Learning Inquiry begins with articles that demonstrate a maturing view of the field. They explore the potential of the Scholarship of Teaching and Learning (SoTL) as a framework for doing more informed, higher quality work in course (re)design, national accreditation processes, and the analysis of problem-solving processes of teachers and learners. They illustrate one of the ways SoTL practitioners serve as what Bernstein calls “Cosmopolitan Assets to an Institution”: specifically, these faculty members habitually “acknowledge a formal body of expertise in their work and . . . make reference to an external community when evaluating quality and seeking innovation in ideas and practices” (2013, p. 37-38). The work in these articles also reflects the “vision of academia’s future” in which “inquiry, evidence, and innovation in teaching and learn- ing are part of the fabric of everyday life”—and not just teaching and learning (Williams, Verwood, Beery, Dalton, McKinnon, Strickland, Pace, & Poole, 2013, p. 50). Williams and colleagues illustrate this future by describing a committee that—instead of relying on assumptions and anecdotes—consults relevant research to more precisely frame, under- stand, and respond to what is perceived as a local campus problem. In this future, and in this issue of TLI, approaching problems through the lens of SoTL is a mark of rigour. Auten and Twigg describe how SoTL informed the design and content of a graduate student pedagogy course to more effectively equip future teach- ers. deBraga, Boyd, and Abdulnour similarly used Felten’s “principles of good practice in SoTL” to guide a course redesign aimed at higher-level thinking skills. They note that this framework, which began with student learning, was tied to relevant contexts, part- nered with students, and opened itself to peer review, challenged students more effec- tively while also improving their course evaluations. Maurer and Kropp also describe increasing rigour—in this case, through a specific testing strategy—without negatively affecting student evaluations. f Lee and Son demonstrate how conversations about teaching, which often feature a comparison of practices, can be taken to a new level of rigour when those comparisons are made more systematically. Wismath, Orr, and MacKay apply the notion of threshold concepts to help us better understand how students approach problem solving. When instructors must become problem solvers, necessity can become the mother of their in- vention. Teaching & Learning Inquiry, Volume 3, Issue 1, pp. 1–2, 2015. Copyright © 2015 The International Society for the Scholarship of Teaching and Learning Gary Poole, UNIVERSITY OF BRITISH COLUMBIA, gary.poole@ubc.ca Nancy Chick, VANDERBILT UNIVERSITY, nancy.chick@ucalgary.ca INTRODUCTION Weaving SoTL into Our Everyday Lives INTRODUCTION Weaving SoTL into Our Everyday Lives Gary Poole is the Associate Director of the School of Population and Public Health and a Senior Scholar in the Centre for Health Education Scholarship at the University of British Columbia. Gary Poole is the Associate Director of the School of Population and Public Health and a Senior Scholar in the Centre for Health Education Scholarship at the University of British Columbia. Nancy Chick was Assistant Director of the Center for Teaching and an aﬃ liated faculty member in the English Department at Vanderbilt University. She is now University Chair in Teaching and Learn ing, Academic Director of the Taylor Institute for Teaching and Learning, and Professor of English at the University of Calgary. Nancy Chick was Assistant Director of the Center for Teaching and an aﬃ liated faculty member in the English Department at Vanderbilt University. She is now University Chair in Teaching and Learn ing, Academic Director of the Taylor Institute for Teaching and Learning, and Professor of English at the University of Calgary. INTRODUCTION Weaving SoTL into Our Everyday Lives Such was the case when Day turned to a clever use of educational technology to keep his course moving smoothly during a time that could have been disruptive. In doing so, he realized he had the makings of a natural experiment to measure the educational impact of his idea. His paper invites us all to find such SoTL opportunities. 1 Poole, Chick Indeed, all the articles in this issue of TLI present the same invitation—to consider SOTL as part of the “fabric of everyday life.” As SoTL becomes more thoroughly woven into this fabric, fascinating questions get asked, research opportunities get pursued, and teaching and learning keep gett ing bett er. Figure 1. After a recent SoTL workshop, a participant submitted Figure 1 as the defi nition of SoTL, hinting at this under- standing of SoTL as signifi cant and useful beyond the classroom. Figure 1. After a recent SoTL workshop, a participant submitted Figure 1 as the defi nition of SoTL, hinting at this under- standing of SoTL as signifi cant and useful beyond the classroom. Gary Poole is the Associate Director of the School of Population and Public Health and a Senior Scholar in the Centre for Health Education Scholarship at the University of British Columbia. Williams, A. L., Verwoord, R., Beery, T. A., Dalton, H., McKinnon, K., Strickland, K., Pace, J., and Poole, G. (2013). The Power of social networks: A model for weaving the Scholarship of Teaching and Learning into institutional culture. Teaching & Learning Inquiry, 1(2). 49- 62. Bernstein, D. (2013). How SoTL- active faculty members can be cosmopolitan assets to an institution. Teaching & Learning Inquiry, 1(1). 35- 40. REFERENCES Bernstein, D. (2013). How SoTL- active faculty members can be cosmopolitan assets to an institution. Teaching & Learning Inquiry, 1(1). 35- 40. Williams, A. L., Verwoord, R., Beery, T. A., Dalton, H., McKinnon, K., Strickland, K., Pace, J., and Poole, G. (2013). The Power of social networks: A model for weaving the Scholarship of Teaching and Learning into institutional culture. Teaching & Learning Inquiry, 1(2). 49- 62. 2 2 TEACHING & LEARNING INQUIRY, VOL. 3.1 2015
https://openalex.org/W2583231386	http://www.nomos-elibrary.de/10.5771/0010-3497-1989-1-2-76.pdf	German	null	Franz von Sales -Patron der Journalisten	Communicatio socialis	1,989	cc-by	5,448	Franz von Sales -Patron der Journalisten von Herber! Wink/ehner OSFS "Man behandelt manchmal die Schriftsteller zu hart. Man fällt sehr rasch ein strenges Urteil über sie und offenbart dabei selbst mehr an Taktlosigkeit, alsjene an Unklugheit, da sie ihre Schriften voreilig veröffentlichen. Dieses unüberlegte Urteilen· gefährdet schwer sowohl das Gewissen der Urteilenden als auch die Unschuld der Angeklagten. Manche schreiben Albernheiten, andere gefallen sich wieder in plumpem Tadel."' Dieses Plädoyer für oft zu Unrecht kritisierte Schriftsteller stammt aus der Feder des hl. Franz von Sales, des Patrons der katholischen Journalisten und Schriftsteller. Am 26. Jänner 1923 erließ Papst Pius XI. anläßlich des dreihundertsten Todestages des Heili· gendie Enzyklika "Rerum omnium". 2 In diesem Rundschreiben stellte der Papst den Bischofvon Genf den katholischen Journalisten und Schriftstellern als ihren Patron zur Seite. Der Papst ergriff die günstige Gelegenheit, "um aufgrund zuverlässiger Sachkenntnis und nach reiflicher Überlegungkraft Unserer apostolischen Gewalt durch dieses Rund· schreiben den heiligen Franz von Sales, Bischof von Genf und Kirchenlehrer, zum himmlischen Schutzpatron aller Redaktoren und Schriftsteller zu bestimmen bzw. zu bestätigen". 3 Seit 1923 gibt es also einen Patron für Journalisten. Dieser Patron heißt Franz von Sa- les, ein Heiliger, der von 1567 bis 1622, also vor rund vierhundert Jahren, lebte. Die Frage ist berechtigt: Wie kann ein Heiliger, der zu einer Zeit lebte, in dervon einem mo· dernen Journalismus noch nicht die Rede ist, einem heutigen Journalisten ein Patron sein? Ist Franz von Sales aktuell genug, um als Journalistenpatron anerkannt werden zu können? Zu diesen Fragen möchte ich zunächst zwei Thesen formulieren, die im Laufe des Bei· trages diskutiert werden sollen: I. These: Der hl. Franz von Sales ist als Patron der Journalisten heute noch aktuell. Er kann auch für heutige Anforderungen Orientierungshilfen für journalistische Tätigkeit geben, wenn versucht wird, sein Leben und Werk hermeneutisch ins Heute zu übertra- gen. 2. These: Der hl. Franz von Sales zählt als Patron der Journalisten zu einer verschütte· ten Tradition der Kirchengeschichte des 20. Jahrhunderts. Angesichts der wachsenden Bedeutung der Massenkommunikation sank die Bedeutung des Journalistenpatrons, weil der Heilige für die heutigen Anforderungen nach Meinung der Journalisten nicht mehr aktuell genug ist. Communicatio Socialis 22 (1989) 1: 76-85 Quelle: www.communicatio-socialis.de Communicatio Socialis 22 (1989) 1: 76-85 Quelle: www.communicatio-socialis.de Communicatio Socialis 22 (1989) 1: 76-85 Quelle: www.communicatio-socialis.de Communicatio Socialis 22 (1989) 1: 76-85 Quelle: www.communicatio-socialis.de 1. Journalistenpatronat des heiligen Franz von Sales - auch heute noch aktuell und bedeutsam Was bewog Pius XI. gerade, den hl. Franz von Sales als Journalistenpatron einzuset· zen? In Kürze geantwortet: Biographische Fakten und Qualitäten seines Lebens und Schreibens. Diese Gründe sollen nun näher ausgefaltet werden, wobei ich zugleich auch deren Bedeutung und Aktualität für den modernen Journalismus aufzuzeigen ver· suche. 76 / – - 1.1. Journalistische Berührungspunkte im Leben des hl. Franz von Sales 4 Der hl. Franz von Sales wurde am 21. August 156 7 auf Schloß Thorens in Savoyen ge- boren. Seine Kindheit wartrotz der Wirrnisse rund um die Reformation und Gegenre- formation nach dem Konzil von Trient ( 1545- 1563) vom tiefen katholischen Glauben seiner Eltern geprägt. Die Auseinandersetzung mit dem Calvinismus, dessen Hoch- burg zu dieser Zeit die Stadt Genf war, wird dennoch das Leben des hL Franz von Sales entscheidend prägen. Der hl. Franz von Sales wurde am 21. August 156 7 auf Schloß Thorens in Savoyen ge- boren. Seine Kindheit wartrotz der Wirrnisse rund um die Reformation und Gegenre- formation nach dem Konzil von Trient ( 1545- 1563) vom tiefen katholischen Glauben seiner Eltern geprägt. Die Auseinandersetzung mit dem Calvinismus, dessen Hoch- burg zu dieser Zeit die Stadt Genf war, wird dennoch das Leben des hL Franz von Sales entscheidend prägen. Nach dem Willen seines Vaters sollte Franz jedoch nichts mit diesen kirchlichen Pro- blemen zu tun bekommen. Dieser plante für seinen Sohn vielmehr eine große Karriere im öffentlichen Frankreich. Er schickte ihn daher 1579 zum Rechtsstudium nach Paris. Dort geriet der hl. Franz von Sales in eine tiefe persönliche Krise - er glaubte sich von Gott verstoßen. In einem bewußten Vertrauensakt an Gott konnte er dieses schmerzli- che Kapitel seines Lebens überwinden. Auf der Basis seiner Erfahrungen in dieser Kri- se reifte in ihm ein optimistisches Gottes- und Weltbild, geprägt durch die Liebe. Diese salesianische Gedankenwelt erhielt in einem seiner späteren Hauptwerke ~Abhand lung über die Gottesliebe"' ihre schriftliche Ausprägung. Schon in Paris und später in Padua studierte Franz von Sales nebenbei und ohne Wissen seines Vaters Theologie. Nach Abschluß dieser Studien entschied sich Franz entgegen dem Willen seines Vaters zum Priesterberuf. Am 18. Dezember 1593 wurde dieser Wunsch durch die Priesterweihe Wirklichkeit. Bereits nach einem Jahr erhielt Franz den Auftrag, das von den Calvinisten bewohnte Gebiet des Chablais zu missionieren. Diese Chablaismission gab den Anlaß für seine journalistische Tätigkeit. 1. Journalistenpatronat des heiligen Franz von Sales - auch heute noch aktuell und bedeutsam Das größte Problem seiner Mission war es nämlich, daß die politischen Kräfte den Be- wohnern des Chablais unter Strafe verboten hatten, die Predigten des hl. Franz von Sa- les auch nur anzuhören. Franz wurde von allen Seiten boykottiert. So bediente er sich des journalistischen Genres des Flugblattes und begann damit in Serie den katholi- schen Glauben darzulegen. An allen Haustüren und Marktplätzen schlug er Schriften an. Und er hatte Erfolg: Die Menschen waren von seinen Gedanken so fasziniert, daß sie im Laufe der Zeit immer häufiger das Verbot der Stadtherrn mißachteten und zu sei- nen Predigten gingen. Mit Hilfe des Flugblattes erreichte der Heilige die Rückkehr der Bewohner des Chablais zum katholischen Glauben" 1602 wird der hl. Franz von Sales Bischofvon Genf. Obwohl mit diesem Datum seine Aufgaben und Arbeiten um vieles zahlreicher wurden, blieb er einer Tätigkeit immer treu: Er schrieb Briefe. Zu diesen Briefen stellt Hubert Wetzler fest: ~Die Korrespon- denz des Heiligen ist nicht ein literarisches Produkt, wie wir es später in der klassischen Literatur als eine Art Kunstbestätigung finden, noch weniger waren es farblose 'Es geht mir gut'-Briefe, wie sie heute die Briefkästen füllen. Zum großen Teil waren es Briefe, wie wir sie heute in den Fragekästen unserer Zeitschriften finden; persönliche Briefe zwar,jedoch von allgemeinem Interesse; Briefe, die Antwort geben aufFragen und Pro- bleme des Lebens, Fragen höchst persönlicher und privater Art wie solche, die in das gesellschaftliche und selbst in das politische Leben tief eingreifen." 7 Das Schreibpotential des hl. Franz von Sales war immens. Während seines 55-jährigen Lebens soll er rund 21.000 Briefe verfaßt haben. Rund 2000 Briefe sind heute noch er- halten. Ein besonderes Produkt dieser Briefliteratur zeigt sich uns in der ~Philothea" oder ~Anleitung zum frommen Leben" . 8 Dieses Werk, das noch heute einen Bestseller der religiösen Literatur darstellt, ist nichts anderes als die Sammlung von Seelenfüh- 77 rungsbriefen. Der "Philothea" war von allem Anfang an ein durchschlagender Erfolg beschieden. Denn mit diesem Werk hatte der hl. Franz von Sales eine Lücke im Zeitbe- dürfnis der Gesellschaft geschlossen. Im Jahre 1610 gründete der Bischofvon Genfzusammen mit der hl. Johanna Franziska von Chantal ( 1572- 1641) den Orden der Heimsuchung Mariens. Ständig um die Sor- gen und Probleme seiner Diözese bemüht und erfüllt mit Aufgaben rund um seine Or- densgründung, konnte er kaum Zeit der Ruhe finden. Die Wahrheitsliebe Der hl. Franz von Sales wurde 1877 zum Kirchenlehrer erhoben. Die Kirche brachte damit zum Ausdruck, daß der Bischofvon Genf ein Lehrer der Wahrheit ist. Er selbst schreibt: "Der Gegenstand unseres Erkenntnisvermögens (ist) die Wahrheit. Seine ganze Freude und Befriedigung besteht darin, die Wahrheit über die Dinge zu entdek- ken und zu erkennen; und je erhabener die Wahrheiten sind, desto freudiger und auf- merksamer gibt sich unser Verstand ihrer Betrachtung hin." 10 Interessant für Journali- sten ist in diesem Zusammenhang eine Aussage in einem Briefdes hl. Franz von Sales an Papst Clemens VIII., in dem er seine Berichterstattung über die Situation seiner Diözese damit rechtfertigt, daß die Wahrheit nicht verdreht werden soll: "Deshalb ist es auch so wichtig," schreibt er, "ihm (dem Papst) [Anm.d.Verf.] einen treuen und gewis· senhaften Bericht über die Ereignisse zu erstatten, welche die Kirche in jedem Land be- treffen. Sonst kann man bei Darlegungen von Tatsachen etwas für wahr erklären, was falsch ist, und für falsch, was wahr ist." 11 Gerade mit dieser Eigenschaft und diesen Forderungen in Bezug auf die Wahrheit, spricht der Journalistenpatron ein akutes Pro- blem 'der heutigen Zeit an: Die Wahrheitstindung entpuppt sich in unserer plu- ralistischen Gesellschaft als ein oft sehr schwieriges, ja fast unmögliches U nterfangen. 12 1. Journalistenpatronat des heiligen Franz von Sales - auch heute noch aktuell und bedeutsam Gegen Ende seines Lebens ent- stand daher in ihm der Wunsch, sich in eine Einsiedelei in den Bergen rund um den See von Annecy zurückzuziehen. Dieser Traum, der aus dem für Journalisten wohl bekann- ten Zeit- und Termindruck und der ständigen Arbeitsüberlastung entsprang, blieb je- doch durch den frühen Tod am 28. Dezember 1622 unerfiillt. Die katholische Kirche würdigte Leben und Werk des Bischofs von Genf durch die Se- ligsprechung 1661, Heiligsprechung 1665, die Ernennung zum Kirchenlehrer 1877 und zum Patron der Journalisten und Schriftsteller 1923. Allein dieser kurze Lebensabriß zeigt uns, daß Franz von Sales sehr wohl mitjournali- stischer Tätigkeit in Berührung kam. In gewissem Sinne könnte man ihn sogar als Pio- nier massenmedialer Kommunikation bezeichnen. 9 Ihn allerdings nach heutigen Maß- stäben als Journalisten zu bezeichnen, wäre sicher ein hermeneutischer Affront und ein Anachronismus. Trotzdem waren diese Berührungspunkte Grund genug, ihn als Pa- tron den Journalisten zur Seite zu stellen. 1.2. Pressequalitäten des hl. Franz von Sales In der Begründung Pius' XI. lassen sich sechs Aspekte herausfiltern, die ihn zur Wahl des hl. Franz von Sales als Journalistenpatron bewogen. Gerade an diesen sechs Aspekten läßt sich besonders gut die Bedeutung des Heiligen für heute aufzeigen. Überzeugungskraft durch zeitgerechte Leserorientierung Überzeugungskraft durch zeitgerechte Leserorientierung "Natürlich berücksichtige ich die Geistesverfassung unserer Zeit. Ich mußte es tun; es ist sehr wichtig zu wissen, in welcher Zeit man schreibt", meint Franz von Sales im Vor· wortseines Buches "Abhandlung über die Gottesliebe". 13 Ein Geheimnis seines großen schriftstellerischen Erfolges war die Leserorientierung. Dieses Motiv veranlaßte ihn, seine Flugschriften in der Landessprache zu schreiben, was damals im katholischen 78 Raum, in dem Latein als die Sprache der Kirche galt, unüblich war. Zudem verstand Franz es ausgezeichnet, schwierigste theologische Inhalte lesergerecht darzulegen, oh- ne Abstriche an der Wahrheit vornehmen zu müssen. Rezipientengerechtes Schreiben isteine der Hauptaufgaben desJournalisten. 14 Der Journalist sollte den Leser immervor Augen haben und fahig sein, die schwierigsten und komplexesten Zusammenhänge in Politik und Gesellschaft klar und deutlich zu formulieren. Auch hier ist der hl. Franz von Sales als Vorbild aktueller denn je. Objektivität Der hl. Franz von Sales wollte in der Zeit der Gegenreformation und kirchlichen Er- neuerung den rechten katholischen Glauben verkünden. Seine Schriften sind also klar von einem Interesse geleitet: von Gott und der wahren Wiedergabe der geoffenbarten göttlichen Heilsbotschaft. Dieses Interesse widersprichtjedoch keinesfalls dem Begriff Objektivität. Objektiv sein heißt ja nicht, vonjedem Wert abzusehen und so über etwas zu berichten, als ob es einen nichts anginge. Franz von Sales machte zudem aus diesem Interesse kein Hehl. Immer wieder bekannte er offen, daß er nichts anderes schreiben wolle als der Lehre der Kirche Entsprechendes. Somit hat er auch nie versucht, seine Zuhörer mit irgendwelchen Verschleierungen zu manipulieren. Die durch den Para- graphen 25 des Österreichischen Mediengesetzes geforderte ~Offenlegung", in der vor- geschrieben wird, die grundlegende Richtung einer periodischen Druckschrift abzu- geben, war für den hl. Franz von Sales selbstverständlich. Er war aber auch auf eine an- dere Weise objektiv: Er wirkte jeder Gefahr der einseitigen Betrachtung eines Themas entgegegen, indem er sich immer wieder über die Meinung seiner Gegner informierte. So bat er seinen Papst um die Erlaubnis, zur besseren Beurteilung der Reformatoren de- ren Bücher lesen zu dürfen, die damals natürlich alle im Index der verbotenen Bücher aufgelistet waren. Die Einseitigkeit der Berichterstattung gilt als einer der Hauptkritik- punkteam modernen Journalismus. 15 Auch in diesem Punkt könnte der hl. Franz von Sales den Journalisten Vorbild sein. Achtung der Personenwürde Dem hl. Franz von Sales ging es in seinen Kontroversschriften, Streitgesprächen oder Predigten, die in seinem Umfeld von reformatorischer wie katholischer Seite ziemlich hitzig und heftig ausfielen, nicht darum, seine Gegner zu erniedrigen. Sein Ziel war das Aufzeigen der Wahrheit und die Bekämpfung jedes Irrtums. Er handelte nach dem Grundsatz: ~Man muß wohl über das Schlechte empört und fest entschlossen sein, sich niemals darauf einzulassen; dennoch muß man dem Nächsten gegenüber ganz mild bleiben. " 16 Von katholischer Seite erntete er manchen Argwohn, da er die Reformato- ren in der Zeit härtester Auseinandersetzungen ~seine Brüder" nannte. 17 Er achtete im- mer die Intimsphäre seiner Gegner und die Würde der Person. Heute, in einer Zeit, in der immer häufiger Menschen durch Medien .,vorverurteilt" werden, in der immer häu- figer Menschen durch die Sensationslust der Medien persönlich diffamiert werden, in der Journalisten immer weniger vor Privatsphäre und Würde der menschlichen Person zurückschrecken, wäre eine Besinnung auf den Journalistenpatron dringend ange- bracht. Bildung und Genauigkeit Bildung und Genauigkeit .Recherchieren, auswählen und aufarbeiten (bearbeiten) sowie präsentieren bzw. publizieren gehören zu den wichtigstenjournalistischen Tätigkeiten in Zeitung, Radio und Fernsehen. " 18 Täglich erreichen den Journalisten Informationen, die er in kürze- ster Zeit nachprüfen, sortieren und verwerten muß. Für eine korrekte und genaue Dar- stellung ist eine hinreichende Faktensammlung unabdingbar notwendig. Die Recher- 79 5 /00 0 3 9 – - ehe wird daher als "das Um und Auf jeder journalistischen Tätigkeit" angesehen. 19 Eine möglichst solide Allgemeinbildung bzw. eine fundierte Spezialbildung fiir Journali- sten, die fiir bestimmte Ressorts festangestellt sind, sowie ein theoretisches Wissen über seinen Beruf erleichtern nicht nur die Arbeit des Artikelschreibens, sondern fördern auch die Qualität der Presseprodukte. Hinsichtlich dieser Problematik kann der hl. Franz von Sales ebenso Vorbild sein. Seine Methode der Informationsbeschaffung trug zwar noch nicht die heute üblichen Namen "Check", "Gegencheck" und "Recheck", lief aber nach einem ähnlichen Schema ab. Seine Schriften erstaunen durch Genauig- keit und Bildung. Die umfassende Darstellung eines Sachverhaltes war ihm ein be- sonderes Anliegen. Franz von Sales verwendete dazu eine Methode, die auch heute noch Gang und Gebe ist. Dem modernen Journalisten wird eine derartige Methode un- ter dem Schlagwort "Die sechs W's" dargelegt. Der erste Absatz einer klassischen Nachricht sollte diese sechs W's- die Fragen Wer, Was, Wo, Wann, Wie und Warum- fiir den Rezipienten beantworten. Franz von Sales rät den Priestern seiner Diözese die- selbe Methode fiir die Gestaltung des Aufbaus ihrer Predigten. Wenn sie predigen, sol- len sie "folgende Punkte erwägen: wer? warum? wie?" .20 Wenn man bedenkt, wieviel Schaden heute durch Oberflächlichkeit in der Berichterstattung angerichtet wird, kann der hl. Franz von Sales zum Mahner fiir soliden Recherchejournalismus werden. Ein Klassiker des Schreibstils Der Bischof von Genf wird heute in der Literaturgeschichte in der Reihe der großen französischen Prosaisten des 16. Jahrhunderts gestellt und zählt "zu den Klassikern der französischen Sprache". 22 Trotz eines schweren Tagesprogrammes und einer ständigen Arbeitsüberlastung brachte es der hl. Franz von Sales zu diesen literarischen Ehren. Wie aktuell er als Vorbild fiir den Journalismus gerade unter diesem Aspekt ist, zeigt sich an einer Stilkritik von Heinz Pürer: "Viele über Zeitung, Radio und Fernsehen ver- mittelte Botschaften erreichen zwar die Öffentlichkeit, kommen beim Leser, Hörer oder Seher jedoch nicht oder nur teilweise an - werden nicht verstanden und erzielen daher auch keine Wirkung. Schuld daran sind oft sprachliche Mängel, die der Verständ- lichkeit entgegenwirken. -1989-1-2-76, am 24.10.2024, 05:57:26 ttps://www.nomos-elibrary.de/agb Bildung und Genauigkeit Mitunter mangelt es auch beträchtlich an den Kenntnissen der Grammatik und der Wortbedeutungslehre (Semantik). Der Zeit- und Termin- druck, unter dem Journalisten täglich, ja stündlich arbeiten, mag durchaus ein Grund fiir sprachliche Mängel im Journalismus sein, er sollte es aber nicht. "23 Zeitdruck 2. Das Journalistenpatronat des heiligen Franz von Sales- eine verschüttete Tradition der Kirchengeschichte des 20. Jahrhunderts Was bewirkte nun das Patronat des hl. Franz von Sales?Welche Auswirkungen hatte es auf das Pressewesen, auf Aussagen der Kirche zu den Massenmedien? Gab es über- haupt irgendwelche Wirkungen oder wurde der hl. Franz von Sales als Patron der Jour- nalisten vergessen? Nach der Durchsicht publizistisch-relevanter Aussagen der Päpste seit PiusXI. und der Wiener Erzbischöfe Kardinal Franz König und Hans Hermann Groer, sowie nach der Forschung in zwei katholischen Verlagen (Don-Bosco-Verlag; Franz-Sales-Verlag), der Katholischen Presseagentur kathpress, der Wochenzeitung .Die Furche", der • Wien er Kirchenzeitung" und der Arbeitsgemeinschaft Katholischer Journalisten Österreichs kam ich zu folgenden Ergebnissen25: -Die Ernennung des hl. Franz von Sales zum Patron der Journalisten hat im 20. Jahr- hundert seine Wirkung gezeigt, wenn auch von Jahr zu Jahr und von Institution zu Insti- tution in unterschiedlicher Intensität. -Die Ernennung des hl. Franz von Sales zum Patron der Journalisten hat im 20. Jahr- hundert seine Wirkung gezeigt, wenn auch von Jahr zu Jahr und von Institution zu Insti- tution in unterschiedlicher Intensität. -Diese Wirkung zeigte sich weniger im Einfluß des hl. Franz von Sales auf die Entwick- Iungen im Pressewesen, vor allem nicht auf ethische Bereiche, sondern vielmehr im Sinne einer Schutz- und Bestätigungsfunktionjener Journalisten, die sich als katholisch bezeichnen. - Dies läßt sich vor allem durch das Faktum erkennen, daß der hl. Franz von Sales nur dann als Patron der Journalisten Erwähnung fand, wenn der Anlaß dafiir der Heilige selbst war. Nicht aber wurde auf den Patron verwiesen, wenn es allein um das Pressewe- sen ging. - Es kann daher festgestellt werden, daß die Reichhaltigkeit und Spiritualität des hl. Franz von Sales fiir Journalisten zu wenig ausgeschöpft wurde. Es fehlt auch an Richtlinien- wie etwa an einem Ehrenkodex fiir Journalisten-, die speziell aus den Schriften des hl. Franz von Sales herausgefiltert worden wären. - Es kann daher festgestellt werden, daß die Reichhaltigkeit und Spiritualität des hl. Franz von Sales fiir Journalisten zu wenig ausgeschöpft wurde. Es fehlt auch an Richtlinien- wie etwa an einem Ehrenkodex fiir Journalisten-, die speziell aus den Schriften des hl. Franz von Sales herausgefiltert worden wären. - Der hl. Franz von Sales wurde zudem nicht immer mit gleicher Intensität als Patron der Journalisten ins Blickfeld gerückt. Es läßt sich ein Höhepunkt in den Sechzigerjah- ren feststellen. Ausschlaggebend dafiir war der 400. Geburtstag des Heiligen im Jahre 1967. Zeitdruck Unter dem Aspekt, der von Pius XI. nicht angefiihrt wird, ist Franz von Sales weniger ein Vorbild als ein Mit -leidender, als einer, der mit den gleichen Problemen zu kämpfen hatte, nämlich unter dem Aspekt des Zeitdruckes. Der hl. Franz von Sales steht in die- sem Punkt den streBgeplagten Journalisten in nichts nach. Der Patron der Journalisten wird so auch der Patron vieler ruheloser und überlasteter Menschen unserer Tage, er wird zum Patron der großstädtischen Streß-Gesellschaft, die ih ihrem Trubel unterzu- gehen droht. In einigen seiner Briefe24 zeigen sich sogar Ansätze einer möglichen StreB- therapie, allerdings aufreligiöser Basis: Ruhe finden in Gott, Gottvertrauen, demütiges Annehmen der Situation- das sind christliche Parallelen fiir autogenes Training, Medi- tation, usw. Die praktische Anweisung des hl. Franz von Sales lautet: Erledigen Sie ein Problem nach dem anderen, damit Sie nicht durcheinander kommen und das unter Ge- brauch des Geistes. Die heutige Welt spricht von Koordination und Konzentration. Nach dieser genaueren Darstellung der Gründe, die Pius XI. veranlaßten, den hl. Franz von Sales zum Patron der Journalisten zu erwählen, wurde hoffentlich deutlich, daß dies nicht zu unrecht erfolgte. Im folgenden möchte ich nun auf die Wirkgeschichte der Patronatsernennung eingehen. 80 2. Das Journalistenpatronat des heiligen Franz von Sales- eine verschüttete Tradition der Kirchengeschichte des 20. Jahrhunderts 2. Das Journalistenpatronat des heiligen Franz von Sales- eine verschüttete Tradition der Kirchengeschichte des 20. Jahrhunderts Wichtig ist es zu sehen, daß der Journalistenpatron injüngster Zeit eher selten in Erscheinung tritt, daß aber wieder eine ansteigende Tendenz festzustellen ist. Dies hängt wahrscheinlich mit der Tatsache zusammen, daß in den letzten Jahren seitens der Kongregation der Oblaten des hl. Franz von Sales verstärkt versucht wird, Kontakte mit Journalisten aufzubauen. -Bedeutsam ist ferner, daß im deutschsprachigen Raum durch den Franz-Sales-Verlag eine Institution existiert, die ihre Existenzberechtigung unter anderem vom Patronat des hl. Franz von Sales herleitet. - Als besondere Früchte des Journalistenpatronats des hl. Franz von Sales gelten die jährlich in verschiedenen Ländern abgehaltenen Journalistenmessen anläßtich des Ge- denktages des hl. Franz von Sales am 24. Jänner und das Franz-von-Sales-Essen, das von der Arbeitsgemeinschaft Katholischer Journalisten in Wien jährlich organisiert ·wird. - Im großen und ganzen kann der Schluß gezogen werden, daß das Journalistenpa- tronat des hl. Franz von Sales zu einer halbverschütteten Tradition in der Kirchenge- SI SI 5771/0010 349 – - schichte des 20. Jahrhunderts gehört. Halbverschüttet deshalb, weil die Wirkung der Patronatsernennung auf einer Ebene blieb, auf der die existentiellen Probleme des Jour· nalisten nicht berührt wurden. Es scheint so, als ob im Bewußtsein der Journalisten der hl. Franz von Sales fiir ihre Probleme fiir nicht kompetent genug gehalten wird. Da dieses Phänomen festzustellen ist, und da es bisher noch keinen erarbeiteten Kata· log von Orientierungskriterien mit salesianischen Aspekten gibt, möchte ich hier einen entsprechenden Versuch vorstellen. Der folgende salesianische Ehrenkodex soll die spezifischen Elemente eines salesianisch-geprägten Journalismus deutlich machen und jeden Journalisten dazu anregen, sich intensiver mit seinem Patron auseinanderzuset· zen. 3. Salesianischer Ehrenkodex für Journalisten Franz von Sales war bemüht, täglich :Zwei Stunden für seine Weiterbildung aufzuwenden, seine Schriften sind geprägt von minutiöser Ge- nauigkeit. (9) Trotz permanentem Zeit- und Termindruck soll sich der Journalist um einen guten sprachlichen Stil und um eine solide Kenntnis der Grammatik und Semantik bemühen. Franz von Sales, der ebenfalls unter akutem Zeitmangel litt, gilt heute als Klassiker der französischen Sprache. Vergleicht man diesen Salesianischen Ehrenkodex für Journalisten mit anderen heuti- gen Kodices, wie etwa dem Ehrencodex des Österreichischen Presserates oder den "Zehn Geboten für Journalisten" von Günther Virt30,läßt sich erkennen, daß dieser ein durchaus brauchbares Werkzeug für eine Bewertung des journalistischen Handeins ist.3' Der Journalist selbst erhält eine viel tiefere Beziehung zu seinem Patron, als dies bisher der Fall war, da er erkennt, daß dieser ein journalistisches Verständnis an den Tag legte, das modernen Anforderungen durchaus gerecht wird. Den hl. Franz von Sales wieder mehr ins Bewußtsein der Journalisten zu rücken, istAn- liegen dieses Artikels. Das bedeutet nicht, daß der Weg des hl. Franz von Sales der ein- zig wahre und richtige Weg für Journalisten ist. Keineswegs. Mir geht es nur darum, zu zeigen, daß der Patron der Journalisten immer noch- also auch heute noch ein Weg ist, den Journalisten gehen können, um ihren Beruf verantwortungsvoll auszuüben. Leider ist aber gerade dieser Weg in den letzten Jahren etwas in Vergessenheit geraten, ver- schüttet und überwuchert worden. Ich hoffe, daß meine Arbeit dazu beitragen kann, diesen Weg etwas zu reinigen, damit er heute wieder ohne größere Anstrengung be- gehbar ist. Für diesen Weg möchte ich allen Journalisten ein Wort des hl. Franz von Sa- les mitgeben: "Mach es wie die kleinen Kinder: Mit der einen Hand halten sie sich am Vater fest, mit der anderen pflücken sie Erdbeeren oder Brombeeren am Wegrain. So sammle und ge- brauche auch du die irdischen Güter mit der einen Hand, mit der anderen halte dich an der Hand des himmlischen Vaters fest. Schau immer wieder zu ihm auf, ob ihm dein Tun und dein Wandel recht ist. Hüte dich vor allem, seine Hand loszulassen und dich seiner Obhut zu entziehen, in der Meinung, du könntest dann mehr zusammenraffen. Hält er dich nicht mehr, dann wirst du keinen Schritt tun, ohne hinzufallen. Hast du nur gewöhnliche Beschäftigungen, die keine gesammelte Aufmerksamkeit verlangen, dann schau mehr auf Gott als auf deine Arbeit. 3. Salesianischer Ehrenkodex für Journalisten Der nun angefiihrte Orientierungskatalog soll keine Konkurrenz zu schon bestehenden darstellen, und schon gar nicht fiir nichtig erklären bzw. ersetzen. Vielmehr soll dieser Salesianische Ehrenkodex fiir Journalisten schon bestehende Kodices mit salesiani· sehen Komponenten ergänzen. ( 1) Oberstes Prinzip jeder journalistischen Tätigkeit ist die Liebe zur Wahrheit. Franz von Sales sagt: "Leben Sie nach den Wahrheiten, die der Glaube uns lehrt, und pflegen Sie die kostbare Gabe, die Sie so sehr zu Ihrem Vorteil empfangen haben. "26 ( 2) Die Strategie des Journalisten, der Wahrheit zu ihrem Sieg zu verhelfen, ist die Me· thode der Liebe, Sanftmut und des Friedens. Franz von Sales sagt: "Wie wunderbar ge· lingt es einem doch, Herzen zu gewinnen und mitzureißen, wenn man eine gute Sache geschickt und liebenswürdig vertritt. "27 (3) Zur journalistischen Tätigkeit gehört die Berücksichtigung der Geistesverfassung der jeweiligen Zeit. Franz von Sales sagt: "Natürlich berücksichtige ich die Geistesver· fassungunserer Zeit. Ich mußte es tun; es ist sehr wichtig zu wissen, in welcher Zeit man schreibt." 28 ( 4) Zur journalistischen Tätigkeit gehört ein leserorientiertes Schreibverhalten, ohne dadurch die Wahrheit zu beeinträchtigen. Franz von Sales verstand es, schwierigste Themen dem einfachen Volk klar und deutlich darzulegen. (5) Objektive Berichterstattung eines Journalisten bedeutet nicht einfach wertneutrale Berichterstattung. Der Journalist soll allerdings seine zugrunde liegenden Wertvorstel· Iungen offenlegen, um der Gefahr ungerechtfertigter Meinungsmanipulation zu ent· gehen. Franz von Sales hat nie ein Hehl daraus gemacht, daß er in seinen Schriften die Lehre der Katholischen Kirche darlegen und verteidigen will. ( 6) Zur objektiven Berichterstattung eines Journalisten gehört das Ziel, jeder Gefahr der einseitigen Betrachtung eines Themas entgegenzuwirken. Franz von Sales war be· müht, auch die Schriften seiner Gegner zu kennen, um bei sichjede Einseitigkeit zu ver· meiden. (7) Der Journalist soll durch seine Arbeit Informationen und Sachverhalte deutlich darstellen und klären, ohne aber dadurch betroffene Menschen zu ruinieren. Franz von Sales sagt: "Man muß wohl über das Schlechte empört und fest entschlossen sein, sich niemals darauf einzulassen; dennoch muß man den Nächsten gegenüber ganz mild bleiben. "29 ( 8) Genauigkeit bei der Recherche und eine umgreifende und zumindest ressortspezi· fisch tiefgreifende Bildung sind unumgängliche Qualitäten einer seriösen und qualifi· 82 zierten journalistischen Tätigkeit. Franz von Sales war bemüht, täglich :Zwei Stunden für seine Weiterbildung aufzuwenden, seine Schriften sind geprägt von minutiöser Ge- nauigkeit. zierten journalistischen Tätigkeit. I Deutsche Ausgabe der Werke des hl. Franz von Sales (DA) Bd.3, S.38 Ü 3. Salesianischer Ehrenkodex für Journalisten Hast du aber eine Arbeit, die deine ganze Auf- merksamkeit beansprucht, dann blicke wenigstens von Zeit zu Zeit zu Gott auf, gleich dem Seemann auf offenem Meer: um seine Richtung einzuhalten, schaut er mehr auf den Himmel als auf das Wasser, auf dem er dahinfahrt. So wird Gott mit dir, in dir und für dich arbeiten, und deine Arbeit wird dir Freude bereiten. " 32 Anmerkungen I Deutsche Ausgabe der Werke des hl. Franz von Sales (DA) Bd.3, S.38 2 AAS XV ( 1923), 49-63. Eine deutsche Übersetzung findet sich im Sammelband: Heilslehre der Kirche. Dokumente von Pius IX. bis Pius XII. Deutsche Ausgabe des französischen Ori- ginals von P. Cattin; H.Th. Conus, hgg. v. Anton Rohrbasser, Fribourg 1953 3 Heilslehre der Kirche, 1227. Der Papst schreibt hier von "bestätigen", da bereits Pius IX. 1877 Franz von Sales als Journalistenpatron proklamierte; ein Faktum, das allerdings völlig in Vergessenheit geriet. 4 Als Grundlage fiir diesen biographischen Grundriß diente: Etienne-Jean Lajeunie: Franz von Sales, Leben-Lehre-Werk, Eichstätt 1980, 2. Aufl. 5 DA I I Deutsche Ausgabe der Werke des hl. Franz von Sales (DA) Bd.3, S.38 2 AAS XV ( 1923), 49-63. Eine deutsche Übersetzung findet sich im Sammelband: Heilslehre der Kirche. Dokumente von Pius IX. bis Pius XII. Deutsche Ausgabe des französischen Ori- ginals von P. Cattin; H.Th. Conus, hgg. v. Anton Rohrbasser, Fribourg 1953 3 Heilslehre der Kirche, 1227. Der Papst schreibt hier von "bestätigen", da bereits Pius IX. 1877 Franz von Sales als Journalistenpatron proklamierte; ein Faktum, das allerdings völlig in Vergessenheit geriet. 4 Als Grundlage fiir diesen biographischen Grundriß diente: Etienne-Jean Lajeunie: Franz von Sales, Leben-Lehre-Werk, Eichstätt 1980, 2. Aufl. 5 DA I 4 Als Grundlage fiir diesen biographischen Grundriß diente: Etienne-Jean Lajeunie: Franz von Sales, Leben-Lehre-Werk, Eichstätt 1980, 2. Aufl. 5 DA I 83 6 Eine Sammlung dieser Flugblätter findet sich in: DA 10 u. 11. 7 Hubert Wetzler: Der Patron der Journalisten, in: Jahrbuch fiir salesianische Studien (JSS) I (1963), s. 57-90 8 DA 1 9 Vgl. Herbert Winklehner: Priester als Presse-Pioniere, in: Multimedia. 1987 Nr. 22, S. 3 10 DA 3, S. 185 11 DA 8 S 89 9 Vgl. Herbert Winklehner: Priester als Presse-Pioniere, in: Multimedia. 1987 Nr. 22, S. 3 10 DA 3 S 185 10 DA 3, S. 185 11 DA 8, S. 89 11 DA 8, S. 89 12 Vgl. etwa Bernd, Engelmann; Alfred Horne; Stephan Lohr u.a.: Anspruch auf Wahrheit. 3. Salesianischer Ehrenkodex für Journalisten Wie werden wir richtig informiert? Göttingen 1981 13 DA 3 S 39 13 DA 3, S. 39 , 14 Vgl. Heinz Pürer: Sprache im Journalismus, in: Praktischer Journalismus in Zeitung, Radio und Fernsehen, Salzburg 1985 2. Aufl., S. 219- 262 15 Vgl. Holger Rust: Entfremdete Elite? Journalisten im Kreuzfeuer der Kritik, Wien 1986, S. 13 -57 16 DA 5, S. 391. , 17 Diese Anrede gebrauchte der hl. Pranz von Sa1es vor allem in seinen apologetischen Predig· ten. Vgl. DA 10, S. 281 - 335 18 Praktischer Journalismus, a.a.O. S. 21 , 19 Vgl. Georg Weiland: Journalistische Tätigkeit in der Zeitung, in: Praktischer Journalismus, a.a.O. S. 21 - 23 20 DA 12, S. 42 20 DA 12, S. 42 21 Meyers Handbuch über die Literatur, Mannheim 1964, S. 355 Vgl. dazu auch: Gisbert Kranz: Lexikon der christlichen Weltliteratur, Freiburg i. Br. 1978, S.838 y , , Vgl. dazu auch: Gisbert Kranz: Lexikon der christlichen Weltliteratur, Freiburg i. Br. 1978, S.838 22 Anton Nobis: Vierzig Jahre .Rerum omnium", in: JSS 1 (1963), S. 15 23 Heinz Pürer: Sprache im Journalismus, a.a.O., S. 219. 24 Vgl. DA 5, S. 145; S. 229; 1, S. 134 25 Eine ausfiihrliche Darlegung der Wirkgeschichte des Patronats in den genannten Medien und Institutionen findet sich in: Herbert Winklehner: Pranz von Sales, der Patron der Joumali· sten, und seine Anregungen fiir eine heutige Journalistenpastoral, Wien 1987, (Dip1omar· beit) s. 84- 186 26 DA 7, S. 100 27 DA 1, S. l72f 28 DA 3, S. 39 29 DA 5, S. 391 30 Vgl. Günter Virt: Ethische Normierung im Bereich der Medien, in: Handbuch der christli· eben Ethik, Bd. 3: Wege ethischer Praxis. Freiburg i. Br. 1982, S. 551 - 555 31 Ein genauer Vergleich wurde durchgefiihrt in: Herbert Winklehner: Pranz von Sales, der Pa· tron der Journalisten, a.a.O., S. 187 - 206 31 Ein genauer Vergleich wurde durchgefiihrt in tron der Journalisten, a.a.O., S. 187 - 206 Resurne Depuis 1923 saint Fran~ois de Sales, Eveque de Geneve, est Je patron desjournalistes et ecrivains catholiques. Uneanalyse de l'influence de ce patronage dans differents media et institutions a re- vele que ce patronage fait partie d'une tradition presque ensevelie de l'histoire de J'Eglise du XXeme siecle, bien que !es mass-media aientgagne toujours plus d'importance. Cela est du au fait que- a J'avis desjournalistes- Fran~ois de Sales, un saint du XVIlerne siecle, n'est plus "actuel" en ce qui concerne !es problemes contemporains de Ja communication de masses. Cet article es- saie donc de relever et actualiser de nouveau !es raisons qui ont amene Pape Pius XI a faire saint Fran~ois de Sales Je patron des journalistes. II s'avere que l'Eveque de Geneve peut donner aux journalistes des aides d'orientation meme aujourd'hui, ce qui est souligne par l'elaboration d'un code d'honeur de caractere salesien pour journalistes. Summary Since 1923 St. Francis de Sales, Bisbop ofGeneva, has been patron ofCatholic journalists and au· thors. An investigation into the etfectiveness oftbis patronage in various media and institutions has shown that it is a badly-shaken tradition oftwentieth-century church history, even though the mass media have constantly been growing in significance. The explanation is that Francis de Sa· !es, a seventeenth century saint, is no Ionger considered by journalists tobe relevant for the pro- blems ofmass communication nowadays. The present paper sets out, therefore, to present anew and to reinterpret for today those arguments and considerations which led Pope Pius XI to choo· se Francis de Sales as patron ofjournalists. It emerges that the Bisbop ofGeneva can even today be ofassisstance to journalists by indicating objectives and directions. Thus a code ofhonour for journalists has been drawn up, based on the Salesian spirit. 84 5 /00 0 3 9 – - Resurnen San Francisco de Sales, Obispo de Ginebra, es el patronodelos periodistas y escritores cat61icos desde 1923. De una investigaci6n de Ia historia de actividades de este patronato en varios medios e instituciones resulto que este patronato es parte de una tradici6n medio sepultada de Ja historia eclesiastica del siglo XX, aunque los medios de masas obtuvieran cada vez mas importancia. Este hecho se debe a que San Francisco de Sales, un santo del siglo XVII, en Ja opini6n de los periodi- stas ya no tiene nada que ver con los tiempos modernos. Por eso, este arttculo trata de mostrar nuevamente y actualizar los motivos que llevaron a Papa Pius XI a nombrar a San Francisco de Sales patronodelos periodistas. Viene a ser evidente que el Obispo de Ginebra, todavia hoy, ofre- ce ayuda de orientaci6n a periodistas. Esto es ilustrado por Ja configuraci6n de un c6digo de ho- nor con caracter salesiano. 85 97-1989-1-2-76, am 24.10.2024, 05:57:26 - https://www.nomos-elibrary.de/agb 5771/0010 –
https://openalex.org/W3184451604	http://jurnal.untag-sby.ac.id/index.php/EL-SAINS/article/download/1858/pdf	Indonesian	null	Rancang Bangun Alat Penjernih Air Berbasis PLC	El Sains/Elsains	2,019	cc-by-sa	1,345	ABSTRAK Pada umumnya air adalah sumber kebutuhan untuk manusia, air juga merupakan salah satu komponen lingkungan yang sangat penting dalam kehidupan. Maka dari itu pengelolaan air perlu dilakukan agar terjadi keseimbangan dalam kebutuhan manusia dan juga ketersediaan air di alam. Untuk itu kami mengaplikasikan Alat Penjernih Air Berbasis PLC kami untuk membantu mengurangi air keruh dan menfaatkannya untuk kebutuhan sehari-hari.Dengan adanya Alat Penjernih Air Berbasis PLC ini di harapkan dapat mengurangi efisiensi terhadap air bersih. Kontruksi atau konsep alat kami memanfaatkan proses sirkulasi secara bertahap dan melewati berbagai proses untuk menghasilkan air bersih. Berawal dari air keruh (raw water) yang didorong menuju proses Koagulasi, lalu di aduk dan di dorong oleh mixer sehingga air terdorong menuju proses Sedimentasi, di proses Sedimentasi air perlahan naik dan menuju ke proses Filtrasi. Dan hasil dari proses Filtrasi ini yang nantinya akan di manfaatkaan sebagai air bersih. vital bagi mahluk hidup diantaranya sebagai air minum atau keperluan rumah tangga lainnya. Oleh karena itu sistem kerja alat penjernih air ini menggunakan sistem pengolahan air limbah dengan Water Treatment Plant yang difungsikan untuk mengolah air dari kualitas air baku (influent) yang kuarang bagus agar mendapatkan kualitas air pengolahan (effluent) standart yang diinginkan,dan siap untuk kebutuhan sehari-sehari. Rancang Bangun Alat Penjernih Air Berbasis PLC Arman Aprilliana1, Agung Prasetyo2, Gatut Budiono3 Jurusan Teknik Elektro, Universitas 17 Agustus 1945 Surabaya Jl. Semolowaru 45 Surabaya 60118 Telp./Fax: 031-5921516 E-mail:Arman_Aprilliana@gmail.com E-mail:Agung_prasetyo@gmail.com E-mail:Gatut_budiono@untag-sby.ac.id Arman Aprilliana1, Agung Prasetyo2, Gatut Budiono3 Jurusan Teknik Elektro, Universitas 17 Agustus 1945 Surabaya Jl. Semolowaru 45 Surabaya 60118 Telp./Fax: 031-5921516 E-mail:Arman_Aprilliana@gmail.com E-mail:Agung_prasetyo@gmail.com E-mail:Gatut_budiono@untag-sby.ac.id Pendahuluan vital bagi mahluk hidup diantaranya sebagai air minum atau keperluan rumah tangga lainnya. Air merupakan salah satu komponen lingkungan yang mempunyai peranan yang cukup besar dalam kehidupan.Bagi manusia air berperan dalam kegiatan pertanian,industri dan pemenuhan kebutuhan rumah tangga.Air yang digunakan harus memenuhi syarat dari segikualitas maupun kuantitasnya.Kualitas dapat ditinjau dari segifisik,kimia,dan biologi,Kualitas air yang baik tidak selamanya di alam.Perkembangan industry dan permukiman dapat mengancam kelestarian air bersih. A.Perancangan Umum Sistem Secara umum terdiri dari dalam 2 sistem yaitu dengan system software dan system hardware.Pada rancangan bangun ini juga dilengkapi PLC sebagai control pengendali dalam system pengolahan air . Metode Peneletian Tujuan dari semua proses pengolahan air yang adaadalah menghilangkan kontaminan dalam air,atau mengurangi konsentrasi kontaminan tersebut sehingga air yang di inginkan sesuai kebutuhan (penggunaakhir)tanpa merugikan dampak ekologis. Dalam perancangan pembuatan alat ini diawali dengan perancangan umum dari system dari keseluruhan.Adapunnperancangan pembuatan rancang bangun system ini terbagi atas beberapa perangkat yang salign berhubungan yaitu perangkat elektonik (hardware) dan perangkat lunak (software) yang berisi intruksi untuk menjalankan progam Upaya pemenuhan kebutuhan air oleh manusia dapat mengambil air dari dalam tanah, air permukaan, atau langsung dari air hujan. Dari ke tiga sumber air tersebut, air tanah yang paling banyak digunakan karena air tanah memiliki beberapa kelebihan di banding sumber-sumber lainnya antara lain karena kualitas airnya yang lebih baik serta pengaruh akibat pencemaran yang relatif kecil.Akan tetapi air yang dipergunakan tidak selalu sesuai dengan syarat kesehatan, karena sering ditemui air tersebut mengandung bibit ataupun zat-zat tertentu yang dapat menimbulkan penyakit yang justru membahayakan kelangsungan hidup manusia. ELSAINS Vol 1. No. 1 Rancang Bangun Alat Penjernih Air Berbasis PLC Arman Aprilliana1, Agung Prasetyo2, Gatut Budiono3 Jurusan Teknik Elektro, Universitas 17 Agustus 1945 Surabaya Jl. Semolowaru 45 Surabaya 60118 Telp./Fax: 031-5921516 E-mail:Arman_Aprilliana@gmail.com E-mail:Agung_prasetyo@gmail.com E-mail:Gatut_budiono@untag-sby.ac.id ISSN: XXXX-XXXX ISSN: XXXX-XXXX ISSN: XXXX-XXXX ELSAINS Vol 1. No. 1 Pengujian sensor LDR Pengujian sensor cahaya (LDR) sebagai pengukur kondisi cahaya yang terhalang dari air keruh, dalam pengujian ini kami mencoba mengukur kondisi cahaya di luar ruangan untuk mengetahui data yang di dapat dari sensor tersebut. Prosedur pengujian 1. Hubungkan power 220v ke PLC LG pada L1 untuk phase dan N untuk netral phase dan N untuk netral 2. Hubungkan PLC LG dengan kabel 2. Hubungkan PLC LG dengan kabel k ifk l d j l k 3. Aktifkan laptop dan jalankan program KGL W Pengujian PLC Pengujian PLC ini untuk memastikan bahwa diagram ladder berkerja dengan baik dan PLC bekerja sesuai dengan program. Sehingga program yang ditanamkan pada PLC mampu menginput dan output sesuai dengan program. Gambar 3. Teknik Pengukuran Tingkat Kekeruhan Air Tabel 1 Hasil Uji Sensor LDR Gambar 3. Teknik Pengukuran Tingkat Kekeruhan Air Tabel 1 Hasil Uji Sensor LDR Pengujian Selenoid dan Timer Pengujian Selenoid dan Timer 1. Hubungkan catu daya 5v sebagai input sensor dan catu daya 24v sebagai output relay Pengujian Selenoid dan timer ini bertujuan untuk mengetahui bahwa timer dapat memerintahkan solenoid sesuai dengan tingkat kekeruhan. Sehingga program yang berada dalam PLC dapat menginput dan output sesuai program. 2. Tempatkan relay pada satu cawan 3. Beri cahaya pada level tertentu Gambar 1. Desain akuarium Pengukuran Kekeruhan Air Prosedur Pengujian 1. Hubungkan catu daya 5v sebagai input sensor dan catu daya 24v sebagai output relay B.Perancangan Perangkat Keras Dalam perencananan alat alat pada perangkat keras (hardware) dilakukan perencanaan pada system mekanik dan juga system perencanaan pada elektronika.Pada perencanaan system elektronika dilakukan beberapa perencanaan terhadap rangkaian elektronika yang digunakan untuk mendriver dan mengontrol dari pada system mekanik. Proses-proses yang terlibat dalam pemisahan kontaminan dapat menggunakan proses fisik seperti menata/(melihat),atau penyaringan kimia seperti Desinfeksi dan koagulasi.Selain itu proses Biologi juga dalam pengolahan air limbah, proses ini meliputi mencampur air dengan udara,saringan pasir padat dan kerikil. Air merupakan faktor penting dalam pemenuhan kebutuhan ISSN: XXXX-XXXX Prosedur Pengujian 1. Hubungkan catu daya 220v ke PLC LG 1. Hubungkan catu daya 220v ke PLC LG 2. Hubungkan catu daya 5V ke Arduino 3. Beri cahaya pada level tertentu 4. Lampu Indikator akan menunjukkan posisi level cahaya 5. Lampu Indikator akan menunjukkan Selenoid bekerja per detik 1.Perancangan Catu Daya Hasil pengujian sensor LDR Hasil pengujian sensor LDR Catu daya merupakan sumber tenaga yang dibutuhkan suatu rangkaian elektronika untuk bekerja.Besarnya suplai daya tergantung spesifikasi alat masing-masing.Pada system pengendalian ini,catu daya digunakan untuk mengaktfikan andruino dimana sebagai perangkat yang berfungsi menerima data yang di hasilkan oleh sensor dan dikirim ke Input PLC. Gambar 3. Teknik Pengukuran Tingkat Kekeruhan Air Hasil Pengujian Selenoid dan Timer Tabel 2 Tingkat kekeruahan air Saran Beberapa saran untuk perbaikan dan pengembangan alat adalah: Beberapa saran untuk perbaikan dan pengembangan alat adalah: 1. Desain untuk penempatan sensor bisa dikembangkan dengan desain yang lebih aman dan baik 2. Dalam hal mekanisme,rangka meja kerang kokoh dalam menopang beban aquarium dengan volume air yang penuh. y g p 3. Rancang bangun alat penjernih air berbasis PLC dapat dikembangkan dengan menambahkan layar HMI dengan SCADA yang dapat memudahkan dalam system monitoring flow air dan perawatan mekanismenya. ` ELSAINS Vol 1. No. 1 8. https://www.scribd.com/doc/154593937/Fungsi- Gearbox KESIMPULAN Dari hasil perancangan alat ini dapat diambil kesimpulan sebagai berikut: 1. Pada air sumur dimana tingkat kekeruhan 20% masuk level LOW (Jernih) Dossing kaporit akan bekerja dalam tempo per 10 second),menghasilkan air dengan tingkat kekeruhan 5 NTU 2. Pada air kolam dimana tingkat kekeruhan 50% masuk level MEDIUM (Setengah Keruh) Dossing kaporit akan bekerja dalam tempo per 2 second,menghasilkan air dengan tingkat kekeruhan 8 NTU. 3. Pada air sungai dimana tingkat kekeruhan 80% masuk level HARD (Keruh) Dossing Kaporit akan bekerja dalam tempo per 1 second,menghasilkan air dengan tingkat kekeruhan 10 NTU. 4. Rancang bangun Alat Penjernih Air berbasis PLC adalah suatu mesin pengolahan air keruh yang di rancang menggunakan system water treadment plant dan menggunakan PLC sebagai control otomatis dalam proses pengolahan berlangsung. 5. Penelitian ini telah berhasil merancang dan membangun system pengolahan air mengunakan water treadment plan. 6. Dalam proses pengolahan alat ini menghasilkan Air bersih yang dihasilkan dalam alat ini Tabel 2 Tingkat kekeruahan air Gambar 1. Desain akuarium Pengukuran Kekeruhan Air Tingkat keruh Timer Selenoid 0-20% 10 detik 10 detik 21-40% 2 detik 2 detik 41-60% 2 detik 2 detik 61-80% 1 detik 1 detik 81-100% 1 detik 1 detik Gambar 2. Akuarium Pengukuran Kekeruhan Air Gambar 2. Akuarium Pengukuran Kekeruhan Air Gambar 2. Akuarium Pengukuran Kekeruhan Air ISSN: XXXX-XXXX 8. https://www.scribd.com/doc/154593937/Fungsi- Gearbox DAFTAR PUSTAKA 1. [Anonim].2012.Pengertian LDR Dan Cara Mengukurnya http://teknikelektronika.com/pengertian-ldr-light- dependent-resistor-cara-mengukur-ldr/ 2. [Anonim].2012.Arduino RTC 3. http://www.instructable.com/id/Arduino-Real- Time-Clock-DS1307/. 4. Anonim, 2012. Elektronika Dasar Sensor Cahaya LDR. http://elektronika- dasar.web.id/komponen/sensor-tranducer/sensor- cahaya-ldr-light-dependent-resistor/ 5. http://indoware.com/komponen-penyusun-plc.html 6. https://anwarmekatronikapens.wordpress.com/2015 /04/22/plc/ 7. http://elektronika-dasar.web.id/sensor-cahaya-ldr- light-dependent-resistor/ ISSN: XXXX-XXXX ISSN: XXXX-XXXX ELSAINS Vol 1. No. 1 ELSAINS Vol 1. No. 1
https://openalex.org/W1979455700	https://www.scielo.br/j/rsp/a/mMpKrY4KLzShwwKWX9B5QcN/?lang=pt&format=pdf	Portuguese	null	Valores de referência para plumbemia em população urbana	Revista de saúde pública/Revista de Saúde Pública	1,997	cc-by	3,029	31 Valores de referência para plumbemia em população urbana Lead reference values in the blood of the urban population 31 Monica M. B. Paolielo, Paulo R. Gutierrez, Conceição A. Turini, Tiemi Matsuo, Leda Mezzaroba, Décio S. Barbosa, Aparecida L. P. Alvarenga, Sandra R. Q. Carvalho, Garcia A. Figueiroa, Viviane G. M. Leite, Amanda C. Gutierrez, Kleber B. Nogueira, Willyan A. S. Inamine e Antonio M. Zavatti 31 Monica M. B. Paolielo, Paulo R. Gutierrez, Conceição A. Turini, Tiemi Matsuo, Leda Mezzaroba, Décio S. Barbosa, Aparecida L. P. Alvarenga, Sandra R. Q. Carvalho, Garcia A. Figueiroa, Viviane G. M. Leite, Amanda C. Gutierrez, Kleber B. Nogueira, Willyan A. S. Inamine e Antonio M. Zavatti 31 Departamento de Patologia Aplicada, Legislação e Deontologia do Centro de Ciências da Saúde da Universidade Estadual de Londrina. Londrina, PR - Brasil (M.M.B.P., C.A.T., L.M., D.S.B., A.L.P.A., S.R.Q.C.); Departamento Materno-Infantil e Saúde Comunitária do Centro de Ciências da Saúde da Universidade Estadual de Londrina. Londrina, PR - Brasil (P.R.G.); Departamento de Matemática Aplicada do Centro de Ciências Exatas da Universidade Estadual de Londrina. Londrina, PR - Brasil (T.M.); Prefeitura do Município de Londrina. Londrina, PR - Brasil (G.A.F.); Universidade Estadual de Londrina. Londrina, PR - Brasil (V.G.M.L., A.C.G., K.B.N., W.A.S.I., A.M.Z.) PAOLIELO, Monica M. B., Valores de referência para plumbemia em população urbana Rev. Saúde Pública, 31 (2): 144-8, 1997. PAOLIELO, Monica M. B., Valores de referência para plumbemia em população urbana. Rev. Saúde Pública, 31 (2): 144-8, 1997. PAOLIELO, Monica M. B., Valores de referência para plumbemia em população urbana. Rev. Saúde Pública, 31 (2): 144-8, 1997. © Copyright Faculdade de Saúde Pública da USP. Proibida a reprodução mesmo que parcial sem a devida autorização do Editor Científico. Proibida a utilização de matérias para fins comerciais. All rights reserved. 144 Rev. Saúde Pública, 31 (2): 144-8, 1997 * Pesquisa financiada pelo Projeto UNI de Londrina - Fundação Kellogg. Apresentado no VII Congresso Internacional de Toxicologia, em Seattle - EUA, 1995. Correspondência para/Correspondence to: Monica M. Bastos Paolielo - Departamento de Patologia Aplicada, Legislação e Deontologia do Centro de Ciências da Saúde da Universidade Estadual de Londrina. Av. Robert Koch, 60 - Vila Operária - 86038-440 Londrina, PR - Brasil. E-mail: prounild@sercomtel.com.br Recebido em 18.12.1995. Reapresentado em 19.8.1996. Aprovado em 23.9.1996. Introdução Os valores de referência utilizados no Brasil, para chumbo em sangue, advêm de estudos realizados em outros países onde as condições socioeconômicas, clínicas, nutricionais e ocupacionais diferem bastante das brasileiras. Para ga- rantir uma correta biomonitorização da população ocupacionalmente exposta ao chumbo, um dos principais problemas identificados no município estudado, foram estabelecidos valores de referência na população não exposta ocupa- cionalmente da região sul do município. Material e Método Diferentes estratégias foram utilizadas para assegurar a qualidade de amostra- gem, que foi dimensionada em 206 sujeitos acima de 15 anos. Sujeitos que apresentaram valores clínicos e laboratoriais fora da faixa de normalidade fo- ram excluídos, bem como os que apresentaram atividades específicas que pu- dessem interferir nos valores de plumbemia. Valores de referência para plumbemia em população urbana* Lead reference values in the blood of the urban population Monica M. B. Paolielo, Paulo R. Gutierrez, Conceição A. Turini, Tiemi Matsuo, Leda Mezzaroba, Décio S. Barbosa, Aparecida L. P. Alvarenga, Sandra R. Q. Carvalho, Garcia A. Figueiroa, Viviane G. M. Leite, Amanda C. Gutierrez, Kleber B. Nogueira, Willyan A. S. Inamine e Antonio M. Zavatti Departamento de Patologia Aplicada, Legislação e Deontologia do Centro de Ciências da Saúde da Universidade Estadual de Londrina. Londrina, PR - Brasil (M.M.B.P., C.A.T., L.M., D.S.B., A.L.P.A., S.R.Q.C.); Departamento Materno-Infantil e Saúde Comunitária do Centro de Ciências da Saúde da Universidade Estadual de Londrina. Londrina, PR - Brasil (P.R.G.); Departamento de Matemática Aplicada do Centro de Ciências Exatas da Universidade Estadual de Londrina. Londrina, PR - Brasil (T.M.); Prefeitura do Município de Londrina. Londrina, PR - Brasil (G.A.F.); Universidade Estadual de Londrina. Londrina, PR - Brasil (V.G.M.L., A.C.G., K.B.N., W.A.S.I., A.M.Z.) Definição da População de Referência e Critérios de Exclusão Em decorrência do desenvolvimento urbano e tecnológico, tem-se observado o aumento da concen- tração de substâncias químicas no ambiente: conse- qüentemente, as diferenças quantitativas entre expo- sição ambiental e ocupacional estão progressivamen- te se reduzindo. Esta condição levará ao surgimento de problemas interpretativos sobre os resultados ob- tidos com os indicadores biológicos pesquisados11. Com o objetivo de estabelecer a população de refe- rência para o presente estudo, foram definidos alguns pa- râmetros relativos às condições de exposição e às condi- ções de saúde da população. Em relação às condições de exposição, os critérios utilizados foram: sujeitos não expostos ocupacionalmente ao chumbo ou não expostos através de hábitos, não fuman- tes ou que fumavam até 10 cigarros por dia e com mora- dia não próxima a indústrias ou outros locais de trabalho que utilizassem o chumbo em seus processos. Para averi- guar tais condições, aplicou-se um questionário onde se detalhavam estas informações4, 5, 6. Portanto, há uma necessidade crescente em se propor valores de referência, fundamentais para uma adequada biomonitorização. Deve-se, então, procu- rar obter pela própria definição, o valor de um deter- minado indicador biológico na população em geral (ou grupos populacionais) não exposta diretamente por razões de trabalho e/ou ambiente ao elemento ou substâncias em estudo1, 12. Quanto às condições de saúde, procurou-se obter “su- jeitos sadios”. Para isto, foram realizados anamnese e exa- me físico, além dos seguintes exames laboratoriais: he- mograma, glicose, uréia, creatinina, gama glutamil trans- ferase, alanina amino transferase, aspartato amino trans- ferase, colesterol total, triglicerídeos e urina I. Em relação aos valores para plumbemia no Bra- sil, praticamente não existe uma base de dados para obtenção de tais valores. Por esse motivo, os valores de referência utilizados advêm de estudos realiza- dos em outros países, onde as condições socioeco- nômicas, clínicas, nutricionais e ocupacionais dife- rem bastante das brasileiras. Embora a exposição ao chumbo tenha sido considera- da o principal critério de exclusão, também foram excluí- dos sujeitos que apresentavam valores clínicos e laborato- riais fora da faixa de normalidade, fumantes que consu- miam mais de 10 cigarros por dia, obesos e hipertensos, além daqueles afetados por doenças crônicas como alcoo- lismo e distúrbios cardiovasculares4, 6. Objetivou-se, no presente trabalho, propor valo- res de referência para chumbo em sangue na popu- lação do Município de Londrina, PR, contribuindo assim à correta biomonitorização de trabalhadores ocupacionalmente expostos ao referido metal. Results Results Lead reference values for blood were found to be from 2.40 to 16.6 µg.dL–1, obtained by the interval x– ± 2s (where x– is the mean and s is the standard deviation form observed values) and the median was 7.9 µg.dL-1. Lead poisoning, blood. Reference values. Lead poisoning, blood. Reference values. Resultados Foram encontrados valores de referência para chumbo em sangue de 2,4 a 16,6 µg.dL-1, obtidos através do intervalo x– ± 2s (onde x– é o valor médio e s é o desvio-padrão dos valores observados) e mediana = 7,9 µg.dL-1. Intoxicação por chumbo, sangue. Valores de referência. Rev. Saúde Pública, 31 (2), 1997 145 Valores de referência para plumbemia Paolielo, M.M.B. et al. Material and Method The sample was composed of 206 subjects of at least 15 years of age. Various strategies were employed to assure good-quality sampling. Subjects who pre- sented values outside clinical or laboratory norms were excluded, as well as those whose specific activities might interfere with the results. Introduction The lead reference values for blood used in Brazil come from studies conducted in other countries, where socioeconomic, clinical, nutritional and occupa- tional conditions are significantly different. In order to guarantee an accurate biomonitoring of the population which is occupationally exposed to lead, a major health concern of the studied community, reference values for individu- als who are not occupationally exposed and who live in the southern region of the city were established. Material and Method Técnica Analítica A plumbemia foi determinada por espectrofotometria de absorção atômica com chama de ar acetileno, através da complexação do elemento chumbo com pirrolidinadi- tiocarbamato de amônio (APDC) e extração com metili- sobutilcetona (MIC)7. RESULTADOS Com o objetivo de determinar o tamanho da amostra, realizou-se um pré-teste com 91 indivíduos, dos quais so- mente 72 foram considerados dentro dos critérios previa- mente definidos, observando-se, portanto, uma perda de 20,8%. A amostra foi dimensionada levando-se em consi- deração o número de habitantes da região sul acima de 15 anos (aproximadamente 32.000)3, a média de plumbemia obtida no pré-teste (= 8,21 µg.dL-1) e desvio-padrão (= 3,07 µg.dL-1). Com base em tais parâmetros, a amostra foi estimada em 224 sujeitos, com nível de confiança de 95% e 5% de erro na estimativa. Com base na perda observada no pré-teste (20,8%), dimensionou-se o tamanho da amos- tra a ser investigada em 289 indivíduos. De acordo com os critérios de exclusão pré-esta- belecidos, dos 289 sujeitos examinados foram ex- cluídos 28,7%, um índice maior do que o observado no pré-teste. Portanto, foram considerados para o presente estudo 206 sujeitos, sendo 145 do sexo fe- minino e 61 do sexo masculino, com idade média igual a 38,4 anos (± 13,1). A Tabela apresenta os dados estatísticos obtidos a partir das análises de chumbo em sangue em sujei- tos saudáveis, segundo o sexo. A Figura 1 apresenta um “box plot” para chum- bo em sangue, com a distribuição dos dados segun- do os quartis2, mostrando um ponto discrepante (23,0 µg.dL2). Segundo Minóia e col.6, 1990, as condições que levam sujeitos a apresentarem valores dentro do intervalo de incerteza, que é o intervalo dos altos valores observados, devem ser precisamente deter- minados. Assim sendo, efetuou-se uma nova análise do caso em relação aos critérios de exclusão, sendo que os dados clínicos, laboratoriais e ocupacionais do mesmo se enquadram nos critérios estabelecidos para a normalidade. Portanto, optou-se por sua per- manência no conjunto de dados. Definição da População de Referência e Critérios de Exclusão A região escolhida para o presente estudo foi a região sul do Município de Londrina. Tal escolha se justifica por ser essa região predominantemente residencial e muito Rev. Saúde Pública, 31 (2), 1997 Valores de referência para plumbemia Paolielo, M.M.B. et al. 146 pouco industrializada sendo, portanto, mais adequada para se obter uma população não exposta profissionalmente ao chumbo ou com moradias próximas a indústrias. b) Intervalo de confiança (95%) - obtidos através da equação x– ± z.s (x), onde x– é o valor médio, z95% é o valor da distribuição normal e s (x) é o erro padrão da média; b) Intervalo de confiança (95%) - obtidos através da equação x– ± z.s (x), onde x– é o valor médio, z95% é o valor da distribuição normal e s (x) é o erro padrão da média; pouco industrializada sendo, portanto, mais adequada para se obter uma população não exposta profissionalmente ao chumbo ou com moradias próximas a indústrias. A população estudada foi formada por voluntários contatados através do Conselho Regional de Saúde da Região Sul e Associações de Moradores da região. As co- lheitas das amostras de sangue e os demais procedimen- tos foram realizados nos meses de julho e agosto de 1993; maio, julho e setembro de 1994 e março de 1995. c) Intervalo experimental - delimitado pelo menor e maior valor observado (x min. e x max., respectivamente); d) Valor de referência - obtido através do intervalo x– ± 2.s, onde x– é o valor médio e s é o desvio-padrão dos valores observados; e) Intervalo de incerteza - delimitado por x– + 2s e o maior valor obtido. Tratamento Estatístico Figura 1 - “Box-and-whisker plot" para níveis de plumbemia no Município de Londrina, PR. Figure 1 - “Box-and-whisker plot” for levels of lead in blood in the county of Londrina, PR, Brazil. Figura 2 - “Box-and-whisker plot" para níveis de plumbemia em função do sexo, Município de Londrina, PR. Figure 2 - “Box-and-whisker plot” for levels of lead in blood by gender, county of Londrina, PR, Brazil. Tratamento Estatístico Após a realização das análises de chumbo em sangue, foram determinados6: a) Valor mínimo, 1º quartil, mediana, 3º quartil e valor máximo; Tabela - Valores de chumbo em sangue (µg.dL-1) em sujeitos saudáveis. Município de Londrina, PR, segundo o sexo. Table - Lead values for blood (µg.dL-1) in health subjects from the southern region of the Municipality of Londrina, by gender. Sexo Estatísticas Feminino Masculino Total (n = 145) (n = 61) (n = 206) Valor mínimo 2,4 2,4 2,4 1º Quartil 5,3 5,4 5,3 Mediana 7,9 9,0 7,9 3º Quartil 10,0 11,0 11,0 Valor máximo 23,0 19,0 23,0 Intervalo de confiança 95% 8,1 ± 0,7 8,7 ± 1,1 8,3 ± 0,6 Intervalo experimental 2,4 – 23,0 2,4 – 19,0 2,4 – 23,0 Valores de referência 2,4 – 16,4 2,4 – 17,1 2,4 – 16,6 Intervalo de incerteza 16,4 – 23,0 17,1 – 19,0 16,6 – 23,0 Tabela - Valores de chumbo em sangue (µg.dL-1) em sujeitos saudáveis. Município de Londrina, PR, segundo o sexo. Table - Lead values for blood (µg.dL-1) in health subjects from the southern region of the Municipality of Londrina, by gend bo em sangue (µg.dL-1) em sujeitos saudáveis. Município de Londrina, PR, segundo o sexo. bo em sangue (µg.dL-1) em sujeitos saudáveis. Município de Londrina, PR, segundo o sexo. ood (µg.dL-1) in health subjects from the southern region of the Municipality of Londrina, by gender. g (µg ) j p , , g dL-1) in health subjects from the southern region of the Municipality of Londrina, by gender. Rev. Saúde Pública, 31 (2), 1997 Valores de referência para plumbemia Paolielo, M.M.B. et al. 147 Figura 1 - “Box-and-whisker plot" para níveis de plumbemia no Município de Londrina, PR. Figure 1 - “Box-and-whisker plot” for levels of lead in blood in the county of Londrina, PR, Brazil. Fi 2 “B d hi k l t" í i d l b i Figura 1 - “Box-and-whisker plot" para níveis de plumbemia no Município de Londrina, PR. Figure 1 - “Box-and-whisker plot” for levels of lead in blood in the county of Londrina, PR, Brazil. Figura 2 - “Box-and-whisker plot" para níveis de plumbemia em função do sexo, Município de Londrina, PR. Figure 2 - “Box-and-whisker plot” for levels of lead in blood by gender, county of Londrina, PR, Brazil. Figura 1 - “Box-and-whisker plot" para níveis de plumbemia no Município de Londrina, PR. DISCUSSÃO Figure 2 - “Box-and-whisker plot” for levels of lead in blood by gender, county of Londrina, PR, Brazil. Os principais resultados de plumbemia obtidos no presente estudo, ou seja, valores de referência = 2,4 – 16,6 µg.dL-1 e mediana = 7,9 µg.dL-1 (Tabela), de um modo geral, encontram-se abaixo daqueles re- latados em estudos semelhantes realizados em outros países. Minóia e col.6 (1990), obtiveram em regiões italianas uma média = 15,77 ± 9,9 µg.dL-1 e valores de referência = 3,97 – 27,57 µg.dL-1. Morisi e col.8 (1989), também na Itália, encontraram valores de plumbemia = 15,3 µg.dL-1. Iyengar e col.4 (1988), obtiveram, na Holanda, uma mediana = 12,3 µg.dL-1. Em Singapura, Ooi e col.9 (1991), obtiveram resul- tados que se aproximaram dos encontrados no pre- sente estudo, ou seja, média = 7,66 µg.dL-1. Entre- tanto, os citados autores obtiveram tal média consi- derando também crianças, o que não ocorreu com os outros trabalhos citados e no presente estudo, que obtiveram tais valores apenas em adultos. Foram obtidos níveis de plumbemia mais baixos por Piomellis e col.10 (1980), na região do Himalaya, onde encontraram média = 3,40 µg.dL-1 de plumbemia em adultos e crianças. Alguns autores confirmaram a influência de de- terminadas variáveis associadas aos níveis de chum- bo em sangue, quais sejam, sexo, idade, consumo de álcool e hábito de fumar1, 8, 9. Entretanto, no presente estudo não foram observadas diferenças entre os ní- veis médios de chumbo sangüíneo entre os sexos. O “box plot” que compara os resultados de plumbemia entre homens e mulheres (Fig. 2) mostra uma distri- buição semelhante dos dados. Os resultados obtidos parecem confirmar os da- dos que demonstram que níveis mais altos de plum- bemia são encontrados em populações residentes em regiões industrializadas, ao contrário dos níveis de plumbemia observados em regiões de pouca indus- trialização, como é o caso da região sul do Municí- pio de Londrina. Rev. Saúde Pública, 31 (2), 1997 Valores de referência para plumbemia Paolielo, M.M.B. et al. 148 REFERÊNCIAS BIBLIOGRÁFICAS values in tissues from habitants of the European. Community: a study of 46 elements in urine, blood and serum of italian subjects. Sci. Total Envirom., 95:89-105, 1990. 1. APOSTOLI, P. & ALESSIO, L. Valori di referimento e controllo dei fattori di variabilitá. In: La Promazione della qualitá dei dati nel monitoraggio biologico. Bologna, Moderna, 1990. p. 111-27. 7. MITCHELL, D.G.; RYAN,R.J.; ALDOUS, K.M. The precise determination of lead in whole blood by solvent extraction atomic spectrometry. Atomic. Abs. News, 11:120-1, 1972. 2. HOAGLIN, D.C.; MOSTELLER, F.; TUKEY, J.W. Análise exploratória de dados: técnicas robustas. Lisboa, Ed. Salamandra, 1992. 8. MORISI, G.; PATRIARCA, M.; CARRIERI, M. P.; FONDI, G.; TAGGI, F. Lead exposure: assessent of the risk for the general italian population. Ann. Ist. Sup. Sanitá, 25:423-6, 1989. 3. INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. Décimo recenseamento geral do Brasil: boletim resumo do Município de Londrina. Rio de Janeiro, 1991. 9. OOI, P.L.; GOH, K.T.; HENG, B.H.; SAM, C.T.; KONG, K.H.; RAJAN, U. Biological monitoring of human exposure to environmental lead in Singapore. Rev. Environ. Health, 9: 207-13, 1991. 4. IYENGAR, V. & WOLTTIEZ, J. Trace elements in humam clinical specimens: evaluation of literature data to identify reference values. Clin.Chem., 34:474-81. 1988. 10. PIOMELLI, S.; CORASH, L.; CORASH, M.B.; SEAMEN, C.; MUSHAK, P.; GLOVER, B.; PADGETT, R. Blood lead concentrations in a remote Himalayan population. Science, 210:1135-7, 1980. 5. MINOIA, C.; POZZOLI, L.; CAVALLERI, A.; CAPODAGLIO, E. Definizione dei valore di referimento di 30 elementi in tracce nei liquid biologici. In: Congresso Nazionale della Societá Italiana Di Medicina Del Lavoro e Igiene Industriale, 48º, Bologna, 1985. Anais, Bologna, 1985. p. 317-23. 11. SALGADO, P.E.T. Valores de referência. In: Congresso Latino-Americano de Toxicologia, 8º, Porto Alegre. 1992. Anais, Porto Alegre, 1992. 6. MINOIA, C.; SABBION, E.; APOSTOLI, P.; PIETRA, R.; POZZOLI, L.; GALLORINI, M.; NICOLAOU, G.; ALESSIO, L., CAPODAGLIO, E. Trace element reference 12. SALGADO, P.E.T. Valores de referência para metais. Rev. Bras. Toxicol., 3:35, 1990.
https://openalex.org/W2010577903	https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.0030085&type=printable	English	null	HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4AVPRBP E3 Ubiquitin Ligase	PLOS pathogens	2,007	cc-by	13,133	Introduction Human immunodeﬁciency virus type 1 (HIV-1) viral protein R (Vpr) accessory protein is a small 96amino acid protein that plays several roles during virus infection (reviewed in [1,2]). In particular, the protein mediates cell cycle arrest at the G2/M transition in various mammalian cells [3–6], a cytostatic ability that is well conserved among the primate lentiviruses [7]. Its biological signiﬁcance is not fully understood but may be related to general activation of virus expression [8] and/or induction of apoptosis [9]. Vpr suppresses cell proliferation by preventing the activation of the p34cdc2/cyclin B complex [3,4]. Accumulating evidence indicates that Vpr-mediated cell cycle arrest depends on DNA damage response, but precise mechanisms of its induction remain obscure. Several groups have reported that Vpr expression induces Rad17- and Hus1-dependent activation of ATR, but not of ATM, and induces the formation of nuclear c-H2AFX/BRCA1 foci [14–16]. However, the mechanism by which Vpr triggers ATR activation is not well understood. Some authors proposed that Vpr would interfere with normal DNA replication, leading to stalled replication forks [15,17], while others suggested that the protein may promote the formation of DNA double-strand breaks by recruiting unknown endo- Editor: Klaus Fru¨h, Oregon Health and Science University, United States of America Phosphatidylinositol 3 kinaselike ATM (ataxia telangiec- tasia mutated) and ATR (ataxia and telangiectasia mutated and Rad3 related) are key components of the G2/M checkpoint. In addition, ATR activates S-phase checkpoint in replication stress response resulting from stalled repli- cation [10]. Depending on the type of stress, ATR- or ATM- mediated checkpoints are fully activated by the coordinated activity of Rad9-Rad1-Hus1 (9-1-1), Rad17-RFC, breast cancerassociated protein (BRCA1), and p53 binding protein (53BP) (reviewed in [11–13]). ATR- or ATM-dependent phosphorylation of the histone variant H2AFX (H2AX) triggers the formation of c-H2AFX/BRCA1 or 53BP foci. These foci are presumed to help in the recruitment and/or retention of DNA repair machinery and checkpoint effectors at the damaged DNA sites, thus promoting checkpoint signal ampliﬁcation [12]. Downstream activation of CHEK1 or CHEK2 kinases by ATM and ATR results in the inactivation Copyright: 2007 Belzile 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. HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4AVPRBP E3 Ubiquitin Ligase Jean-Philippe Belzile1[, Ghislaine Duisit2[¤, Nicole Rougeau1, Johanne Mercier1, Andre´s Finzi1, E´ric A. Cohen1,2* 1 Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montre´al, Montreal, Quebec, Canada, 2 Department of Microbiology and Immunology, Universite´ de Montre´al, Montreal, Quebec, Canada Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)—components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4AVPRBP—were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest–defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4AVPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest. Citation: Belzile JP, Duisit G, Rougeau N, Mercier J, Finzi A, et al. (2007) HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase. PLoS Pathog 3(7): e85. doi:10.1371/journal.ppat.0030085 Citation: Belzile JP, Duisit G, Rougeau N, Mercier J, Finzi A, et al. (2007) HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 doi:10.1371/journal.ppat.0030085 of Cdc25 phosphatase and increased expression of both WEE1 kinase and the 14-3-3 family of proteins. Inactivation of cdc2/cyclin by hyperphosphorylation and cytoplasmic retention prevents entry into mitosis before the completion of DNA repair [10,12]. PLoS Pathogens \| www.plospathogens.org Author Summary We demon- strated that association of Vpr with this ubiquitinating complex might be responsible for the defect in cell growth. Further characterization of this protein complex and the elucidation of the mechanism by which it affects cell growth might lead to the development of new anti-HIV drugs and an improved under- standing of the basic cellular processes controlling cell division. We sought to conﬁrm the interaction of Vpr with DDB1 and VPRBP in HEK293T cells transfected with TAP or TAP- Vpr expression plasmids. TAP pull-down experiments were performed on cell lysates using IgG-coated sepharose beads. Co-precipitated endogenous DDB1 and VPRBP were de- tected by Western blot using speciﬁc antibodies. As shown in Figure 1A, endogenous DDB1 and VPRBP could be pulled down when co-expressed with TAP-Vpr (lane 3), but not when the protein was in the presence of the native TAP tag (lane 2), indicating that DDB1 and VPRBP binding was speciﬁc to TAP-Vpr. These interactions could be detected in conditions containing 1% NP40 (unpublished data) as well as 0.5% Triton X-100 (Figure 1A). nucleases to the chromatin [18]. Both models imply direct Vpr interactions with host chromatin [15,18]. On the other hand, blockade of the proliferation might rely on the mislocation of key cell cycle regulators, because of alterations in the nuclear envelope induced by membrane-anchored Vpr [19]. To conﬁrm the speciﬁcity of the interaction between Vpr and DDB1, we performed pull-down assays in cells co- transfected with TAP-Vpr and hemagglutinin (HA)-tagged DDB1–encoding plasmids (Figure 1B). We were able to observe that HA-DDB1 could be co-precipitated speciﬁcally in the presence of TAP-Vpr (lane 6), but not in the presence of the empty plasmid (lane 2) or a TAP-expressing plasmid (lane 5). We constructed TAP-DDB1 as well as green ﬂuorescent protein (GFP)–tagged DDB1 expression plasmids to verify whether the interaction could be observed in the reversed orientation. However, immunoprecipitation using endogenous, TAP-tagged, HA-tagged, or GFP-fused DDB1 as bait and wild-type or HA-tagged Vpr yielded inconsistent results (unpublished data). These discrepancies between HA- Vpr and TAP-Vpr abilities to bind to DDB1 are reminiscent of the versatile association between DDB1 and the DNA replication licensing factor CDT1. In that case, detection of DDB1-CDT1 complexes in absence of chromatin was depend- ent on the amount of antibody used for the immunopreci- pitation [20]. Author Summary tion time-of-ﬂight mass spectrometric analysis revealed that the upper band corresponded to VPRBP, a 180-kDa protein that had been isolated as a Vpr-binding factor a decade ago, but whose function still remained obscure [21,22]. The other 120-kDa protein was identiﬁed as DDB1, initially considered part of a heterodimeric complex containing damaged DNA binding protein 2 (DDB2), involved in a cellular response to UV-induced DNA damages [24,25]. However, the protein is now emerging as a central scaffolding factor in the DDB1- CUL4A-RBX1 E3 ubiquitin ligase complex associated with the COP9 signalosome [26]. Importantly, recently the WD40 protein VPRBP has been demonstrated to interact with DDB1 and probably serves as an adapter to confer substrate speciﬁcity to the DDB1-CUL4A-RBX1 E3 ubiquitin ligase complex [20]. tion time-of-ﬂight mass spectrometric analysis revealed that the upper band corresponded to VPRBP, a 180-kDa protein that had been isolated as a Vpr-binding factor a decade ago, but whose function still remained obscure [21,22]. The other 120-kDa protein was identiﬁed as DDB1, initially considered part of a heterodimeric complex containing damaged DNA binding protein 2 (DDB2), involved in a cellular response to UV-induced DNA damages [24,25]. However, the protein is now emerging as a central scaffolding factor in the DDB1- CUL4A-RBX1 E3 ubiquitin ligase complex associated with the COP9 signalosome [26]. Importantly, recently the WD40 protein VPRBP has been demonstrated to interact with DDB1 and probably serves as an adapter to confer substrate speciﬁcity to the DDB1-CUL4A-RBX1 E3 ubiquitin ligase complex [20]. Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, encodes several proteins termed accessory, which play a critical role in viral pathogenesis. Collectively, HIV-1 accessory proteins Vif, Vpr, Vpu, and Nef have been shown to manipulate host cell biology to promote viral replication, immune evasion, and AIDS progression. In this context, Vpr has been found to block normal cell growth. This interruption of cell division by Vpr is thought to increase viral replication and induce programmed cell death. However, how Vpr is able to block cell division remains unknown. To understand this mechanism, we have studied the interaction between Vpr and novel cellular proteins. We identified a novel complex comprising Vpr and cellular proteins involved in a process called ubiquitination. Ubiquitination is a mechanism by which a small protein, ubiquitin, is conjugated to cellular proteins to modulate their activity or induce their degradation. Author Summary Given that CDT1 interacts indirectly with DDB1 via the adapter protein CDT2 [27,28], it is tempting to hypothesize that Vpr would likewise interact indirectly with DDB1 through an adapter protein, perhaps VPRBP, and that the TAP/IgG bead complexes may somehow stabilize the interaction. To identify Vpr-interacting cellular proteins responsible for the initial events leading to the induction of G2 arrest, we used the proteomic tandem afﬁnity puriﬁcation (TAP) procedure followed by mass spectrometry. Native complexes containing TAP-Vpr were puriﬁed from human cells by two consecutive afﬁnity chromatographic steps under mild conditions. Here, we identify a novel protein complex comprising Vpr, the damaged DNA binding protein 1 (DDB1), the E3 ubiquitin ligase scaffold protein cullin 4A (CUL4A), and the newly identiﬁed DDB1-CUL4A–associated factor 1 (DCAF1), which is also designated as viral protein R binding protein (VPRBP) [20–22]. We provide functional evidence indicating that Vpr interaction with this E3 ubiquitin ligase complex is involved in induction of G2 cell cycle arrest. PLoS Pathogens \| www.plospathogens.org Introduction Abbreviations: 53BP, p53 binding protein; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia mutated and Rad3 related; BRCA1, breast cancer– associated protein; CBP, calmodulin-binding peptide; CUL4A, cullin 4A; DDB1, damaged DNA binding protein 1; DDB2, damaged DNA binding protein 2; GFP, green fluorescent protein; HA, hemagglutinin; HEK, human embryonic kidney; HIV- 1, human immunodeficiency virus type 1; RBX1, ring-box 1; siRNA, small interfering RNA; TAP, tandem affinity purification; TEV, tobacco etch virus; UNG2, uracil-DNA glycosylase; Vpr, viral protein R; VPRBP, viral protein R binding protein; VSV-G, vesicular stomatatis virus glycoprotein G; XPC, xeroderma pigmentosum group C protein * To whom correspondence should be addressed. E-mail: eric.cohen@ircm.qc.ca * To whom correspondence should be addressed. E-mail: eric.cohen@ircm.qc.ca [ These authors contributed equally to this work. ¤ Current address: Centre de The´rapie Ge´nique, Faculte´ de Me´decine, Marseille, France PLoS Pathogens \| www.plospathogens.org July 2007 \| Volume 3 \| Issue 7 \| e85 July 2007 \| Volume 3 \| Issue 7 \| e85 0882 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest Author Summary Vpr Interacts with DDB1 and VPRBP To purify cellular protein complexes interacting with HIV Vpr, Vpr was fused to a TAP tag containing two immuno- globulin-binding domains of protein A from Staphylococcus aureus, a cleavage site for the tobacco etch virus (TEV) protease, and the calmodulin-binding peptide (CBP). Since Vpr C-terminal modiﬁcations have been reported to alter its cytostatic abilities [23], the bipartite tag was introduced N- terminally. Puriﬁcation of TAP-Vpr–containing complexes was then conducted in human embryonic kidney (HEK) 293T cells, although the tagged protein induced cell cycle arrest less efﬁciently than wild-type Vpr (unpublished data). After electrophoresis and silver staining, two bands corresponding to high–molecular weight proteins were repeatedly observed (unpublished data). Matrix-assisted laser desorption/ioniza- To conﬁrm the speciﬁcity of interaction between Vpr and VPRBP in our system, we performed co-immunoprecipitation experiments in the presence of endogenous VPRBP and over- expressed HA-Vpr (Figure 1C). We could speciﬁcally detect co-immunoprecipitated VPRBP in the presence of HA-Vpr (lane 2), but not in the presence of the empty plasmid (lane 1). Finally, we observed an interaction between over-expressed HA-Vpr and Myc-tagged VPRBP (Figure 1D), conﬁrming the speciﬁcity of the interaction between Vpr and VPRBP. July 2007 \| Volume 3 \| Issue 7 \| e85 July 2007 \| Volume 3 \| Issue 7 \| e85 0883 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest Figure 1. Immunoprecipitation of DDB1/Vpr and VPRBP/Vpr Complexes (A) HEK293T cells were mock transfected (lanes 1) or transfected with either TAP (lanes 2) or TAP-Vpr–expressing plasmids (lanes 3). Two days later, immunoprecipitations of TAP tag were performed on cell lysates using IgG-coupled beads and purified complexes were eluted by cleavage with TEV protease. The levels of endogenous VPRBP and DDB1 were monitored in crude lysates and pulled-down fractions by Western blot using specific antibodies. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide. (B) HEK293T cells were mock transfected (lanes 1 and 2) or transfected with either TAP (lanes 3 and 5) or TAP-Vpr–expressing plasmids (lanes 4 and 6). Cells were transcomplemented with the empty vector (lanes 1, 3, and 4) or HA-DDB1–encoding plasmid (lanes 2, 5, and 6). (C) HEK293T cells were mock transfected (lanes 1) or transfected with HA-Vpr–expressing plasmid (lanes 2). Immunoprecipitations using anti-HA antibodies were performed on cell extracts using protein A–sepharose beads. DDB1 Appears to Bind to Vpr Indirectly observe DDB1 expression by Western blot in Vpr-expressing yeast cells (Figure 2A). Finally, an association between Vpr and DDB1 could not be detected in co-immunoprecipitation experiments using in vitro–translated T7-tagged Vpr and HA-DDB1 proteins (Figure 2B) in conditions that have demonstrated an association between Vpr and VPRBP [21]; this suggests that the binding observed in human cells might be indirect and may involve a bridging factor. To further investigate the apparent association between TAP-Vpr and DDB1, Vpr, TAP, and TAP-Vpr sequences were subcloned in yeast two-hybrid expression constructs. Saccha- romyces cerevisae cells were co-transformed with each combi- nation of plasmids. Interaction afﬁnities were determined by measurement of the b-galactosidase activity. As expected, Vpr appeared to form homodimers, leading to strong reporter gene activity (Figure 2A). Surprisingly, no binding could be detected between Vpr and DDB1, a result consistent with the lack of interaction observed with some co-immunoprecipita- tion experiments. We found that dimerization of TAP-Vpr was three times weaker than that of the wild-type protein. Hence, N-terminal addition of large peptide appears to disturb the tertiary structure of Vpr, explaining at least in part the reduction of TAP-Vpr cytostatic abilities. Impor- tantly, we found that b-galactosidase activity remained undetectable in cells co-expressing TAP-Vpr and DDB1. This lack of stable interaction between Vpr and DDB1 did not result from lack of DDB1 expression in yeast, since we could Vpr Associates with a DDB1-CUL4AVPRBP E3 Ubiquitin Ligase Complex Vpr Interacts with DDB1 and VPRBP (B) HEK293T cells were mock transfected (lanes 1 and 2) or transfected with either TAP (lanes 3 and 5) or TAP-Vpr–expressing plasmids (lanes 4 and 6). Cells were transcomplemented with the empty vector (lanes 1, 3, and 4) or HA-DDB1–encoding plasmid (lanes 2, 5, and 6). (C) HEK293T cells were mock transfected (lanes 1) or transfected with HA-Vpr–expressing plasmid (lanes 2). Immunoprecipitations using anti-HA antibodies were performed on cell extracts using protein A–sepharose beads. The levels of HA-Vpr and endogenous VPRBP were monitored in cell extracts as well as immunoprecipitated fractions by Western blot using specific antibodies. (D) HEK293T cells were mock transfected (lanes 1 and 3) or transfected with a HA-Vpr–expressing plasmid (lanes 2 and 4). Cells were transcomplemented with the empty vector (lanes 1 and 2) or Myc-VPRBP–encoding plasmid (lanes 3 and 4). Anti-HA immunoprecipitations were performed as described above. d i 10 1371/j l t 0030085 001 p p y g p (D) HEK293T cells were mock transfected (lanes 1 and 3) or transfected with a HA-Vpr–expressing plasmid (lanes 2 and 4). Cells were transcomplemented with the empty vector (lanes 1 and 2) or Myc-VPRBP–encoding plasmid (lanes 3 and 4). Anti-HA immunoprecipitations were performed as described above. doi:10.1371/journal.ppat.0030085.g001 Vpr Interacts with DDB1 and VPRBP The levels of HA-Vpr and endogenous VPRBP were monitored in cell extracts as well as immunoprecipitated fractions by Western blot using specific antibodies. (D) HEK293T cells were mock transfected (lanes 1 and 3) or transfected with a HA-Vpr–expressing plasmid (lanes 2 and 4). Cells were transcomplemented with the empty vector (lanes 1 and 2) or Myc-VPRBP–encoding plasmid (lanes 3 and 4). Anti-HA immunoprecipitations were performed as described above. doi:10.1371/journal.ppat.0030085.g001 C l Figure 1. Immunoprecipitation of DDB1/Vpr and VPRBP/Vpr Complexes gure 1. Immunoprecipitation of DDB1/Vpr and VPRBP/Vp g (A) HEK293T cells were mock transfected (lanes 1) or transfected with either TAP (lanes 2) or TAP-Vpr–expressing plasmids (lanes 3). Two days later, immunoprecipitations of TAP tag were performed on cell lysates using IgG-coupled beads and purified complexes were eluted by cleavage with TEV protease. The levels of endogenous VPRBP and DDB1 were monitored in crude lysates and pulled-down fractions by Western blot using specific antibodies. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide. (B) HEK293T cells were mock transfected (lanes 1 and 2) or transfected with either TAP (lanes 3 and 5) or TAP-Vpr–expressing plasmids (lanes 4 and 6). Cells were transcomplemented with the empty vector (lanes 1, 3, and 4) or HA-DDB1–encoding plasmid (lanes 2, 5, and 6). (C) HEK293T cells were mock transfected (lanes 1) or transfected with HA-Vpr–expressing plasmid (lanes 2). Immunoprecipitations using anti-HA antibodies were performed on cell extracts using protein A–sepharose beads. The levels of HA-Vpr and endogenous VPRBP were monitored in cell extracts as well as immunoprecipitated fractions by Western blot using specific antibodies. (D) HEK293T cells were mock transfected (lanes 1 and 3) or transfected with a HA-Vpr–expressing plasmid (lanes 2 and 4). Cells were transcomplemented with the empty vector (lanes 1 and 2) or Myc-VPRBP–encoding plasmid (lanes 3 and 4). Anti-HA immunoprecipitations were (A) HEK293T cells were mock transfected (lanes 1) or transfected with either TAP (lanes 2) or TAP-Vpr–expressing plasmids (lanes 3). Two days later, immunoprecipitations of TAP tag were performed on cell lysates using IgG-coupled beads and purified complexes were eluted by cleavage with TEV protease. The levels of endogenous VPRBP and DDB1 were monitored in crude lysates and pulled-down fractions by Western blot using specific antibodies. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide. PLoS Pathogens \| www.plospathogens.org Vpr Associates with a DDB1-CUL4AVPRBP E3 Ubiquitin Ligase Complex One possible explanation for the apparent association between Vpr and DDB1 is that Vpr would associate with the DDB1-CUL4A-RBX1 ubiquitin ligase complex through a direct interaction with VPRBP. Indeed, recently the WD40 protein VPRBP has been demonstrated to interact with DDB1 and probably serves as an adapter to confer substrate speciﬁcity to the DDB1-CUL4A-RBX1 E3 ubiquitin ligase complex [20]. To assess the possibility that the Vpr-VPRBP-DDB1 complex might be part of an ubiquitin E3 ligase complex, 0884 July 2007 \| Volume 3 \| Issue 7 \| e85 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest Figure 2. Absence of Direct Vpr Binding to DDB1 in Yeast (A) The EGY48 reporter strain containing LexA-TAP, LexA-Vpr, or LexA-TAP-Vpr (‘‘bait’’) was transformed with B42, B42-DDB1, or B42-Vpr–expressing plasmid (‘‘prey’’). The binding affinity between the different proteins was assessed by assaying b-galactosidase activity using the o-nitrophenyl-b-D- galactopyranoside method. Histograms represent averaged data from 2–4 different clones and are representative of two independent assays. Western blot analysis of induced and non-induced B42-HA-DDB1 expression in the B42 and B42-DDB1-transformed EGY48/LexA-Vpr reporter strain is shown below. (B) In vitro–translated T7-Vpr was immunoprecipitated with an anti-T7 antibody in the presence or absence of in vitro–translated HA-DDB1. Amounts of protein initially added to the assay (input) are shown in the left panel. * represents non-specific proteins immunoprecipitated by the anti-T7 antibody. doi:10.1371/journal.ppat.0030085.g002 Figure 2. Absence of Direct Vpr Binding to DDB1 in Yeast Figure 2. Absence of Direct Vpr Binding to DDB1 in Yeast (A) The EGY48 reporter strain containing LexA-TAP, LexA-Vpr, or LexA-TAP-Vpr (‘‘bait’’) was transformed with B42, B42-DDB1, or B42-Vpr–expressing plasmid (‘‘prey’’). The binding affinity between the different proteins was assessed by assaying b-galactosidase activity using the o-nitrophenyl-b-D- galactopyranoside method. Histograms represent averaged data from 2–4 different clones and are representative of two independent assays. Western blot analysis of induced and non-induced B42-HA-DDB1 expression in the B42 and B42-DDB1-transformed EGY48/LexA-Vpr reporter strain is shown below. (B) In vitro–translated T7-Vpr was immunoprecipitated with an anti-T7 antibody in the presence or absence of in vitro–translated HA-DDB1. Amounts of protein initially added to the assay (input) are shown in the left panel. * represents non-specific proteins immunoprecipitated by the anti-T7 antibody. doi:10.1371/journal.ppat.0030085.g002 cytostatic properties. Cells transfected with VPRBP siRNA displayed a major reduction of VPRBP at the mRNA level (Figure 4A) as well as at the protein level (Figure 4B) compared with scrambled siRNA-transfected cells. Vpr Associates with a DDB1-CUL4AVPRBP E3 Ubiquitin Ligase Complex We thus transfected HEK293 cells with siRNA against VPRBP and, 24 h later, transduced these cells with a lentiviral vector co- expressing GFP and native Vpr. We observed that cells transfected with VPRBP siRNA displayed strongly attenuated Vpr-mediated G2 arrest as compared with cells that received scrambled control siRNA (Figure 5A). This difference in the induction of G2 arrest was not due to a defect in infectivity potentially resulting from VPRBP knockdown, because the levels of transduction were equivalent in all the conditions tested (unpublished data). To verify that this defect in the induction of G2 arrest in VPRBP-depleted cells was the result of the abrogation of the Vpr–VPRBP interaction rather than a defect in cell growth, we treated these cells with nocodazole with the rationale that properly cycling cells should accumu- late in mitosis because of the effect of the drug on microtubule polymerization (Figure 5B). In response to nocodazole, cells transfected with VPRBP siRNA alone or with the concomitant expression of Vpr accumulated at the G2/M phase, indicating that knockdown of VPRBP speciﬁcally impaired Vpr-mediated G2 arrest functions. Moreover, the knockdown of VPRBP did not produce any observable defects in the activation of the ATR-mediated checkpoints, since treatment with low concentrations of aphidicolin, a DNA replication inhibitor known to produce DNA double-strand breaks at fragile chromosomal sites and to activate the ATR- mediated intra-S checkpoint [29–31], blocked cell cycle progression at the S-phase in scrambled as well as VPRBP siRNA-transfected cells (Figure 5C). Finally, we analyzed the we investigated whether Vpr could associate with the E3 ligase scaffold protein CUL4A. HEK293T cells were trans- fected with plasmids expressing TAP and TAP-Vpr, and pull- down assays were performed on cell extracts containing endogenous CUL4A. These assays demonstrated that endog- enous CUL4A could speciﬁcally associate with TAP-Vpr but not with native TAP (Figure 3A, lane 3), suggesting that Vpr associates with a CUL4A-scaffolded E3 ligase complex. Moreover, anti-CUL4A immunoprecipitation experiments on cells transfected with an empty plasmid or an HA-Vpr– expressing plasmid revealed that HA-Vpr co-immunopreci- pitated with a CUL4A-containing complex (Figure 3B, lane 2), conﬁrming that Vpr can associate with an E3 ligase complex with potential ubiquitinating activities. Importantly, VPRBP could be co-immunoprecipitated along with CUL4A (Figure 3B). Vpr Associates with a DDB1-CUL4AVPRBP E3 Ubiquitin Ligase Complex Therefore, given the mutual association of Vpr (Figure 1A, 1C, and 1D therein and [21,22]) and CUL4A (Figure 3B therein and [20]) with VPRBP, these results suggest that Vpr interacts with the DDB1-CUL4AVPRBP E3 ubiquitin ligase in a single complex and that this association might occur via the intermediary of VPRBP. The Interaction between DDB1-CUL4AVPRBP and Vpr Is Required for the Induction of G2 Arrest PLoS Pathogens \| www.plospathogens.org The Interaction between DDB1-CUL4AVPRB Required for the Induction of G2 Arrest TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide. (B) Ten million HEK293T cells were transfected with 80 lg of empty plasmid (lanes 1) or with HA-Vpr–expressing plasmid (lanes 2). Immunoprecipitation of endogenous CUL4A was performed using a goat polyclonal anti-CUL4A antibody and protein A–sepharose beads. The levels of endogenous CUL4A, VPRBP, and over-expressed HA-Vpr were monitored in crude lysates and immunoprecipitated fractions by Western blot using, respectively, a polyclonal goat anti-CUL4A antibody, a polyclonal rabbit anti-VPRBP antibody, and a monoclonal mouse anti-HA antibody. * represents a non-specific protein detected by the anti-CUL4A antibody. The anti-CUL4A antibody generally recognized a doublet of CUL4A when the gel resolution was sufficiently high. In the TAP pull-down fractions, only the upper band of CUL4A was detected. doi:10.1371/journal.ppat.0030085.g003 g g (A) Five million HEK293T cells were transfected with 40 lg of empty (lanes 1), TAP-expressing (lanes 2), or TAP-Vpr–ex eight hours after transfection, TAP pull-downs were performed on cell lysates using IgG-coupled beads, and pu cleavage with TEV protease. The levels of endogenous CUL4A were monitored in crude lysates and pulled-down polyclonal goat anti-CUL4A antibody. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibod peptide peptide. (B) Ten million HEK293T cells were transfected with 80 lg of empty plasmid (lanes 1) or with HA-Vpr–expressing plasmid (lanes 2). Immunoprecipitation of endogenous CUL4A was performed using a goat polyclonal anti-CUL4A antibody and protein A–sepharose beads. The levels of endogenous CUL4A, VPRBP, and over-expressed HA-Vpr were monitored in crude lysates and immunoprecipitated fractions by Western blot using, respectively, a polyclonal goat anti-CUL4A antibody, a polyclonal rabbit anti-VPRBP antibody, and a monoclonal mouse anti-HA antibody. * represents a non-specific protein detected by the anti-CUL4A antibody. The anti-CUL4A antibody generally recognized a doublet of CUL4A when the gel resolution was sufficiently high. In the TAP pull-down fractions, only the upper band of CUL4A was detected. doi:10.1371/journal.ppat.0030085.g003 role of VPRBP in the induction of G2 arrest by Vpr in the context of viral infection. SiRNA-transfected cells were infected with vesicular stomatatis virus glycoprotein G (VSV-G)–pseudotyped fully infectious isogenic viruses defec- tive (NL4–3DVpr-GFP, vpr) or proﬁcient (NL4–3-GFP, vprþ) for Vpr expression. Cells were analyzed for their cell cycle proﬁle 48 h after infection. As expected, cells transfected with scrambled control siRNA accumulated in G2 after infection with the vprþ but not with the vpr virus (Figure 5D). The Interaction between DDB1-CUL4AVPRB Required for the Induction of G2 Arrest We next sought to study the potential role of DDB1- CUL4AVPRBP E3 ubiquitin ligase in the induction of G2 cell cycle arrest by Vpr. DDB1 depletion by small interfering RNA (siRNA) has been reported to induce the accumulation of cells in G2/M due to DNA re-replication [28]; consequently, this strategy could not be used to demonstrate the involve- ment of DDB1-CUL4AVPRBP in the induction of Vpr- mediated G2 arrest. As an alternative strategy, we analyzed the effect of siRNA-mediated VPRBP knockdown on Vpr 0885 July 2007 \| Volume 3 \| Issue 7 \| e85 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest Figure 3. Vpr Associates with the Ubiquitin Ligase Scaffold Protein CUL4A (A) Five million HEK293T cells were transfected with 40 lg of empty (lanes 1), TAP-expressing (lanes 2), or TAP-Vpr–expressing plasmids (lanes 3). Forty- eight hours after transfection, TAP pull-downs were performed on cell lysates using IgG-coupled beads, and purified complexes were eluted by cleavage with TEV protease. The levels of endogenous CUL4A were monitored in crude lysates and pulled-down fractions by Western blot using a polyclonal goat anti-CUL4A antibody. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide. (B) Ten million HEK293T cells were transfected with 80 lg of empty plasmid (lanes 1) or with HA-Vpr–expressing plasmid (lanes 2). Immunoprecipitation of endogenous CUL4A was performed using a goat polyclonal anti-CUL4A antibody and protein A–sepharose beads. The levels of endogenous CUL4A, VPRBP, and over-expressed HA-Vpr were monitored in crude lysates and immunoprecipitated fractions by Western blot using, respectively, a polyclonal goat anti-CUL4A antibody, a polyclonal rabbit anti-VPRBP antibody, and a monoclonal mouse anti-HA antibody. * represents a non-specific protein detected by the anti-CUL4A antibody. The anti-CUL4A antibody generally recognized a doublet of CUL4A when the gel resolution was sufficiently high. In the TAP pull-down fractions, only the upper band of CUL4A was detected. doi:10.1371/journal.ppat.0030085.g003 Figure 3. Vpr Associates with the Ubiquitin Ligase Scaffold Protein CUL4A g p q g (A) Five million HEK293T cells were transfected with 40 lg of empty (lanes 1), TAP-expressing (lanes 2), or TAP-Vpr–expressing plasmids (lanes 3). Forty- eight hours after transfection, TAP pull-downs were performed on cell lysates using IgG-coupled beads, and purified complexes were eluted by cleavage with TEV protease. The levels of endogenous CUL4A were monitored in crude lysates and pulled-down fractions by Western blot using a polyclonal goat anti-CUL4A antibody. The Interaction between DDB1-CUL4AVPRB Required for the Induction of G2 Arrest We analyzed whether the L64A and Q65R mutations in this domain of Vpr could abrogate binding to VPRBP. Using our TAP-Vpr pull- down assay, we observed a very strong reduction of binding to To examine whether the association of Vpr to the E3 ligase complex is required, we tested the ability of several TAP- tagged Vpr mutants to associate with VPRBP and DDB1 (Figure 6A and 6B) and assessed their effect on Vpr-induced G2 arrest (Figure 6C). HEK293T cells were transfected with Figure 4. Depletion of VPRBP Using siRNA HEK293 cells were transfected with 300 pmol of VPRBP-targeting siRNA or control scrambled siRNA using Oligofectamine. (A) At 24, 48, and 72 h post-transfection, RNA was extracted and analyzed by RT-PCR to determine the extent of VPRBP downregulation at the mRNA level. PCR products were analyzed in the exponential phase of amplification. Actin levels were used as a control for RNA quality and reverse transcription efficiency. (B) Forty-eight hours after siRNA transfection, cells lysates were harvested and analyzed by Western blot using a polyclonal rabbit anti-VPRBP antibody Fi D l i f VPRBP U i iRNA Figure 4. Depletion of VPRBP Using siRNA g p g HEK293 cells were transfected with 300 pmol of VPRBP-targeting siRNA or control scrambled siRNA using Oligofectamine. (A) At 24, 48, and 72 h post-transfection, RNA was extracted and analyzed by RT-PCR to determine the extent of VPRBP downregulation at the mRNA level. PCR products were analyzed in the exponential phase of amplification. Actin levels were used as a control for RNA quality and reverse transcription efficiency. HEK293 cells were transfected with 300 pmol of VPRBP-targeting siRNA or control scrambled siRNA using Oligofectamine. (A) At 24, 48, and 72 h post-transfection, RNA was extracted and analyzed by RT-PCR to determine the extent of VPRBP downregulation at the mRNA level. PCR products were analyzed in the exponential phase of amplification. Actin levels were used as a control for RNA quality and reverse transcription efficiency transcription efficiency. (B) Forty-eight hours after siRNA transfection, cells lysates were harvested and analyzed by Western blot using a polyclonal rabbit anti-VPRBP antibody to demonstrate the downregulation of VPRBP at the protein level. Actin levels were used as a control for protein loading. The Interaction between DDB1-CUL4AVPRB Required for the Induction of G2 Arrest Knockdown of VPRBP almost abrogated the accumulation of cells in G2 in response to vprþ virus infection, but did not signiﬁcantly affect the cell cycle proﬁle of vpr virus–infected cells. Again, these differences in G2 arrest were not the result of differential infectivity (unpublished data). Overall, these results indicate that VPRBP is necessary for Vpr-induced G2 arrest, suggesting that the DDB1-CUL4AVPRBP E3 ligase complex might be involved in this Vpr biological activity. role of VPRBP in the induction of G2 arrest by Vpr in the context of viral infection. SiRNA-transfected cells were infected with vesicular stomatatis virus glycoprotein G (VSV-G)–pseudotyped fully infectious isogenic viruses defec- tive (NL4–3DVpr-GFP, vpr) or proﬁcient (NL4–3-GFP, vprþ) for Vpr expression. Cells were analyzed for their cell cycle proﬁle 48 h after infection. As expected, cells transfected with scrambled control siRNA accumulated in G2 after infection with the vprþ but not with the vpr virus (Figure 5D). Knockdown of VPRBP almost abrogated the accumulation of cells in G2 in response to vprþ virus infection, but did not signiﬁcantly affect the cell cycle proﬁle of vpr virus–infected cells. Again, these differences in G2 arrest were not the result of differential infectivity (unpublished data). Overall, these results indicate that VPRBP is necessary for Vpr-induced G2 arrest, suggesting that the DDB1-CUL4AVPRBP E3 ligase complex might be involved in this Vpr biological activity. plasmids expressing mutants of TAP-Vpr, and TAP pull-down experiments were performed on these transfectant cellular extracts. As previously observed, TAP-Vpr was able to pull down endogenous VPRBP and DDB1 (Figure 6A, lane 3). Interestingly, the classical Vpr mutants S79A and R80A that are attenuated for the induction of G2 arrest (Figure 6C; [32,33]) could still associate with VPRBP and DDB1 at levels comparable with that of the wild-type protein (Figure 6A, compare lane 3 with lanes 5 and 6), suggesting that the association between Vpr and the DDB1-VPRBP complex, per se, is not sufﬁcient to block cell cycle progression. Moreover, W54R, a mutant of Vpr that was previously shown to be defective for the interaction and degradation of uracil-DNA glycosylase (UNG2/CCNU) [34], was still capable of associating with the DDB1-CUL4AVPRBPcomplex (Figure 6A, lane 4) and mediating G2 arrest (Figure 6C), suggesting that UNG2 and VPRBP bind to two distinct domains on Vpr. Zhao et al. previously mapped the domain of interaction of VPRBP to the leucine/isoleucine–rich domain of Vpr [22]. Discussion The induction of G2 arrest by the HIV-1 accessory protein Vpr was described more than ten years ago [3–6]; however, the mechanism by which Vpr can accomplish this function has remained elusive. Several recent reports have demon- strated that expression of Vpr leads to activation of ATR, as well as to the formation of DNA repair foci containing BRCA1 and c-H2AFX [14–16]. Nonetheless, the initial events leading to ATR checkpoint signaling are not known. Herein, we have used the TAP method to isolate cellular protein complexes interacting with HIV-1 Vpr to identify cellular factors that would be involved in Vpr-mediated ATR activation and subsequent G2 arrest. Using this strategy, we have identiﬁed DDB1 and VPRBP as cellular factors forming a complex with Vpr. In our system, an interaction between Vpr and DDB1 was observed when Vpr was fused to a TAP tag (TAP experiments, Figure 1A and 1B). However, co-precipitations in the reverse direction with DDB1 fused to other tags (TAP, HA, or GFP) only yielded inconsistent interaction results (unpublished data). The lack of apparent interaction between DDB1 and Vpr in that context may be due to our experimental conditions or to the type of association that engages Vpr and DDB1. In that regard, the lack of interaction between Vpr and DDB1 in the yeast two-hybrid system (Figure 2A) as well as in in vitro co-precipitation experiments (Figure 2B) argued for an indirect interaction between these two proteins, though deﬁnite demonstration of this will require a more thorough analysis of this association in the future. These results were in contrast to SV5 V protein, which can directly interact with the DDB1 bPA–bPC domain [35,49]. Through the use of TAP procedures (unpublished data), immunoprecipitation experiments (Figure 1), and several Vpr mutants (Figure 6), we identiﬁed VPRBP as a cellular factor also in a complex with Vpr and DDB1 that possibly permits the recruitment of a DDB1-CUL4A E3 ubiquitin ligase complex. The cellular function of VPRBP has recently been uncovered through different proteomic approaches. It is a WD40 protein linked to the DDB1-CUL4A-RBX1 complex and probably serves as an adapter presenting protein DDB1 is part of the DDB1-CUL4A-RBX1 E3 ubiquitin ligase complex that targets proteins for degradation via the COP9 signalosome [26]. In this complex, DDB1 serves as a scaffold protein presenting substrate to the E3 ubiquitin ligase. The Interaction between DDB1-CUL4AVPRB Required for the Induction of G2 Arrest (D) T d i if VPRBP k kd ld l b h i d i f G2 i h f i l i f i iRNA f d ll treatment. (D) To determine if VPRBP knockdown could also abrogate the induction of G2 arrest in the context of viral infection, siRNA-transfected cells were treatment. (D) To determine if VPRBP knockdown could also abrogate the induction of G2 arrest in the context of viral infection, siRNA-transfected cells were infected with NL4.3-GFP and NL4–3DVpr-GFP at a concentration of 100 cpm/cell and cell cycle profiles were analyzed 48 h later. (D) To determine if VPRBP knockdown could also abrogate the induction of G2 arrest in the context of viral infection, siRNA-transfected cells were infected with NL4.3-GFP and NL4–3DVpr-GFP at a concentration of 100 cpm/cell and cell cycle profiles were analyzed 48 h later. Percentages of G1 and G2/M cell populations were determined using the ModFit software. These results are representative of the data obtained in at least two independent experiments. infected with NL4.3-GFP and NL4–3DVpr-GFP at a concentration of 100 cpm/cell and cell cycle profiles were analyzed 48 h later. Percentages of G1 and G2/M cell populations were determined using the ModFit software. These results are representative of the data obtained in at least two independent experiments. p p doi:10.1371/journal.ppat.0030085.g005 VPRBP and DDB1 with the Q65R mutant (under longer exposure VPRBP could be detected) (Figure 6B, compare lanes 3 and 4). A residual association with DDB1 was observed under these conditions, most likely reﬂecting difference in antibody afﬁnities. Additionally, the reduction of TAP-Vpr binding to VPRBP and DDB1 could be observed with the L64A mutant, though it was less pronounced (Figure 6B, compare lanes 5 and 6). Interestingly, with both mutants, the reduced afﬁnity for VPRBP was accompanied by a concom- itant decrease in the association with DDB1, again suggesting that VPRBP and DDB1 are likely to form a single complex with Vpr (Figure 6B, compare lane 3 with lane 4 and lane 5 with lane 6). Importantly, we found that Vpr (L64A) and Vpr (Q65R) were strongly attenuated for the induction of G2 arrest (Figure 6C). Indeed, the residual level of G2 arrest observed with these two mutants was comparable with the attenuated G2 arrest produced by the R80A mutation. The Interaction between DDB1-CUL4AVPRB Required for the Induction of G2 Arrest Together, these results suggest that the interaction of Vpr with the DDB1-CUL4AVPRBP E3 ubiquitin ligase complex is necessary but not sufﬁcient to induce G2 arrest. a few cellular proteins have been found to be regulated by the DDB1-CUL4A-RBX1 complex. In human cells, DDB1-CU- L4ADDB2 promotes the ubiquitylation of histone 2A [38], histone 3, histone 4 [39], and the xeroderma pigmentosum group C protein (XPC) to regulate their activity [40]. In contrast, DDB1-CUL4ACSA and DDB1-CUL4ADET1-COP1 pro- mote proteolysis of Cockayne syndrome type B gene product (CSB) [26] and c-JUN [41]. Recently, DDB1, via the WD40 adapter CDT2, has been shown to prevent DNA re-repli- cation during normal S-phase progression or in response to S-phase accumulation of DNA lesions by regulating the degradation of the replication licensing factor CDT1 [27,28]. Interestingly, DDB1 also forms complexes with two other viral proteins, namely hepatitis B virus X protein [42–44] and simian paramyxovirus SV5 V protein [45,46]. Whereas the mechanisms underlying the DDB1-dependent cytotoxicity induced by hepatitis B virus X protein remain poorly understood, it has been established that SV5 V protein facilitates the ubiquitination and subsequent signalosome- mediated degradation of STAT1 [45]. Several data argue in favor of a functional involvement of DDB1-CUL4A-RBX1 in Vpr functions. CUL4A has been implicated in Vpr-induced degradation of UNG2 and SMUG1 proteins [47], and Vpr was shown to interact with VIP/mov34/CNS6, one of the subunits of CUL4A-associated signalosome [48]. PLoS Pathogens \| www.plospathogens.org July 2007 \| Volume 3 \| Issue 7 \| e85 The Interaction between DDB1-CUL4AVPRB Required for the Induction of G2 Arrest d i 10 1371/j l t 0030085 004 p y (B) Forty-eight hours after siRNA transfection, cells lysates were harvested and analyzed by Western blot using a polyclonal rabbit anti-VPRBP antibody to demonstrate the downregulation of VPRBP at the protein level. Actin levels were used as a control for protein loading. doi:10 1371/journal ppat 0030085 g004 PLoS Pathogens \| www.plospathogens.org July 2007 \| Volume 3 \| Issue 7 \| e85 July 2007 \| Volume 3 \| Issue 7 \| e85 0886 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest PLoS Pathogens \| www.plospathogens.org July 2007 \| Volume 3 \| Issue 7 \| 0887 PLoS Pathogens \| www.plospathogens.org PLoS Pathogens \| www.plospathogens.org July 2007 \| Volume 3 \| Issue 7 \| e85 July 2007 \| Volume 3 \| Issue 7 \| e85 July 2007 \| Volume 3 \| Issue 7 \| e85 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest ure 5. Effect of VPRBP Depletion on Vpr-Induced G2 Arres (A) HEK293 cells were transfected with 300 pmol of VPRBP-targeting siRNA or control scrambled siRNA using Oligofectamine, followed by the same transfection 24 h later. Twenty-four hours after the second siRNA transfection, cells were transduced at a multiplicity of infection of 1 with lentiviral vectors expressing Vpr (WPI-Vpr) or the empty vector (WPI). Cell cycle profiles were analyzed 24 h after transduction by flow cytometry using propidium iodide staining. iodide staining. (B and C) To determine if cell growth (B) or checkpoint activation (C) was affected by VPRBP knockdown, HEK293 cells were transfected once with siRNA, as described above, and treated respectively with 1 lg/ml nocodazole and 0.5 lM aphidicolin 24 h later. Cell cycle profiles were analyzed 24 h after drug treatment g (B and C) To determine if cell growth (B) or checkpoint activation (C) was affected by VPRBP knockdown, HEK293 cells as described above, and treated respectively with 1 lg/ml nocodazole and 0.5 lM aphidicolin 24 h later. Cell cycle pro treatment. (B and C) To determine if cell growth (B) or checkpoint activation (C) was affected by VPRBP knockdown, HEK293 cells were transfected once with siRNA, as described above, and treated respectively with 1 lg/ml nocodazole and 0.5 lM aphidicolin 24 h later. Cell cycle profiles were analyzed 24 h after drug treatment. Discussion Following TAP pull-down using IgG- coupled beads, the levels of endogenous VPRBP and DDB1 were monitored in crude lysates and pulled-down fractions by Western blot using specific antibodies. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide. (B) HEK293T cells were transfected with TAP-Vpr plasmids encoding for wild-type Bru Vpr (lanes 3) and wild-type NL4–3 Vpr (lanes 5), or Vpr mutants Bru Q65R (lanes 4) and NL4–3 L64A (lanes 6). As control, cells were mock transfected (lanes 1) or transfected with a TAP-expressing plasmid (lanes 2). TAP pull-downs and immunodetection of VPRBP, DDB1, TAP, and Vpr were performed as described for (A). (C) HEK293T cells were co-transfected with 1 lg of GFP-expressing plasmid and 15 lg of TAP-Vpr plasmids expressing wild-type or mutant proteins. Cell cycle analysis was performed using propidium iodide staining on the GFPþ cell population as described in Materials and Methods. Percentages of G1 and G2/M cell populations were determined using the ModFit software. doi:10.1371/journal.ppat.0030085.g006 (A) HEK293T cells were transfected with TAP-Vpr plasmids encoding for wild-type Vpr (lanes 3) or Vpr mutants W54R (lanes 4), S79A (lanes 5), and R80A (lanes 6). As control, cells were mock transfected (lanes 1) or transfected with a TAP-expressing plasmid (lanes 2). Following TAP pull-down using IgG- coupled beads, the levels of endogenous VPRBP and DDB1 were monitored in crude lysates and pulled-down fractions by Western blot using specific antibodies. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide. (B) HEK293T cells were transfected with TAP-Vpr plasmids encoding for wild-type Bru Vpr (lanes 3) and wild-type NL4–3 Vpr (lanes 5), or Vpr mutants Bru Q65R (lanes 4) and NL4–3 L64A (lanes 6). As control, cells were mock transfected (lanes 1) or transfected with a TAP-expressing plasmid (lanes 2). TAP pull-downs and immunodetection of VPRBP, DDB1, TAP, and Vpr were performed as described for (A). (C) HEK293T cells were co-transfected with 1 lg of GFP-expressing plasmid and 15 lg of TAP-Vpr plasmids expressing wild-type or mutant proteins. Cell cycle analysis was performed using propidium iodide staining on the GFPþ cell population as described in Materials and Methods. Percentages of G1 and G2/M cell populations were determined using the ModFit software. doi:10.1371/journal.ppat.0030085.g006 substrate for degradation [20]. PLoS Pathogens \| www.plospathogens.org Discussion The protein is structurally complex and contains three seven-bladed b propellers (bPA, bPB, and bPC) [35]. The bPB propeller is involved in the interaction with CUL4A, whereas the bPA–bPC double-propeller fold is responsible for substrate presentation via interaction with WD40-repeat proteins [35]. Over 15 different WD40-containing substrate receptors, including DDB2, CSA, DET1-COP1, and CDT2, have been shown to interact with DDB1 and are thought to confer substrate speciﬁcity [20,35–37]. However, to date, only PLoS Pathogens \| www.plospathogens.org July 2007 \| Volume 3 \| Issue 7 \| e85 0888 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest Figure 6. DDB1 and VPRBP Binding Affinities of TAP-Tagged Vpr Mutants (A) HEK293T cells were transfected with TAP-Vpr plasmids encoding for wild-type Vpr (lanes 3) or Vpr mutants W54R (lanes 4), S79A (lanes 5), and R80A (lanes 6). As control, cells were mock transfected (lanes 1) or transfected with a TAP-expressing plasmid (lanes 2). Following TAP pull-down using IgG- coupled beads, the levels of endogenous VPRBP and DDB1 were monitored in crude lysates and pulled-down fractions by Western blot using specific antibodies. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide. (B) HEK293T cells were transfected with TAP-Vpr plasmids encoding for wild-type Bru Vpr (lanes 3) and wild-type NL4–3 Vpr (lanes 5), or Vpr mutants Bru Q65R (lanes 4) and NL4–3 L64A (lanes 6). As control, cells were mock transfected (lanes 1) or transfected with a TAP-expressing plasmid (lanes 2). TAP pull-downs and immunodetection of VPRBP, DDB1, TAP, and Vpr were performed as described for (A). (C) HEK293T cells were co-transfected with 1 lg of GFP-expressing plasmid and 15 lg of TAP-Vpr plasmids expressing wild-type or mutant proteins. Cell cycle analysis was performed using propidium iodide staining on the GFPþ cell population as described in Materials and Methods. Percentages of G1 and G2/M cell populations were determined using the ModFit software. doi:10.1371/journal.ppat.0030085.g006 i d d ff f d Figure 6. DDB1 and VPRBP Binding Affinities of TAP-Tagged Vpr Mutants Figure 6. DDB1 and VPRBP Binding Affinities of TAP-Tagg (A) HEK293T cells were transfected with TAP-Vpr plasmids encoding for wild-type Vpr (lanes 3) or Vpr mutants W54R (lanes 4), S79A (lanes 5), and R80A (lanes 6). As control, cells were mock transfected (lanes 1) or transfected with a TAP-expressing plasmid (lanes 2). July 2007 \| Volume 3 \| Issue 7 \| e85 Discussion Interestingly, no VPRBP ortholog has been identiﬁed in yeast [21], perhaps explaining why we did not observe an interaction between Vpr and human DDB1 in S. cerevisae (Figure 2A) and why the DDB1 ortholog was not found among the putative Vpr-interacting proteins isolated by TAP assay in Schizosaccharomyces pombe (unpublished data). associate with Vpr. Interestingly, we could demonstrate the formation of a complex containing Vpr and CUL4A using two different approaches: by TAP-Vpr pull-down (Figure 3A) and by anti-CUL4A immunoprecipitation (Figure 3B). There- fore, it appears that Vpr can indeed recruit an E3 ligase complex with potential ubiquitinating activity. In these experiments, VPRBP was found in respective association with Vpr (TAP experiments, Figure 1A, 1C, and 1D), as well as with CUL4A (Figure 3B). Considering these interaction results and previous reports demonstrating the association To further characterize the possibility that Vpr might interact with a DDB1-CUL4AVPRBP E3 ligase complex, we investigated whether the scaffold protein CUL4A could 0889 July 2007 \| Volume 3 \| Issue 7 \| e85 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest between Vpr and DDB1-CUL4AVPRBP through the Vpr leucine/isoleucine-rich domain could permit the hypothetical interaction of Vpr through its C-terminal domain with other cellular factors whose ubiquitination would induce the initial events leading to cell cycle arrest. This model is consistent with recent reports [54,55] demonstrating the involvement of VPRBP and DDB1 in Vpr-induced G2 cell cycle arrest. between Vpr and VPRBP [21,22] as well as the association between CUL4A and VPRBP [20], it is most likely that Vpr interacts with the DDB1-CUL4A E3 ligase complex via the intermediary of VPRBP, although direct proof will require further analysis of the protein complex architecture. Importantly, we observed that not only VPRBP but also the formation of a Vpr-DDB1-CUL4AVPRBP complex was re- quired for Vpr-mediated induction of G2 arrest. Indeed, siRNA targeting VPRBP strongly impaired the induction of G2 arrest in the context of a lentiviral vector expressing Vpr and of a fully infectious provirus (Figure 5A and 5D). Knockdown of VPRBP did not produce cell cycle aberrations (Figure 5B and 5C) or reduce viral/vector infection efﬁciency, suggesting that the observed defect in G2 arrest was speciﬁc to the association of Vpr with VPRBP. In conclusion, we presented biochemical and functional evidence suggesting that Vpr is likely the third protein encoded by HIV-1, besides Vif and Vpu, to interact with the ubiquitination machinery. Materials and Methods Cells and antibodies. HEK293 and HEK293T cells were maintained as described elsewhere [56]. The anti-HA tag and anti-Myc tag monoclonal antibodies were clones 12CA5 and 9E10, respectively. Rabbit polyclonal antibody against VPRBP was distributed by Accurate Chemical and Scientiﬁc Corporation (http://www. accuratechemical.com/). The mouse monoclonal antibody against DDB1 was obtained from BD Biosciences (http://www.bdbiosciences. com/). The goat polyclonal antibody against CUL4A was obtained from Santa Cruz Biotechnology (http://www.scbt.com/). Vpr was detected using a rabbit polyclonal antibody directed against a Vpr N-terminal peptide [57]. 2 y One possible model to explain how Vpr induces G2 arrest via the DDB1-CUL4AVPRBP complex is that Vpr could cause a generalized defect in the activity of the whole DDB1 complex. In this context, other investigators have shown that abroga- tion of DDB1 function using siRNA resulted in the failure to degrade CDT1, thus leading to the accumulation of re- replicated DNA fragment and the induction of an ATR- dependent G2 arrest [28]. Therefore, Vpr, through a potential sequestration of DDB1, might be capable of inducing the defective regulation of CDT1 or other DDB1-CUL4A E3 ligase complex substrates leading to ATR-mediated G2 arrest. However, we did not observe any obvious effect of Vpr on the S-phase degradation of CDT1 (unpublished data), which suggests that the DDB1-CUL4A-RBX1 complex was fully functional. Another possible model is that Vpr could block the proper ubiquitination of natural substrates of DDB1- CUL4AVPRBP, thereby affecting their biological activities or preventing their degradation. However, this possibility is unlikely because siRNA-mediated knockdown of VPRBP did not produce G2 arrest (Figure 5). The most probable model is that Vpr triggers the degradation of a yet-unknown modu- lator of cell cycle progression by targeting it to the substrate receptor VPRBP, itself linked to the DDB1-CUL4A-RBX1 E3 ligase complex. This situation would be highly similar to the mechanism by which Vpu induces the degradation of CD4 by recruiting directly CD4 to the SCF b-TRCP E3 ubiquitin ligase complex [53]. However, we cannot exclude the possibility that the Vpr–VPRBP interaction might modulate the activity of substrates through ubiquitination, as was described for XPC and histones [38–40]. Therefore, the formation of a complex p p Plasmid constructions. For the construction of mammalian expression plasmid psvCMV-TAP, N-terminal TAP tag was ﬁrst PCR ampliﬁed from the pBS1479 plasmid [58] using primers 59-TCTA- GACATATGGCAGGCCTTGCGCAAC-39 and 59-GGATCCTCAC- TACTCGAA-TCGTCATCATCAAGTGCC-39, the last one containing two stop codons between the XhoI and BamHI restriction sites. Discussion Vpr was found to associate with DDB1 and CUL4A, which are components of the DDB1- CUL4A-RBX1 E3 ligase complex, possibly through the intermediary of the WD40 substrate receptor VPRBP. Importantly, the interaction between Vpr and the VPRBP- DDB1 complex was shown to be required for the induction of G2 arrest. Vpr could act as a connector, bridging the DDB1- CUL4AVPRBP E3 ubiquitin ligase to an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and cause G2 arrest. It has been extensively shown that Vpr C-terminal residues 7896 are important for G2 arrest [23,50–52], but we found that TAP-tagged Vpr mutants S79A and R80A attenuated for cell cycle arrest were nevertheless competent for association with DDB1 (Figure 6). This was not surprising given that Zhao et al. showed that VPRBP binding required the leucine/ isoleucine-rich domain of Vpr [22]. In fact, L64A and Q65R, two mutations in the leucine/isoleucine-rich domain, reduced to different extents the binding of Vpr to VPRBP (Figure 6B). We also observed a concomitant decrease of afﬁnity between Vpr and DDB1 as a result of these mutations, suggesting that a single complex comprised of VPRBP and DDB1 is binding to the leucine/isoleucine-rich domain of Vpr. Importantly, these two mutations strongly attenuated Vpr-induced G2 arrest (Figure 6C). Overall, these results suggest that recruit- ment of DDB1-CUL4AVPRBP by Vpr is necessary but not sufﬁcient to induce G2 cell cycle arrest. PLoS Pathogens \| www.plospathogens.org Materials and Methods Forty-eight hours after infection, cells were harvested for ﬂow cytometry analysis of cell cycle proﬁling and GFP expression. siRNA-mediated protein knockdown. siRNA targeting VPRBP (siGenome SMARTpool M-021119–00) and scrambled control siRNA (non-targeting siRNA #2) were obtained from Dharmacon (http:// www.dharmacon.com/). siRNA was transfected using Oligofectamine (Invitrogen Canada) according to the manufacturer instructions. Brieﬂy, 300 pmol of siRNA were pre-incubated with 15 ll of Oligofectamine and overlayed on cells at 50% conﬂuence (the ﬁnal concentration of siRNA was 125 nM). Cells were transduced with lentiviral vectors 24 h after transfection. In some experiments, cells were subjected to two sequential transfections of 300 pmol of siRNA, 24 h apart Transfection and immunoprecipitation. Cells were transfected using the calcium phosphate precipitation method. Cells were harvested 2 d later, washed, and lysed in Triton lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% Triton X-100, and EDTA-free complete protease inhibitors). TAP pull-down assays were performed using 10 ll of IgG-coupled beads. After extensive washes in Triton lysis buffer, beads were resuspended in 100 ll of TEV cleavage buffer with 2 units of TEV and incubated for 16 h at 4 8C. Cleaved proteins were diluted in Laemmli buffer, heat-denatured, and loaded onto 12.5% acrylamide gels for SDS-PAGE. After protein transfer onto Hybond-ECL membrane (Amersham BioSciences Canada), proteins were detected by Western blot using speciﬁc antibodies. Immuno- precipitation experiments were performed on cell extracts lysed in the Triton lysis buffer as described previously [60]. p Reverse transcriptase polymerase chain reaction (RT-PCR). RNA was extracted from siRNA-transfected cells at the given time using the Qiagen RNeasy kit (Qiagen, http://www.qiagen.com/) according to the manufacturer instructions. After elution, RNA was stored at 80 8C. All reagents for RT-PCR were purchased from Invitrogen Canada. RNA was reverse transcribed into cDNA using 500 ng of RNA, which in a ﬁnal volume of 12 ll supplemented with 1 ll of oligo (dT)12–18 (500 lg/ml) and 1 ll of 10 mM dNTP mix, were incubated at 65 8C for 5 min. The mixture was cooled on ice and supplemented with 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 2 units/ll RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen Cana- da), and 10 units/ll M-MLV RT. Finally, the reverse transcription reaction mixture was incubated at 37 8C for 50 min and heat- inactivated at 70 8C for 15 min. Materials and Methods Bands detected after silver staining were cut and sent to Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, Massachusetts, United States) for MALDI-TOF. y p p Tandem afﬁnity puriﬁcation. Puriﬁcation was done according to previously published procedures [58]. Brieﬂy, HEK293T cells were seeded onto ﬁve to ten 150 mm-diameter plates. The next day, cells were transfected with 10 lg of psvCMV TAP-Vpr plasmid. Cells were collected 48 h later, washed, and lysed in IPP150 buffer (10 mM Tris- Cl [pH 8.0], 150 mM NaCl, 0.1% NP40) supplemented with EDTA-free complete protease inhibitors (Roche Diagnostics Canada, http://www. rochediagnostics.ca/). Cell debris was removed by low-speed centri- fugation, and cleared supernatants were loaded onto IgG sepharose columns (Amersham BioSciences Canada, http://www.gelifesciences. com/). After extensive washes with IPP150 buffer and a ﬁnal wash with TEV cleavage buffer (10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 0.1% NP40, 0.5 mM EDTA, 1 mM DTT), TAP-containing complexes were eluted by overnight digestion using 100 units of TEV protease (Invitrogen Canada, http://www.invitrogen.com/) diluted in 1 ml of TEV buffer. Columns were eluted and washed with 3 ml of IPP150- calmodulin buffer (10 mM b-mercaptoethanol, 10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM Mg-acetate, 1 mM imidazole, 2 mM CaCl2, 0.1% NP40) supplemented with 3 ml of 1 M CaCl2 and EDTA-free complete protease inhibitors. Fractions were passed through calm- odulin columns (Stratagene, http://www.stratagene.com/), washed in IPP150 buffer, and ﬁnally eluted with a minimal volume of IPP150 elution buffer (10 mM b-mercaptoethanol, 10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM Mg-acetate, 1 mM imidazole, 2 mM EGTA, 0.1% NP40). Recovered proteins were resolved by denaturing 12.5% SDS- PAGE. Bands detected after silver staining were cut and sent to Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, Massachusetts, United States) for MALDI-TOF. Viral clones and infection. The infectious molecular clones pNL4.3-GFP and pNL4–3DVpr-GFP were obtained from Juan Lama (Department of Medicine, University of California San Diego, La Jolla, California, United States) and described in [62]. Virus was produced by transfecting 5 3 106 HEK293T cells with 40 lg of proviral DNA and 10 lg of the VSV-G–expressing plasmid psvCMV- IN-VSV-G as described previously [56]. Virus-containing supernatant was titered by a standard RT assay as previously described [56]. Virions, at a concentration of 100 cpm/cell, were used to infect HEK293 cells in 1 ml of culture medium, in absence of polybrene. Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession num- bers for the proteins discussed in this paper are CUL4A (NM_001008895), DDB1 (NM_001923), and VPRBP (NM_014703). p p y Lentiviral vector production, titration, and transduction. VSV-G– pseudotyped viral particles were produced by transient transfection of 40 lg of vector (pWPI or pWPI-Vpr), 30 lg of the packaging construct psPAX2, and 12 lg of the VSV-G–expressing plasmid psvCMV-IN-VSV-G in ﬁve million HEK293T cells using the calcium phosphate precipitation method. The vector-containing supernatant was harvested 48 and 72 h post-transfection, 0.45 lm–ﬁltered, and concentrated by ultracentrifugation on a sucrose cushion. Concen- Materials and Methods The XbaI/BamHI-digested PCR fragment was then inserted into psvCMV plasmid [56] linearized with XbaI/BglII. For the construction of the psvCMV-TAP-Vpr expression plasmid, wild-type and mutant Vpr genes were PCR-ampliﬁed from the respective psvCMV-HA.Vpr plasmids [56] using oligonucleotides 59-CTCGAGATGGAA- CAAGCCCCAG-39 and 59-GGATCCCTAGGATCTACTGGCT-39. XhoI/BamHI Vpr fragments were ﬁnally fused to the XbaI/XhoI TAP sequence within psvCMV by 3-fragment ligation. Mammalian expression plasmids pSRAS-3HADDB1 and pSRAS-GFP.DDB1 [44] were kindly provided by M. Strubin (University of Geneva, Geneva, Switzerland). The TAP-DDB1 expression plasmid was constructed by inserting the XbaI/XhoI TAP fragment in Xba/SalI linearized pSRAS- 3HA.DDB1 plasmid. The Myc-VPRBP expression plasmid was constructed by PCR ampliﬁcation of the VPRBP cDNA from Image clone ID 5547856 (American Type Culture Collection, http://www. atcc.org/). The resulting PCR product was then subcloned in pCMV- Myc (Clontech, http://www.clontech.com/) at the SalI and NotI sites. For the construction of the yeast expression plasmids, TAP and TAP- Vpr sequences were subcloned after PCR ampliﬁcation in the BamHI sites within pEG202. The DDB1 sequence was extracted from pSRAS- 3HADDB1 by SalI/NotI digestion and introduced in pJG4–5. Vpr- expressing pJG4–5 and pEG202 plasmids have been described previously [59]. The second-generation self-inactivating lentiviral vectors pWPI and pWPXL, as well as the packaging plasmid psPAX2 were obtained from D. Trono (School of Life Sciences, Swiss Federal Institute of Technology, Lausanne, Switzerland). A lentiviral vector tranducing Vpr and GFP (pWPI-Vpr) was generated from pWPI. Vpr was PCR-ampliﬁed from psvCMV-Vpr [56], using primers 59AAGGATCCATGGAACAAGCCCCAGAAGACC-39 and 59-TAC- GACTAGTCTAGGATCTACTGGCTCCATTT-39, which contain a BamHI site at the 59 end and a SpeI site at the 39 end, respectively. PLoS Pathogens \| www.plospathogens.org 0890 July 2007 \| Volume 3 \| Issue 7 \| e85 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest The BamH1- and SpeI-cleaved PCR product was then ligated into pWPXL linearized at the same sites. A fragment containing the Vpr- coding sequence and the EF1-alpha promoter was excised with SpeI (Klenow-treated) and NotI and then ligated into pWPI at the PmeI and NotI sites to yield pWPI-Vpr. trated vectors were resuspended in culture medium and stored at80 8C. Vectors were titered as described previously with some modiﬁcations [61]. Brieﬂy, 5 3 104 HEK293T cells were transduced with serial dilutions of the vector preparations in absence of polybrene. Twenty-four hours later, cells were ﬁxed with 2% PFA for 30 min and the percentage of GFP-expressing cells was determined by ﬂow cytometry. Materials and Methods One microliter of the reverse transcription reaction was subsequently used for PCR ampliﬁcation of b-actin and VPRBP sequences. Brieﬂy, the PCR ampliﬁcation mix was composed of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 50 lM dNTPs, 1 lM sense and antisense primers, and 0.05 units/ll Taq DNA polymerase. Primers 59-GCTCGTCGTCGA- CAACGG-CTC-39 and 59-CAAACATGATCTGGGTCATCTTCTC-39 were used for b-actin ampliﬁcation and primers 59-AGGCCATCCA- CAAGTTTGAC-39 and 59-TCATCTGCCTGCAACATAGC-39 were used for VPRBP ampliﬁcation. The PCR ampliﬁcation conditions for VPRBP were as follows: 94 8C for 2 min; 25 cycles of (94 8C for 45 sec; 57 8C for 30 sec; 72 8C for 1 min); 72 8C for 5 min. The conditions of ampliﬁcation for b-actin were the same except that 18 cycles of ampliﬁcation were used. In vitro translation. T7-Vpr and HA-DDB1 were in vitro translated using the Active Pro In Vitro Translation kit (Ambion, http://www. ambion.com/) according to manufacturer instructions. Cell cycle analyses. Cell cycle analysis of HEK293 cells transduced by lentiviral vectors was performed as previously described in [56]. The mathematical model MODFIT was used to calculate the proportions of cells in the G2/M phases and G1 phase of the cell cycle. For analysis of cell cycle proﬁle in the context of TAP-Vpr, HEK293T cells were co-transfected with 1 lg of a GFP-expressing plasmid and 15 lg of TAP-Vpr–expressing plasmids. Forty-eight hours after transfection, cells were ﬁxed with 1% paraformaldehyde for 15 min followed by ﬁxation/permabilization with 70% ethanol for 10 min. The rest of the procedure was as described previously [56] except that ﬂow cytometry analysis was performed on the GFP- positive cell population. Yeast two-hybrid assay. The yeast ‘‘bait’’ strains were made by transforming the EGY48 yeast strain with a URA3 lacZ (b- galactosidase) reporter plasmid and the different bait plasmids (expressing the HIS3 gene) by the lithium acetate method. The yeast ‘‘bait’’ strains harboring the bait and reporter plasmids were transformed with different prey plasmids and selected for the tryptophan autotrophy phenotype (in addition to the His and Ura nutritional markers for the bait and LacZ reporter plasmids, respectively). Determination of the respective interactions was performed as previously described [59]. Materials and Methods The vector titer was calculated as the number of transduction units in the linear range of transduction (5%–10% of GFP-positive cells). For transduction experiments, 1 3 105 HEK293 cells seeded in the wells of a 6-well plate were incubated for 24 h with WPI or WPI-Vpr vectors (at a multiplicity of infection of 1) in presence of 8 lg/ml polybrene, typically achieving a trans- duction efﬁciency of 90%–95%. Cells were harvested after 24 h for ﬂow cytometry analysis of cell cycle proﬁling and GFP expression. The BamH1- and SpeI-cleaved PCR product was then ligated into pWPXL linearized at the same sites. A fragment containing the Vpr- coding sequence and the EF1-alpha promoter was excised with SpeI (Klenow-treated) and NotI and then ligated into pWPI at the PmeI and NotI sites to yield pWPI-Vpr. y p p Tandem afﬁnity puriﬁcation. Puriﬁcation was done according to previously published procedures [58]. Brieﬂy, HEK293T cells were seeded onto ﬁve to ten 150 mm-diameter plates. The next day, cells were transfected with 10 lg of psvCMV TAP-Vpr plasmid. Cells were collected 48 h later, washed, and lysed in IPP150 buffer (10 mM Tris- Cl [pH 8.0], 150 mM NaCl, 0.1% NP40) supplemented with EDTA-free complete protease inhibitors (Roche Diagnostics Canada, http://www. rochediagnostics.ca/). Cell debris was removed by low-speed centri- fugation, and cleared supernatants were loaded onto IgG sepharose columns (Amersham BioSciences Canada, http://www.gelifesciences. com/). After extensive washes with IPP150 buffer and a ﬁnal wash with TEV cleavage buffer (10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 0.1% NP40, 0.5 mM EDTA, 1 mM DTT), TAP-containing complexes were eluted by overnight digestion using 100 units of TEV protease (Invitrogen Canada, http://www.invitrogen.com/) diluted in 1 ml of TEV buffer. Columns were eluted and washed with 3 ml of IPP150- calmodulin buffer (10 mM b-mercaptoethanol, 10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM Mg-acetate, 1 mM imidazole, 2 mM CaCl2, 0.1% NP40) supplemented with 3 ml of 1 M CaCl2 and EDTA-free complete protease inhibitors. Fractions were passed through calm- odulin columns (Stratagene, http://www.stratagene.com/), washed in IPP150 buffer, and ﬁnally eluted with a minimal volume of IPP150 elution buffer (10 mM b-mercaptoethanol, 10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM Mg-acetate, 1 mM imidazole, 2 mM EGTA, 0.1% NP40). Recovered proteins were resolved by denaturing 12.5% SDS- PAGE. 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Niida H, Nakanishi M (2006) DNA damage checkpoints in mammals. Mutagenesis 21: 3–9. 12. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73: 39–85. 38. Kapetanaki MG, Guerrero-Santoro J, Bisi DC, Hsieh CL, Rapic-Otrin V, et al. (2006) The DDB1-CUL4ADDB2 ubiquitin ligase is deﬁcient in xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites. Proc Natl Acad Sci U S A 103: 2588–2593. 13. McGowan CH, Russell P (2004) The DNA damage response: Sensing and signaling. Curr Opin Cell Biol 16: 629–633. DNA sites. Proc Natl Acad Sci U S A 103: 2588–2593. 39. Wang H, Zhai L, Xu J, Joo HY, Jackson S, et al. (2006) Histone H3 and H4 ubiquitylation by the CUL4-DDB-ROC1 ubiquitin ligase facilitates cellular response to DNA damage. Mol Cell 22: 383–394. 14. Roshal M, Kim B, Zhu Y, Nghiem P, Planelles V (2003) Activation of the ATR-mediated DNA damage response by the HIV-1 viral protein R. J Biol Chem 278: 25879–25886. response to DNA damage. Mol Cell 22: 383–394. 15. References 27. Higa LA, Banks D, Wu M, Kobayashi R, Sun H, et al. (2006) L2DTL/CDT2 interacts with the CUL4/DDB1 complex and PCNA and regulates CDT1 proteolysis in response to DNA damage. Cell Cycle 5: 1675–1680. 1. Le Rouzic E, Benichou S (2005) The Vpr protein from HIV-1: Distinct roles along the viral life cycle. Retrovirology 2: 11. 28. Lovejoy CA, Lock K, Yenamandra A, Cortez D (2006) DDB1 maintains genome integrity through regulation of Cdt1. Mol Cell Biol 26: 7977–7990. 2. Andersen JL, Planelles V (2005) The role of Vpr in HIV-1 pathogenesis. Curr HIV Res 3: 43–51. 29. Casper AM, Nghiem P, Arlt MF, Glover TW (2002) ATR regulates fragile site stability. Cell 111: 779–789. 3. Jowett JB, Planelles V, Poon B, Shah NP, Chen ML, et al. (1995) The human immunodeﬁciency virus type 1 vpr gene arrests infected T cells in the G2 þ M phase of the cell cycle. J Virol 69: 6304–6313. 30. Hammond EM, Denko NC, Dorie MJ, Abraham RT, Giaccia AJ (2002) Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol 22: 1834–1843. 4. He J, Choe S, Walker R, Di Marzio P, Morgan DO, et al. (1995) Human immunodeﬁciency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 69: 6705–6711. 31. Nghiem P, Park PK, Kim Y, Vaziri C, Schreiber SL (2001) ATR inhibition selectively sensitizes G1 checkpoint-deﬁcient cells to lethal premature chromatin condensation. Proc Natl Acad Sci U S A 98: 9092–9097. 5. Rogel ME, Wu LI, Emerman M (1995) The human immunodeﬁciency virus type 1 vpr gene prevents cell proliferation during chronic infection. J Virol 69: 882–888. 32. Forget J, Yao XJ, Mercier J, Cohen EA (1998) Human immunodeﬁciency virus type 1 vpr protein transactivation function: Mechanism and identiﬁcation of domains involved. J Mol Biol 284: 915–923. 6. Re F, Braaten D, Franke EK, Luban J (1995) Human immunodeﬁciency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2-cyclin B. J Virol 69: 6859–6864. 33. Zhou Y, Ratner L (2000) Phosphorylation of human immunodeﬁciency virus type 1 Vpr regulates cell cycle arrest. J Virol 74: 6520–6527. p y J 7. Planelles V, Jowett JB, Li QX, Xie Y, Hahn B, et al. (1996) Vpr-induced cell cycle arrest is conserved among primate lentiviruses. J Virol 70: 2516–2524. 34. mutant L64A. Author contributions. JPB, GD, and EAC conceived and designed the experiments and analyzed the data. JPB, GD, NR, JM, and AF performed the experiments. JPB, GD, and EAC wrote the paper. Competing interests. The authors have declared that no competing interests exist. Funding. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Fonds de la Recherche Funding. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Fonds de la Recherche References Andrejeva J, Poole E, Young DF, Goodbourn S, Randall RE (2002) The p127 subunit (DDB1) of the UVDNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5. J Virol 76: 11379–11386. 21. Zhang S, Feng Y, Narayan O, Zhao LJ (2001) Cytoplasmic retention of HIV- 1 regulatory protein Vpr by proteinprotein interaction with a novel human cytoplasmic protein VprBP. Gene 263: 131–140. 22. Zhao LJ, Mukherjee S, Narayan O (1994) Biochemical mechanism of HIV-I Vpr function. Speciﬁc interaction with a cellular protein. J Biol Chem 269: 15577–15582. 47. Schrofelbauer B, Yu Q, Zeitlin SG, Landau NR (2005) Human immunode- ﬁciency virus type 1 Vpr induces the degradation of the UNG and SMUG uracil-DNA glycosylases. J Virol 79: 10978–10987. 23. Di Marzio P, Choe S, Ebright M, Knoblauch R, Landau NR (1995) Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeﬁciency virus type 1 Vpr. J Virol 69: 7909– 7916. 48. Mahalingam S, Ayyavoo V, Patel M, Kieber-Emmons T, Kao GD, et al. (1998) HIV-1 Vpr interacts with a human 34-kDa mov34 homologue, a cellular factor linked to the G2/M phase transition of the mammalian cell cycle. Proc Natl Acad Sci U S A 95: 3419–3424. p Proc Natl Acad Sci U S A 95: 3419–3424. 24. Wood RD, Mitchell M, Sgouros J, Lindahl T (2001) Human DNA repair genes. Science 291: 1284–1289. 49. Li T, Chen X, Garbutt KC, Zhou P, Zheng N (2006) Structure of DDB1 in complex with a paramyxovirus V protein: Viral hijack of a propeller cluster in ubiquitin ligase. Cell 124: 105–117. g 25. Fujiwara Y, Masutani C, Mizukoshi T, Kondo J, Hanaoka F, et al. (1999) Characterization of DNA recognition by the human UV-damaged DNA- binding protein. J Biol Chem 274: 20027–20033. q g 50. Macreadie IG, Castelli LA, Hewish DR, Kirkpatrick A, Ward AC, et al. (1995) A domain of human immunodeﬁciency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects. Proc Natl Acad Sci U S A 92: 2770–2774. 26. Groisman R, Polanowska J, Kuraoka I, Sawada J, Saijo M, et al. (2003) The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell 113: 357–367. structural defects. Acknowledgments We want to thank M. Strubin for providing us with plasmids expressing DDB1, and D. Trono for the lentiviral vectors, pWPI and pWPXL, and the packaging construct psPAX2. We also thank S. Benichou for the Vpr mutant W54R and W. Greene for the Vpr July 2007 \| Volume 3 \| Issue 7 \| e85 0891 July 2007 \| Volume 3 \| Issue 7 \| e85 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest en Sante´ du Que´bec (FRSQ) to EAC. JPB and AF are recipients of studentships from CIHR and the CIHR strategic training program in cancer research (IRCM), respectively. EAC is recipient of the Canada Research Chair in Human Retrovirology. mutant L64A. mutant L64A. Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest ciency virus type 1 and simian immunodeﬁciency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest. J Virol 71: 4842–4846. ciency virus type 1 and simian immunodeﬁciency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest. J Virol 71: 4842–4846. 57. Cohen EA, Terwilliger EF, Jalinoos Y, Proulx J, Sodroski JG, et al. (1990) Identiﬁcation of HIV-1 vpr product and function. J Acquir Immune Deﬁc Syndr 3: 11–18. g y J 52. Mahalingam S, Ayyavoo V, Patel M, Kieber-Emmons T, Weiner DB (1997) Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeﬁciency virus type 1 Vpr. J Virol 71: 6339–6347. y 58. Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, et al. (2001) The tandem afﬁnity puriﬁcation (TAP) method: A general procedure of protein complex puriﬁcation. Methods 24: 218–229. y virus type 1 Vpr. J Virol 71: 6339–6347. yp p J 53. Binette J, Cohen EA (2004) Recent advances in the understanding of HIV-1 Vpu accessory protein functions. Curr Drug Targets Immune Endocr Metabol Disord 4: 297–307. p p 59. Bachand F, Yao XJ, Hrimech M, Rougeau N, Cohen EA (1999) Incorpo- ration of Vpr into human immunodeﬁciency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor. J Biol Chem 274: 9083–9091. 54. Le Rouzic E, Belaidouni N, Estrabaud E, Morel M, Rain JC, et al. (2007) HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle 6: 182–188. 60. Levesque K, Zhao YS, Cohen EA (2003) Vpu exerts a positive effect on HIV- 1 infectivity by down-modulating CD4 receptor molecules at the surface of HIV-1producing cells. J Biol Chem 278: 28346–28353. 55. Schrofelbauer B, Hakata Y, Landau NR (2007) HIV-1 Vpr function is mediated by interaction with the damage-speciﬁc DNA-binding protein DDB1. Proc Natl Acad Sci U S A 104: 4130–4135. 61. Zufferey R, Donello JE, Trono D, Hope TJ (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73: 2886–2892. 56. Yao XJ, Mouland AJ, Subbramanian RA, Forget J, Rougeau N, et al. (1998) Vpr stimulates viral expression and induces cell killing in human immunodeﬁciency virus type 1-infected dividing Jurkat T cells. J Virol 72: 4686–4693. y J 62. References Proc Natl Acad Sci U S A 92: 2770–2774 51. Selig L, Benichou S, Rogel ME, Wu LI, Vodicka MA, et al. (1997) Uracil DNA glycosylase speciﬁcally interacts with Vpr of both human immunodeﬁ- PLoS Pathogens \| www.plospathogens.org 0892 July 2007 \| Volume 3 \| Issue 7 \| e85 Vpr Recruits DDB1-CUL4AVPRBP to Induce G2 Arrest Arganaraz E, Cortes MJ, Leibel S, Lama J (2002) Human immunodeﬁciency virus type 1 Vpr protein does not modulate surface expression of the CD4 receptor. J Virol 76: 4125–4130. PLoS Pathogens \| www.plospathogens.org July 2007 \| Volume 3 \| Issue 7 \| e85 July 2007 \| Volume 3 \| Issue 7 \| e85 0893
https://openalex.org/W4280512473	https://www.nature.com/articles/s41598-022-11577-y.pdf	English	null	A mechanical and simplified model for RC elements subjected to combined shear and axial tension	Scientific reports	2,022	cc-by	10,046	A mechanical and simplified model for RC elements subjected to combined shear and axial tension OPEN A. Deifalla1* & F. M. Mukhtar2,3 Very little is known about the shear behavior of elements, in particular those subjected to axial tension. The shear accompanied by tensile forces could cause premature failure of reinforced concrete, which is sudden with minimal warning. Therefore, understanding the shear behavior of reinforced concrete (RC) elements, including those subjected to axial tension, is an ultimate goal of the worldwide research community. In the current study, a new shear mechanical model for RC elements subjected to axial tension is developed, which makes physical sense and explains the behavior. The model is strain-based, inspired by the critical crack theory model (CSCT). In addition, the proposed model extended CSCT (ECSCT) quantifies the effect of axial tension forces on the shear strength in terms of reduction in the compression zone depth and increase in the longitudinal strain. Moreover, the nonlinear trend observed in the literature was implemented using nonlinear multi-variable regression. The ECSCT is validated and compared with available design methods with respect to an extensive database, including 180 elements tested under shear and tension from 18 different research investigations. The ECSCT provided an accurate and physically sound model yet safe to an acceptable extent. Last but not least, a simplified model for the purpose of design is proposed. The simplified model was chosen based on the mechanical model and calibrated using the extensive experimental database. The simplified model provided an accurate and simple model, yet safe to an acceptable extent. Shear failure of reinforced concrete (RC) elements is sudden and should be carefully considered, in particular, those without stirrups or with stirrups1–21. In addition, RC elements subjected to shear combined with axial ten- sion are still a dilemma22–26, which occurs in many situations. Many studies have been conducted to understand the effect of axial tension on the shear strength of RC. However, the physical significance of the tensile force on the shear design is not well defined yet. Shear behavior of RC elements is a complex problem that involves many mechanisms, as shown in Fig. 127. Those mechanisms include but are not limited to: (1) direct shear through the compression zone, (2) friction along the sides of the diagonal shear cracks, (3) dowel action through longitudinal reinforcements crossing the diagonal cracks, (4) residual tensile stresses transferred across the diagonal, (5) the aggregate interlock across the diagonal crack. www.nature.com/scientificreports www.nature.com/scientificreports A mechanical and simplified model for RC elements subjected to combined shear and axial tension OPEN Suppose the beam is subjected to tensile axial forces, the crack width and the longitudinal strain increase. On the other hand, the compression zone depth, the residual tensile strength, and the aggregate interlock decrease. Recently, Deifalla26 recommended using the observed behavior to improve and simplify the current physically sound-based models. The current study aims to develop and propose a mechanical model for RC elements under combined shear and tension. Inspired by the critical shear crack theory28, a mechanical model named extended critical shear crack theory (ECSCT) was developed to include the effect of tensile forces. The strength of the experimental database was calculated using the proposed model and compared with existing design codes. In addition, a simplified model was proposed, which was found to be better for design purposes. 1Department of Structural Engineering and Construction Management, Future University in Egypt, New Cairo City 11835, Egypt. 2Department of Civil and Environmental Engineering, King Fahd University of Petroleum & Minerals, Dhahran 31261, Saudi Arabia. 3Interdisciplinary Research Center for Construction and Building Materials, King Fahd University of Petroleum & Minerals, Dhahran, Saudi Arabia. *email: ahmed.deifalla@fue.edu.eg \| https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 Scientific Reports \| (2022) 12:7863 www.nature.com/scientificreports/ Figure 1. Shear failure mechanisms27. Figure 1. Shear failure mechanisms27. Recent findings h l d Recent findings For the last seven decades, several pioneering studies26,29–33 have been conducted to investigate the shear strength of elements under axial tensile forces; a brief recount of the most recent findings is as follows. • The significant variables that affect the shear strength are element dimensions, reinforcement configuration, loading configuration, and boundary conditions; thus, the conclusions of different studies were inconsistent with each other31. For example, the device used to apply the axial tension could cause accidental restraint at the ends of the tested element33.hif • The significant variables that affect the shear strength are element dimensions, reinforcement configuration, loading configuration, and boundary conditions; thus, the conclusions of different studies were inconsistent with each other31. For example, the device used to apply the axial tension could cause accidental restraint at the ends of the tested element33.hif • The angle of inclination of shear cracking is significantly affected by the axial tension, which makes it steeper33. On the other hand, the shear strength of beams with well-detailed longitudinal reinforcements is not affected by axial tension. This is due to the aggregate interlock mechanism. In addition, the compression longitudinal steel reinforcements decrease the effect of axial tension forces on shear, if any29. • The angle of inclination of shear cracking is significantly affected by the axial tension, which makes it steeper33. On the other hand, the shear strength of beams with well-detailed longitudinal reinforcements is not affected by axial tension. This is due to the aggregate interlock mechanism. In addition, the compression longitudinal steel reinforcements decrease the effect of axial tension forces on shear, if any29. gf y • A nonlinear relationship between the axial everyday tensile stresses and the shear strength was found by several researchers32. This contradicts the long-standing linear relation implemented by both the ACI34 and the EC235. f • A nonlinear relationship between the axial everyday tensile stresses and the shear strength was found by several researchers32. This contradicts the long-standing linear relation implemented by both the ACI34 and the EC235. • Pham31 observed a decrease in the compression zone depth with the increase in the axial tensile forces, as shown in Fig. 2. • Pham31 observed a decrease in the compression zone depth with the increase in the axial tensile forces, as shown in Fig. 2. Recent findings h l d g • In general, design codes, including but not limited to the ACI and the EC2, are overly conservative, especially for cases of high tensile forces26. g • An extensive experimental database of elements tested under shear and tension was gathered, combining the database complied by Deifalla26 and Ehmann30. A total of 180 elements from 17 different research investiga- tions. The data covered a wide range of all influential variables, as shown in Table 1 and Fig. 330–33,36–49. The effective parameters included the axial tension, the size, the shear-span to depth ratio, the concrete strength, flexure reinforcement ratio, and the width to depth ratio were gathered. Model development d ih p Introduction. This work is inspired by the Critical Shear Crack Theory (CSCT)28, which was first introduced in the 1990s and later implemented in the swiss design code, MC, and the new draft of the Eurocode26,50. The shear strength ( vu ) is calculated such that: (1) vu f ′c = f(ω, ddg) (1) while ω is the crack width, f ′ c is the cylinder compressive strength and ddg is the maximum nominal aggregate size. (2) ω ∝εd while ω is the crack width, f ′ c is the cylinder compressive strength and ddg is the maximum nominal aggregate size while ω is the crack width, f ′ c is the cylinder compressive strength and ddg is the maximum nominal aggregate size. (2) d (2) https://doi.org/10.1038/s41598-022-11577-y https://doi.org/10.1038/s41598-022-11577-y https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Figure 2. Experimentally observed behavior of RC beams under combined sh Figure 2. Experimentally observed behavior of RC beams under combined shear and tension31. Figure 2. Experimentally observed behavior of RC beams under combined shear and tension31. where d is the effective depth, and ε is the longitudinal strain, which is taken at 60% of the effective depth from extreme compression fibers28. In addition, the following assumptions were implemented, which are similar to the work by Deifalla24,25 for slabs under combined punching shear and tension: (1) Plane cross-sections before deformation remain plain after deformation,m while maintaining small deformation. (2) Concrete in compres- sion is linear elastic behavior. (3) Concrete in tension is neglected. (4) Steel reinforcements reached yield. (5) The superposition principle applies to the longitudinal strains from flexure and tension. Scientific Reports \| (2022) 12:7863 \| Model development d ih Figure 4a–c shows the https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ References Label d (mm) b (mm) fc′ (MPa) ρ (–) fy (MPa) a/d (–) N (kN) V (kN) 36 9 284 175 23 0.41% 343.4 3.69 86 19.68 10 284 175 23 0.41% 343.4 3.69 68 24.13 37 4 254 152 46 1.03% 399.9 3.00 29 44.48 5 254 152 16 2.07% 399.9 3.00 29 33.36 11 254 152 15 3.10% 399.9 3.00 61 42.26 16 254 152 30 1.03% 399.9 5.40 48 28.02 19 254 152 19 2.07% 399.9 5.40 29 40.03 20 254 152 48 2.07% 399.9 5.40 29 57.83 21 254 152 51 2.07% 399.9 5.40 61 56.93 23 254 152 19 3.10% 399.9 5.40 29 42.26 25 254 152 28 3.10% 399.9 5.40 48 51.15 26 254 152 29 1.00% 399.9 5.40 80 42.26 29 254 152 53 3.10% 399.9 5.40 29 66.72 38 A1T 381 178 28 3.78% 517.3 2.50 144 122.55 C1T 381 178 29 3.78% 517.3 3.38 144 120.21 J1T 381 178 29 3.78% 517.3 2.50 144 87.00 39 N3 272 152 33 1.46% 427 2.80 120 42.00 N4 272 152 34 1.46% 427 2.80 90 42.00 N5 272 152 32 1.46% 427 2.80 60 48.00 N6 272 152 32 1.46% 427 2.80 70 50.00 N7 272 152 35 1.46% 427 2.80 130 45.00 N9 272 152 31 1.46% 427 2.80 85 42.00 N11 272 152 33 0.97% 427 2.80 75 37.00 N12 272 152 28 1.46% 628 5.61 30 48.00 N13 272 152 31 1.46% 628 5.61 40 50.00 N14 272 152 31 1.46% 427 2.80 40 50.00 N15 272 152 32 1.46% 427 2.80 20 50.00 N16 272 152 31 1.46% 628 1.96 40 52.00 N18 272 152 31 1.46% 427 2.80 60 45.00 N19 272 152 29 1.46% 427 2.80 80 40.00 N20 272 152 46 1.46% 427 2.80 60 42.00 N21 272 152 15 1.46% 427 2.80 60 40.00 N22 272 152 32 1.46% 427 1.96 60 85.00 N23 272 152 35 1.46% 427 1.96 20 75.00 N24 272 152 22 1.46% 427 2.80 60 37.00 40 M5 250 760 21 0.4% 602 4.00 295 137.30 M6 250 760 27 0.5% 643 4.00 393 137.30 41 T4 262 200 53 1.8% 534 2.50 327 94.00 T5 262 200 53 1.8% 534 2.50 439 81.90 T6 262 200 53 1.8% 534 2.50 223 126.50 42 PB4 890 70 16 1.1% 423 N/A 72 72.30 PB6 890 70 17 1.1% 425 N/A 72 71.60 PB7 890 70 20 1.1% 425 N/A 102 53.60 PB8 890 70 20 1.1% 425 N/A 148 49.20 PB10 890 70 24 1.1% 425 N/A 148 34.90 PB16 890 70 42 1.1% 502 N/A 181 90.30 PB14 890 70 42 2.0% 489 N/A 288 95.90 PB17 890 70 25 2.0% 502 N/A 449 76.00 PB19 890 70 20 2.0% 402 N/A 80 79.70 PB20 890 70 22 2.0% 411 N/A 177 88.50 PB28 890 70 23 2.0% 424 N/A 191 95.30 PB21 890 70 22 2.0% 426 N/A 274 88.50 PB22 890 70 18 2.0% 402 N/A 392 64.20 PB29 890 70 42 2.0% 433 N/A 186 92.80 PB30 890 70 40 2.0% 496 N/A 277 92.20 https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ References Label d (mm) b (mm) fc′ (MPa) ρ (–) fy (MPa) a/d (–) N (kN) V (kN) 43 ZS2 164 600 40 4.0% 500 3.05 1200 356.00 44 P1 178 600 35 0.2% 477 3.82 60 92.00 P2 178 600 35 0.2% 477 3.82 60 92.00 P3 178 600 43 0.2% 477 3.82 60 92.00 P4 178 600 43 0.6% 506 3.82 60 92.00 P5 178 600 35 0.6% 506 3.82 60 92.00 45 ST9 278 290 46 1.95% 536 3.60 280 69.90 ST10 278 290 46 1.95% 536 3.60 525 65.60 ST11 278 290 46 1.95% 536 3.60 776 65.60 ST12 278 290 46 1.95% 536 3.60 1507 47.10 ST13 278 290 46 1.95% 536 3.60 1050 65.60 ST25 278 290 59 1.00% 484 3.60 165 82.00 ST26 278 290 59 1.00% 484 3.60 191 58.90 46 S1 204 80 37 0.87% 356.5 2.00 30 32.75 S2 204 80 37 0.87% 356.5 2.00 50 28.85 S3 204 80 37 0.87% 356.5 2.00 60 27.95 S4 204 80 37 0.87% 356.5 2.00 70 23.85 S5 204 80 37 0.87% 356.5 2.50 30 27.00 S6 204 80 37 0.87% 356.5 2.50 50 23.80 S7 204 80 37 0.87% 356.5 2.50 60 23.35 S8 204 80 37 0.87% 356.5 2.50 70 22.30 S10 204 100 37 1.26% 356.5 2.00 20 30.03 S11 204 100 37 1.26% 356.5 2.00 30 24.34 S12 204 100 37 1.26% 356.5 2.00 40 23.00 S14 204 100 37 1.26% 356.5 2.50 20 24.15 S15 204 100 37 1.26% 356.5 2.50 30 19.42 S16 204 100 37 1.26% 356.5 2.50 40 15.43 Continued https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ References Label d (mm) b (mm) fc′ (MPa) ρ (–) fy (MPa) a/d (–) N (kN) V (kN) 30 A1 250 400 47 1.6% 579 3.00 450 150.00 A2 250 400 47 1.6% 579 3.00 340 146.60 A2′ 250 400 47 1.6% 579 5.00 340 122.90 A3 250 400 49 1.6% 579 3.00 560 119.80 A3′ 250 400 49 1.6% 579 5.00 560 124.50 A4 250 400 49 2.5% 559 3.00 340 162.50 A4′ 250 400 49 2.5% 559 5.00 340 134.10 A5 250 400 49 1.0% 585 3.00 340 146.30 B2 250 400 46 2.0% 558 3.00 200 122.50 B3 250 400 46 2.0% 558 3.00 400 164.40 B3′ 250 400 46 2.0% 558 5.00 400 132.70 B4 250 400 46 2.0% 558 3.00 600 109.80 B4′ 250 400 46 2.0% 558 5.00 600 125.20 B5 250 400 48 2.0% 558 3.00 800 139.40 B5′ 250 400 48 2.0% 558 5.00 800 113.30 B6 250 400 46 1.0% 572 3.00 200 137.30 B7 250 400 44 1.6% 546 3.00 200 144.60 B7′ 250 400 44 1.6% 546 5.00 200 109.00 B8 250 400 45 2.5% 570 3.00 200 150.20 B9 250 400 45 2.8% 566 3.00 200 150.80 B9′ 250 400 45 2.8% 566 5.00 200 143.60 B10 250 400 48 1.0% 572 3.00 600 94.10 B11 250 400 47 1.6% 546 3.00 600 160.10 B11′ 250 400 47 1.6% 546 5.00 600 126.30 B12 250 400 47 2.5% 570 3.00 600 174.00 B12′ 250 400 47 2.5% 570 5.00 600 140.70 C1 250 400 43 1.6% 559 3.00 200 249.60 C1′ 250 400 43 1.6% 559 5.00 200 149.70 C2 250 400 43 1.6% 559 3.00 600 136.10 C2′ 250 400 43 1.6% 559 5.00 600 153.20 C4 250 400 44 1.6% 559 4.00 150 144.20 C4′ 250 400 44 1.6% 559 4.00 150 136.10 C5 250 400 44 1.6% 559 4.00 340 138.50 C7 250 400 44 1.6% 559 4.00 150 129.80 C7′ 250 400 44 1.6% 559 4.00 150 125.10 C8 250 400 45 1.6% 559 4.00 340 127.60 C8′ 250 400 45 1.6% 559 4.00 340 116.70 C9 250 400 27 2.0% 554 4.00 500 105.20 C10 250 400 52 2.0% 554 3.00 500 146.00 C11 250 400 45 1.5% 550 3.00 340 146.40 C12 250 400 45 1.5% 550 3.00 600 151.20 C12′ 250 400 45 1.5% 550 5.00 600 143.00 C13 250 400 46 2.0% 554 3.00 900 111.20 C13′ 250 400 46 2.0% 554 5.00 900 134.50 Continued https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ References Label d (mm) b (mm) fc′ (MPa) ρ (–) fy (MPa) a/d (–) N (kN) V (kN) 47 ST-1 165 200 25 1.1% 1027 2.25 427 39.50 ST-2 165 200 26 1.1% 1027 2.25 97 43.50 ST-3 165 200 26 1.1% 1027 2.25 200 45.40 ST-6 165 200 27 1.1% 1027 2.25 300 43.00 ST-7 165 200 27 1.1% 1027 2.25 401 40.80 ST-8 165 200 27 1.1% 1027 2.25 499 39.80 ST-9 165 200 27 1.1% 1027 2.25 299 45.40 ST-10 165 200 28 1.1% 1027 2.25 401 36.90 ST-12 165 200 28 1.1% 1027 2.25 200 44.00 ST-13 165 200 29 1.1% 1027 2.25 100 33.90 ST-14 165 200 29 1.1% 1027 2.75 201 40.70 ST-15 165 200 29 1.1% 1027 2.75 301 44.60 ST-17 165 200 30 1.1% 1027 2.75 100 39.90 ST-18 165 200 30 1.1% 1027 2.75 200 32.80 ST-19 165 200 30 1.1% 1027 2.75 300 32.00 ST-20 165 200 30 1.1% 1027 2.75 500 30.20 ST-22 165 200 30 1.1% 1027 2.75 501 37.50 ST-23 165 200 30 1.1% 1027 2.25 602 34.50 ST-24 165 200 30 1.1% 1027 2.25 600 35.20 48 V8-1 164 140 36 1.00% 495 1.97 30 57.37 V8-2 164 140 82 1.00% 495 1.97 51 75.73 V8-3 164 140 34 1.00% 495 1.97 50 45.13 V8-4 164 140 34 1.00% 495 1.97 102 50.91 V9-1 164 140 31 1.51% 487 1.97 27 68.94 V9-2 164 140 74 1.51% 487 1.97 47 71.95 V9-3 164 140 36 1.51% 487 1.97 60 52.83 V9-4 164 140 74 1.51% 495 1.97 69 109.90 V9-5 164 140 33 1.51% 495 1.97 109 58.09 V9-6 164 140 82 1.51% 487 1.97 154 52.63 33 ST1 267 4000 34 1.15% 500 4.59 600 711.00 ST2 267 4000 35 1.15% 500 4.59 780 742.00 ST3 267 4000 34 1.15% 500 4.59 1200 539.00 ST4 267 4000 34 1.15% 500 4.59 1440 555.00 49 SC8 267 4000 35 1.15% 500 4.59 1200 801.00 SC9 267 4000 33 1.15% 500 4.59 1800 792.00 32 N1-1 255 300 38 1.00% 957 4.53 259 73.00 N1-2 255 300 39 1.00% 957 4.53 258 70.00 N2-1 255 300 38 1.00% 957 4.53 195 102.00 N2-2 255 300 39 1.00% 957 4.53 195 55.00 N3-1 255 300 38 1.00% 957 4.53 317 68.00 N3-2 255 300 39 1.00% 957 4.53 317 116.00 31 4 280 200 33 1.65% 550 3.57 147 68.00 5 280 200 33 1.65% 550 3.57 147 51.00 6 280 200 35 1.65% 550 3.57 148 59.00 7 280 200 33 1.65% 550 3.57 298 60.00 8 280 200 33 1.65% 550 3.57 297 48.00 9 280 200 35 1.65% 550 3.57 297 56.00 10 280 200 34 1.65% 550 3.57 397 43.00 11 280 200 34 1.65% 550 3.57 397 61.00 12 280 200 35 1.65% 550 3.57 397 62.00 13 280 200 34 1.65% 550 3.57 596 63.00 14 280 200 34 1.65% 550 3.57 596 60.00 15 280 200 35 1.65% 550 3.57 594 73.00 Average 296 370 37 1.5% 563 3.35 292 100.05 Minimum 164 70 15 0.2% 343 1.96 20 15.43 % https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ distribution diagrams for a) flexure only, b) tensile forces only, and c) both, respectively; thus, the tensile s at 60% of the effective depth from extreme compression fibers (ε) , which is calculated such that: Figure 3. Model development d ih The profile of data base. Figure 3. The profile of data base. distribution diagrams for a) flexure only, b) tensile forces only, and c) both, respectively; thus, the tensile strain at 60% of the effective depth from extreme compression fibers (ε) , which is calculated such that: (3) ε = 1 bdρEs   M d 1 −ρ · Es Ec 1 + 2Ec ρ·Es −1 /3 + N 2   0.6d −c d −c (3) https://doi.org/10.1038/s41598-022-11577-y https://doi.org/10.1038/s41598-022-11577-y https://doi.org/10.1038/s41598-022-11577-y https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ where b is the element width, ρ is the flexure reinforcement ratio, N is the axial force ( positive is tension and negative is compression), M is the bending moment at the critical section for shear, Es is the steel reinforcements young’s modulus (210,000 MPa), Ec is the concrete young’s modulus (10,000 3 f ′ c ), c is the compression zone depth, which is calculated such that: Figure 5 shows the variation of the compression zone depth (Eq. 4) versus the tensile stress for different flexure ′ (4) c d = ρ · Es Ec 1 + 2Ec ρ · Es −1 − Nd/M 2 1−ρ· Es Ec 1+ 2Ec ρ·Es −1 /3 + Td M Figure 4. Strain diagram for (a) flexure only, (b) tension only, and (c) flexure and tension. Figure 5. The effect of tension forces on the compression zone depth of elements with different reinforcement ratios (fc′ = 30 MPa and M/Vd = 4). Figure 4. Strain diagram for (a) flexure only, (b) tension only, and (c) flexure and tension. Figure 4. Strain diagram for (a) flexure only, (b) tension only, and (c) flexure and tension. Figure 4. Strain diagram for (a) flexure only, (b) tension only, and (c) flexure and tension. Figure 5. The effect of tension forces on the compression zone depth of elements with different reinforcement ratios (fc′ = 30 MPa and M/Vd = 4). Figure 5. The effect of tension forces on the compression zone depth of elements with different reinforceme ratios (fc′ = 30 MPa and M/Vd = 4). Model development d ih Where vu = V bd , ddg = dg0 + dg (the value of the reference aggregate size dg0 = 16 mm is used), and dg is the maximum nominal aggregate size. Table 2 shows the parameter table for the coefficient α1 and α2. Simplified model pi Model development. In this section, a simplified model is developed. Based on the proposed mechani- cal model presented in Eqs. (3), (4) and (6), the following parameters are identified to be effective in the shear strength of elements subjected to axial tensile forces: (1) M/Nd , (2) N/(bdρfy) , (3) M/Vd or a/d , (4) d , (5) f ′ c , and (6) ρ . Thus, nonlinear multi-variable regression was performed using the following power form: (7) vu = c1 N/(bdρfy) c2(M/Nd)c3ρc4f ′ c c5(M/Vd)c6dc7 (7) vu = c1 N/(bdρfy) c2(M/Nd)c3ρc4f ′ c c5(M/Vd)c6dc7 (7) Power form was implemented in several investigations (Ali et al., 2021; Deifalla et al., 2021; Deifalla, 2020b; 2020c; 2021b; 2021c). Table 3 shows the parameter table, including the probability of each parameter. It is worth noting that the power coefficient of the variable N/(bdρfy) failed the hypotheses test, and it was found to be insignificant. Therefore, it is proposed that shear strength is calculated such that: (8) vu = 17.22(M/Nd)0.077ρ0.34f ′ c 0.4(M/Vd)−0.7d−0.39 vu = 17.22(M/Nd)0.077ρ0.34f ′ c 0.4(M/Vd)−0.7d−0.39 (8) Model development d ih where b is the element width, ρ is the flexure reinforcement ratio, N is the axial force ( positive is tension and negative is compression), M is the bending moment at the critical section for shear, Es is the steel reinforcements young’s modulus (210,000 MPa), Ec is the concrete young’s modulus (10,000 3 f ′ c ), c is the compression zone depth, which is calculated such that: (4) c d = ρ · Es Ec 1 + 2Ec ρ · Es −1 − Nd/M 2 1−ρ· Es Ec 1+ 2Ec ρ·Es −1 /3 + Td M (4) Figure 5 shows the variation of the compression zone depth (Eq. 4) versus the tensile stress for different flexure reinforcement ratios. Figure 5 was based on f ′ c value of 30 MPa and M/Vd value of 4, where V is the shear force at the critical section for shear. It is clear that the model captured the reduction in the compression zone depth observed by Pham31. Proposed failure criteria for combined shear and tension. Based on data shown in Table 1 and Fig. 3 as well as the failure criteria of the CSCT, the following form was proposed: (5) vu f ′c = α1 1 + α2 εd ddg (5) Using nonlinear multi-variable regression, thus, the shear stress is calculated such that: ttps://doi.org/10.1038/s41598-022-11577-y https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Table 2. Parameter table for ECSCT. Parameter Estimate Standard error t-Statistic p-value α1 0.27 0.019 13.7 7.79E − 30 α2 22 8.47 2.56 0.011 Table 2. Parameter table for ECSCT. Parameter Estimate Standard error t-statistic p-value Lower 95% Upper 95% c1 17.22 0.530 5.37 2.53E − 07 6.047 49.05 c2 0.010 0.042 0.24 0.81 − 0.074 0.094 c3 0.077 0.017 4.41 1.81E − 05 0.042 0.111 c4 0.34 0.042 8.00 1.65E − 13 0.258 0.427 c5 0.40 0.063 6.29 2.39E − 09 0.273 0.523 c6 − 0.70 0.064 − 11 1.56E − 21 − 0.83 − 0.576 c7 − 0.39 0.054 − 7.1 2.31E − 11 − 0.495 − 0.281 Table 3. Parameter table for simplified model. Table 3. Parameter table for simplified model. Table 3. Parameter table for simplified model. (6) vu f ′c = 0.27 1 + 22 εd ddg (6) for shear and tension. Table 3. Parameter table for simplified model. Models validation The r is the degree of association, which is such that: r = n i=1 (xi −x) yi −y n i=1 (xi −x)2 n i=1 yi −y 2 (9) where, x and y are average values of variables xi and yi , respectively, and n is the number of tested specimens. The correlation coefficient is measured on a scale that varies from ±1 to 0. For example, ±1 , ±0.70 , ±0.50 , ±0.30 , and zero indicate exact, strong, moderate, weak, and no dependence, respectively. Thus, if the r value is less than ±0.30 shows that the model captured the effect of such parameter, while a coefficient ranged between ±0.50 and ±0.30 indicates a need for refinements in the modeling of this parameter. It is worth noting that the correlation coefficient is not directly related to the data scattering. Because the data scattering is dependent on the overall effect of the considered parameters in the specific model, while the correlation coefficient is indicative of the relation between the accuracy and a specific parameter. It is an indication not conclusive depending on the value as mentioned before. Overall. Figure 6 shows the calculated shear strength versus the measured ones for the ECSCT, the simplified model, the ACI, and the EC2. While the strength is calculated in terms of stress taken as the ratio between the shear force and the concrete cross area. In addition, the line represents the actual performance and the linear fit- ted line for the model performance. Moreover, the inverse of the best-fitted line slope ( χ ) is indicated in the plots. The closer this value to unity is, the better accuracy and less divergence. The χ value for the ECSCT, the simpli- fied model, the ACI, and the EC2 is 0.99, 0.88, 0.50, and 0.65. Thus, the strength calculated using the ECSCT and the simplified model is significantly less scattered than using the ACI and EC2.i pi gi y g Moreover, Fig. 6 shows the histogram of the SR values calculated using the ECSCT, the simplified model, the ACI, and the EC2. The SR is calculated using the simplified model, and the ECSCT is normally distributed around the ratio of unity compared to that using the ACI and the EC2. Models validation Last but not least, Table 4 shows the average, coefficient of variation, and lower 95% limit for SR calculated using the ECSCT, the simplified model, the ACI, and the EC2 for each study. It is clear that the performance of the ECSCT and the simplified model is more accurate and consistent than that of the ACI and EC2. However, it is safe with a lower 95% value of 0.96, higher than 0.85 targeted by most design codes. The simplified model and ECSCT SR values have a narrow range compared to that calculated using the ACI and the EC2, as shown in Table 4. Effect of axial tension (N). The effect of the axial tension was examined using several parameters, namely N/(bdfct), N/(ρbdfy), N/V and M/Nd. The SR is plotted against the N/(bdfct), N/(ρbdfy), N/V and M/Nd as shown in Figs. 7, 8, 9 and 10, respectively, where fct is the tensile concrete strength taken as 0.65 2 f ′ c , fy is the yield stress of the steel reinforcements. From Figs. 7, 8, 9 and 10, the safety of the strength calculated using the ECSCT and the simplified model is more consistent with the axial tension’s effect than the ACI and EC2. The correlation coefficient (r) was calculated as 0.51–0.67, 0.54–0.73, 0.02–0.14, and 0.04–0.25 for the ACI, the EC2, the ECSCT, the simplified model, respectively. In addition, the slope of the best fit line for the SR calculated using the ACI, the EC2, is much higher than that calculated using the ECSCT, the simplified model. Thus, it is clear that SR cal- culated using the ECSCT and the simplified model are weakly correlated to the axial tension, while the ACI and EC2 are highly correlated. The variation of the tensile axial force does not affect the ECSCT and the simplified model compared to the ACI and the EC2 with respect to the experimental database. Effect of shear span to depth ratio (a/d). The SR is plotted against the specimen size in the shear span to depth ratio (a/d), as shown in Fig. 11. From Fig. 11, the safety of the strength calculated using the ECSCT and the simplified model is more consistent with the effect of the a/d compared with the ACI and EC2. However, the safety for the ECSCT is higher for non-slender elements with a/d value less than 3. Models validation For simplicity, two design codes were selected for comparison: the ACI and the EC2. However, it is worth noting that there is other design model that are more accurate with various levels of approximation, for example fib model code50. For simplicity, the model code was not selected as it requires a detailed calculation compared to the ACI and EC2. The strength calculated using the ECSCT and the simplified model were assessed against those calculated using the selected design codes with respect to the experimentally measured strength.i Several types of figures were implemented to compare the performance of the proposed models with the selected design codes, which were implemented in several investigations12,51–54. Firstly: a scatter plot between the measured and calculated strength was plotted for all models, which was assessed using the ideal 45-degree line and the inverse of the slope of the best-fitted line. While the strength in terms of stress is taken as the ratio between the shear force and the concrete cross area. Secondly: a histogram figure for the distribution of the ratio between measured and calculated strength (SR), which is assessed based on the distribution and being far from the ideal ratio of unity and lower coefficient of variation. The unity value for SR indicates the closeness of the calculated value to the measured one (i.e., the model accuracy). At the same time, the coefficient of variation of the SR distribution indicates the consistency of the model. In addition, the lower 95% of the SR indicates the safety of the model. It is the minimum SR value obtained using the model with a 95% confidence level. Therefore, the higher value of the lower 95% limit above the safety factor of design codes (approximately 0.85), the safer the model is. The confidence interval is calculated assuming a standard normal distribution. In addition, a significant level value of 0.05 represents the 95% confidence level. Thus, the lower 95% confidence limit is calculated using the following expression: https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ (8) Lower 95% = Average −1.96 Standard deviation number of samples (8) Thirdly: a scatter plot for the SR against various effective parameters is plotted, which is assessed using the inclination of the best-fitted line and the correlation coefficient (r). The closer the slope to zero is the more negligible effect of the variable on the accuracy and safety of the model. Models validation This is because the original CSCT model was not developed for non-slender. In addition, the correlation coefficient (r) was calculated as 0.46, 0.49, 0.40, and 0.09 for the ACI, the EC2, the ECSCT, the simplified model, respectively. Thus, it is clear that SR calculated using the simplified model is weakly correlated to the a/d, while the ECSCT, the ACI, and EC2 are highly correlated to the a/d.i g y Moreover, the slope of the best fit line for the SR calculated using the ACI, the EC2, the ECSCT, and the simplified model is 0.46, 0.56, 0.10, and 0.02, respectively. The slope for the SR calculated using the ECSCT and simplified model is significantly lower than that calculated using the ACI and the EC2. The simplified model showed quite an improvement with respect to the effect of the arch mechanism in terms of the shear span to depth ratio (a/d). Effect of specimen size (d). Plots of the SR versus the specimen size in terms of effective depth (d) are shown in Fig. 12. From Fig. 12, the safety of the strength calculated using the ECSCT and the simplified model is more consistent with the effect of the d compared with the ACI and EC2. In addition, the correlation coef- https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ fi i t ( ) l l t d 0 26 0 32 0 04 d 0 25 f th ACI th EC2 th ECSCT d th i lifi d Figure 6. Measured Strength versus calculate strength using various methods. Figure 6. Measured Strength versus calculate strength using various methods. ficient (r) was calculated as 0.26, 0.32, 0.04, and 0.25 for the ACI, the EC2, the ECSCT, and the simplified model, respectively. Thus, it is clear that SR calculated using the simplified model, the ECSCT is weakly correlated to the d. Moreover, the slope of the best fit line for the SR calculated using the ACI, the EC2, the ECSCT, and the simplified model is 2300E-6, 2900E-6, 70E-6, and 1E-6, respectively. Thus, the slope for the SR calculated using the ECSCT and simplified model is significantly lower than that calculated using the ACI and the EC2. There- fore, the ECSCT model and the simplified model account for the effect of size in terms of d much better than the ACI and the EC2. Models validation ficient (r) was calculated as 0.26, 0.32, 0.04, and 0.25 for the ACI, the EC2, the ECSCT, and the simplified model, respectively. Thus, it is clear that SR calculated using the simplified model, the ECSCT is weakly correlated to the d. Moreover, the slope of the best fit line for the SR calculated using the ACI, the EC2, the ECSCT, and the simplified model is 2300E-6, 2900E-6, 70E-6, and 1E-6, respectively. Thus, the slope for the SR calculated using the ECSCT and simplified model is significantly lower than that calculated using the ACI and the EC2. There- fore, the ECSCT model and the simplified model account for the effect of size in terms of d much better than the ACI and the EC2. Effect of flexural reinforcement ratio ( ρ). The SR is plotted versus the size in terms of the flexure rein- forcement ratio ( ρ ), as shown in Fig. 13. From Fig. 13, the safety of the strength calculated using the ECSCT and https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Table 4. Statistical measures for the SR for shear with axial tension. Refs. Models validation Measure Number ACI-19 EC2 ECSCT Simplified model 36 Mean 2 1.80 0.95 0.48 0.74 Coefficient of variation 2% 6% 14% 12% 37 Mean 11 1.49 1.03 0.93 1.04 Coefficient of variation 22% 17% 16% 20% 38 Mean 3 2.11 1.40 1.25 0.95 Coefficient of variation 19% 19% 19% 24% 39 Mean 19 1.86 1.23 0.92 0.87 Coefficient of variation 23% 21% 24% 22% 40 Mean 2 3.00 1.58 0.85 1.19 Coefficient of variation 10% 7% 13% 12% 41 Mean 3 5.00 5.00 1.27 1.04 Coefficient of variation 0% 0% 21% 18% 42 Mean 16 5.00 4.62 1.16 1.23 Coefficient of variation 0% 13% 21% 21% 43 Mean 1 5.00 5.00 2.50 1.71 Coefficient of variation N/A N/A N/A N/A 44 Mean 5 2.84 1.53 0.73 1.02 Coefficient of variation 23% 20% 15% 19% 45 Mean 14 3.17 1.89 1.06 0.90 Coefficient of variation 48% 43% 28% 27% 46 Mean 7 3.25 2.75 0.56 0.64 Coefficient of variation 59% 77% 13% 14% 30 Mean 44 3.16 2.23 0.95 1.02 Coefficient of variation 43% 50% 18% 18% 47 Mean 19 4.73 4.46 1.11 0.93 Coefficient of variation 33% 27% 14% 11% 48 Mean 10 4.19 3.00 1.81 1.25 Coefficient of variation 18% 27% 22% 17% 33 Mean 4 0.83 0.59 0.44 0.62 Coefficient of variation 10% 11% 16% 12% 49 Mean 2 1.16 0.81 0.58 0.80 Coefficient of variation 11% 16% 3% 3% 32 Mean 6 2.80 1.70 0.88 1.15 Coefficient of variation 44% 38% 34% 27% 31 Mean 12 4.24 3.22 0.88 0.93 Coefficient of variation 32% 53% 18% 14% Overall Mean 180 3.3 2.5 1.01 0.99 Coefficient of variation 46% 62% 35% 24% Minimum 0.73 0.5 0.37 0.50 Maximum 5 5 2.58 1.71 Lower 95.0% 3.07 2.3 0.96 0.96 Table 4. Statistical measures for the SR for shear with axial tension. the simplified model is more consistent with the effect of the ρ compared with the ACI and EC2. In addition, the correlation coefficient (r) was calculated as 0.08, 0.00, 0.18, and 0.06 for the ACI, the EC2, the ECSCT, and the simplified model, respectively. Thus, it is clear that SR calculated using the ACI, the EC2, the ECSCT, and the simplified model is weakly correlated to the ρ . Moreover, the slope of the best fit line for the SR calculated using https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ p Figure 7. Models validation SR using various methods versus the N/(bdfct). Figure 7. SR using various methods versus the N/(bdfct). Figure 7. SR using various methods versus the N/(bdfct). https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Figure 8. SR using various methods versus the N/(ρbdfy). Figure 8. SR using various methods versus the N/(ρbdfy). Figure 8. SR using various methods versus the N/(ρbdfy). https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Figure 9. SR using various methods versus the N/V. Figure 9. SR using various methods versus the N/V. Figure 9. SR using various methods versus the N/V. https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Figure 10. SR using various methods versus the M/Nd. Figure 10. SR using various methods versus the M/Nd. Figure 10. SR using various methods versus the M/Nd. https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Figure 11. SR using various methods versus the d. Fi 11 SR i i th d th d Figure 11. SR using various methods versus the d. https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Figure 12. SR using various methods versus the a/d. Figure 12. SR using various methods versus the a/d. https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Figure 13. SR using various methods versus the ρ. Figure 13. SR using various methods versus the ρ. https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ Figure 14. SR using various methods versus the fc′. Figure 14. SR using various methods versus the fc′. https://doi.org/10.1038/s41598-022-11577-y Scientific Reports \| (2022) 12:7863 \| www.nature.com/scientificreports/ the ACI, the EC2, the ECSCT, and the simplified model is 18.4, 0.137, 9.6, and 2.2, respectively. Thus, the sl for the SR calculated using all models is similar. Therefore, it is clear that all models account for the dowel ac mechanism in terms of the flexure reinforcement ratio. Figure 15. SR using various methods versus the b/d. h d h b d the ACI, the EC2, the ECSCT, and the simplified model is 18.4, 0.137, 9.6, and 2.2, respectively. Thus, the slope for the SR calculated using all models is similar. Therefore, it is clear that all models account for the dowel action mechanism in terms of the flexure reinforcement ratio. Effect of concrete compressive strength (fc′). The SR is plotted versus the size in terms of the con crete compressive strength (fc′), as shown in Fig. 14. From Fig. www.nature.com/scientificreports/ In addition, the correlation coefficient (r) was calculated as 0.08, 0.04, 0.04, and 0.01 for the ACI, the EC2, the ECSCT, and the simplified model, respectively. Thus, it is clear that SR calculated using the ACI, the EC2, the ECSCT, and the simplified model is weakly correlated to the fc′. Moreover, the slope of the best fit line for the SR calculated using the ACI, the EC2, the ECSCT, and the simplified model is 103E-4, 96E-4, 3E-4, and 11E-4, respectively. Thus, the slope for the SR calculated using the ECSCT and simplified model is significantly lower than that calculated using the ACI and the EC2. Therefore, it is clear that the ECSCT and the simplified model are more consistent and accurate with respect to the direct shear mechanism and the residual tensile stresses in terms of the concrete strength (fc′). Effect of width to depth ratio ( b/d). The SR is plotted versus the size in terms of the width to depth ratio ( b/d ), as shown in Fig. 15. From Fig. 15, the safety of the strength calculated using the ECSCT and the simplified model is more consistent with the effect of the b/d compared with the ACI and EC2. In addition, the correlation coefficient (r) was calculated as 0.29, 0.24, 0.29, and 0.22 for the ACI, the EC2, the ECSCT, and the simplified model, respectively. Thus, it is clear that SR calculated using the ACI, the EC2, the ECSCT, and the simplified model is moderately correlated to the b/d . Moreover, the slope of the best fit line for the SR calculated using the ACI, the EC2, the ECSCT, and the simplified model is 17E-2, 15E-2, 4E-2, and 2E-2, respectively. The slope for the SR calculated using the ACI and the EC2 is significantly higher than that calculated using the ECSCT and simplified model. Therefore, it is clear that the ECSCT and the simplified model are more consistent with respect to the aspect ratio. Data availability All data generated or analysed during this study are included in this published article. y All data generated or analysed during this study are included in this published article. Received: 11 February 2022; Accepted: 26 April 2022 Conclusions A physically sound mechanical model capable of accurately reproducing the actual behavior of reinforced con- crete members under combined shear and tension is proposed. In addition, the model is accurate and simple for design. The effect of axial tensile forces on the compression zone depth and longitudinal strain and, ultimately, on the shear strength is accounted for. The proposed model is based on the principles of mechanics and its appli- cability to reinforced concrete elements under shear combined with tension. In addition, a simplified model is proposed for the purpose of design. The two proposed models were found to be more accurate, consistent, and reasonably safe compared to selected design codes. Moreover, the effect of basic parameters on the safety of the proposed models and the selected design codes was assessed. For all basic variables, including (1) the axial ten- sion; (2) the shear span to depth ratio; (3) the flexure reinforcement ratio; (4) the concrete compressive strength; and (5) the width to dept ratio, the following conclusions were reached. – The correlation relation with the safety factor calculated using the American and European design code was very strong, while that for the proposed models was very weak. Thus, the proposed models captured the effect of all basic variables much better than the selected design models.hi – The correlation relation with the safety factor calculated using the American and European design code was very strong, while that for the proposed models was very weak. Thus, the proposed models captured the effect of all basic variables much better than the selected design models.hi f – The slope of the best fit line for the safety factor calculated using the proposed models is very small compared to that using the selected design codes. Thus, the proposed models are more consistent with the basic vari- ables than existing design codes. f – The slope of the best fit line for the safety factor calculated using the proposed models is very small compared to that using the selected design codes. Thus, the proposed models are more consistent with the basic vari- ables than existing design codes. References References 1. Hu Biao, Wu. & Fei, Y. Quantification of shear cracking in reinforced concrete beams. Eng. Struct. 147(2017), 666–678 (2017).f 1. Hu Biao, Wu. & Fei, Y. Quantification of shear cracking in reinforced concrete beams. Eng. Struct. 147(2017), 666–678 (2017). 2 Bi H & Y F i W Eff t f h t d th ti h t th t f RC b E St t 168(2018) Qi g g ( ) ( ) 2. Biao, H. & Yu-Fei, W. Effect of shear span-to-depth ratio on shear strength components of RC beams. Eng. Struct. 168(2018) 770–783 (2018). 3. Yu-Fei, W. & Biao, H. Shear strength components in reinforced concrete members. J. Struct. Eng. 143(9), 04017092–04017101. https://doi.org/10.1061/(ASCE)ST.1943-541X.0001832 (2017). 3. Yu-Fei, W. & Biao, H. Shear strength components in reinforced concrete members. J. Struct. Eng. 143(9), 04017092–04017101 https://doi.org/10.1061/(ASCE)ST.1943-541X.0001832 (2017). p g 4. Deifalla, A. & Ghobarah, A. Full torsional behavior of RC beams wrapped with FRP: Analytical model. ACSE Compos. Construct 14(3), 289–300 (2010). 5. Deifalla, A. & Ghobarah, A. Strengthening RC T-beams subjected to combined torsion and shear using FRP fabrics—Experimenta study. ASCE Compos. Construct. 14(3), 301–311 (2010). y p 6. Deifalla, A. & Ghobarah, A. Behavior and analysis of inverted T-shaped RC beams under shear and torsion. Eng. Struct. Elsevier 62, 776–786 (2014). 7. Deifalla, A., Hamed, M., Saleh, A. & Ali, T. Exploring GFRP bars as reinforcement for rectangular and L-shaped beams subjected to significant torsion: An experimental study. Eng. Struct. Elsevier 59, 776–786 (2014).i i 8. 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Oct. 2024 2D Materials You may also like A molecular dynamics study of heterogeneous nucleation in generic liquid/substrate systems with positive lattice misfit Z Fan and H Men - Enhancement of open-circuit voltage in InGaP solar cells grown by solid source molecular beam epitaxy Ryuji Oshima, Yuki Nagato, Yoshinobu Okano et al. - Polariton condensate and Landau-Zener- Stückelberg interferometry transition in multilayer transition metal dichalcogenides C M Ekengoue, C Kenfack-Sadem, J E Danga et al. - You may also like A molecular dynamics study of heterogeneous nucleation in generic liquid/substrate systems with positive lattice misfit Z Fan and H Men - Enhancement of open-circuit voltage in InGaP solar cells grown by solid source molecular beam epitaxy Ryuji Oshima, Yuki Nagato, Yoshinobu Okano et al. - Polariton condensate and Landau-Zener- Stückelberg interferometry transition in multilayer transition metal dichalcogenides C M Ekengoue, C Kenfack-Sadem, J E Danga et al. - PAPER • OPEN ACCESS This content was downloaded from IP address 131.155.94.127 on 04/04/2022 at 13:36 PAPER • OPEN ACCESS You may also like Abstract The unique optical and electronic properties of two-dimensional transition metal dichalcogenide (2D TMDs) make them promising materials for applications in (opto-)electronics, catalysis and more. Specifically, alloys of 2D TMDs have broad potential applications owing to their composition-controlled properties. Several important challenges remain regarding controllable and scalable fabrication of these alloys, such as achieving control over their atomic ordering (i.e. clustering or random mixing of the transition metal atoms within the 2D layers). In this work, atomic layer deposition is used to synthesize the TMD alloy Mo1−xWxS2 with excellent composition control along the complete composition range 0 ⩽x ⩽1. Importantly, this composition control allows us to control the atomic ordering of the alloy from well-mixed to clustered while keeping the alloy composition fixed, as is confirmed directly through atomic-resolution high-angle annular dark-field scanning transmission electron micrography imaging. The control over atomic ordering leads to tuning of the bandgap, as is demonstrated using optical transmission spectroscopy. The relation between this tuning of the electronic structure and the atomic ordering of the alloy was further confirmed through ab-initio calculations. Furthermore, as the atomic ordering modulates from clustered to well-mixed, the typical MoS2 and WS2 A1g vibrational modes converge. Our results demonstrate that atomic ordering is an important parameter that can be tuned experimentally to finely tune the fundamental properties of 2D TMD alloys for specific applications. Controlling transition metal atomic ordering in two-dimensional Mo1−xWxS2 alloys eff J P M Schulpen1, Marcel A Verheijen1,2, Wilhelmus M M (Erwin) Kessels1, Vincent Vandalon1 1 Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands 2 Eurofins Materials Science BV, High Tech Campus, Eindhoven, The Netherlands ∗Author to whom any correspondence should be addressed. Supplementary material for this article is available online Controlling transition metal atomic ordering in two- dimensional Mo1−xWxS2 alloys Polariton condensate and Landau-Zener- Stückelberg interferometry transition in multilayer transition metal dichalcogenides C M Ekengoue, C Kenfack-Sadem, J E Danga et al - View the article online for updates and enhancements. https://doi.org/10.1088/2053-1583/ac54ef 2D Mater. 9 (2022) 025016 OPEN ACCESS RECEIVED 10 September 2021 REVISED 9 February 2022 ACCEPTED FOR PUBLICATION 14 February 2022 PUBLISHED 3 March 2022 Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. 2.1.1. In-situ growth characterization g The growth of the Mo1−xWxS2 films by ALD was characterized in-situ by spectroscopic ellipsometry (SE). Figure 2(a) shows the deposited film thickness per ALD cycle (GPC) as a function of the cycle frac- tion n/(n + m). The GPC of the pure WS2 and MoS2 and films are determined to be 0.08 and 0.13 nm respectively, in line with our previously reported characterization of these ALD processes [31, 33]. The GPC of the alloy increases linearly with the molyb- denum content, which is in line with the rule of mix- tures for alloys [35]: GPCalloy = n n + m × GPCMoS2 + m n + m × GPCWS2, (1) (1) demonstrating well-behaved alloying behavior free from the non-idealities commonly observed when depositing alloys by ALD [35]. 2.1. Control over alloy composition y p A series of six Mo1−xWxS2 samples was prepared using supercycle fractions of 0.0 (pure WS2), 0.2, 0.4, 0.6, 0.8 and 1.0 (pure MoS2), each for a total of 50 ALD cycles. We first investigate the growth and chem- ical composition of the alloys, after which we charac- terize their composition-dependent vibrational spec- trum and electronic structure. In this work, we introduce a plasma-enhanced ALD (PE-ALD) process for one-step deposition of Mo1−xWxS2 alloys at a low substrate temperature of 350 ◦C without the need for post-deposition sul- furization. Using this process, we demonstrate fine control over the alloy composition x by manipulat- ing the relative number of MoS2 and WS2 deposition cycles in a supercycle scheme, which is an established method of growing alloys of e.g. oxides by ALD [9]. Furthermore, we demonstrate for the first time con- trol over the atomic ordering within the Mo1−xWxS2 layers, which is confirmed directly through high- angle annular dark-field scanning transmission elec- tron micrography (HAADF-STEM). The control over the atomic ordering of the alloys is achieved by manipulating the supercycle period of the ALD pro- cess (i.e. the total number of ALD cycles per super- cycle). Beyond HAADF-STEM imaging, we report the effects of atomic ordering on the electronic struc- ture and phonon spectrum of the alloy, which are probed through optical transmission spectroscopy and Raman spectroscopy respectively. Finally, dens- ity functional theory (DFT) calculations are carried out to provide fundamental insight into how the elec- tronic structure of the alloys is modulated by their atomic ordering. 1. Introduction The cycle fraction n/(n + m) determines the composition x of the alloy, whereas the number of cycles per supercycle n + m (i.e. the supercycle length) is exploited to control the atomic ordering of the alloy. The total number of cycles N × (n + m) determines the film thickness. Atomic layer deposition (ALD) is a thin film deposition technique that exploits self-limiting sur- face reactions of vapor-phase precursors to achieve sub-nanometer growth control at low deposition temperatures (<450 ◦C). While direct ALD growth of pure TMDs has been reported [31–33], efforts on ALD of TMD alloys have focused on high- temperature sulfurization of metal (oxide) parent films, with the parent film synthesized by ALD [34]. While this approach allows a graded compos- ition profile of the alloy [34], the high-temperature sulfurization step nevertheless diminishes the low- temperature benefit of ALD, such that there remains a need for a fully low-temperature ALD synthesis process for better control over the composition and atomic ordering of the TMD alloys. ALD cycles followed by m WS2 ALD cycles are repeated, as illustrated in figure 1. The supercycle fraction n / (n + m) determines the composition of the alloy (the Mo/W ratio), whereas the supercycle length n + m determines the atomic ordering of the alloy. The total number of ALD cycles N × (n + m) determines the thickness of the deposited film. 1. Introduction nanoelectronics [5], versatile nanophotonics [6], and highly efficient photovoltaics [7]. Two-dimensional (2D) materials consist of molecu- lar layers whose mutual interaction is weak compared to the bonding within the layers [1], such that these materials can be thinned down to a single molecu- lar layer while maintaining predictable properties [2] which often include interesting physical phenom- ena induced by quantum confinement [3, 4]. Trans- ition metal dichalcogenides (TMDs) are a particularly important group of 2D materials since many of them, including the well-studied materials MoS2 and WS2, are semiconductors. This makes them interesting for a wide array of applications such as ultimately-scaled Alloying of semiconducting 2D TMDs further expands the possible applications of these materi- als [8]. For example, their electronic bandgap can be tuned continuously over a wide energy range both through chalcogen substitution [9–17] and through metal substitution [18–20]. Furthermore, alloying can improve the quality of TMD films, such as by reducing the density of chalcogen vacancy defects [21]. Important challenges remain in achieving con- trollable and scalable synthesis of TMD alloys. Tech- niques such as chemical vapor deposition (CVD) and © 2022 The Author(s). Published by IOP Publishing Ltd 2D Mater. 9 (2022) 025016 J J P M Schulpen et al Figure 1. The ALD supercycle scheme used for growth of Mo1−xWxS2 alloys by alternating ALD cycles of MoS2 and WS2. The cycle fraction n/(n + m) determines the composition x of the alloy, whereas the number of cycles per supercycle n + m (i.e. the supercycle length) is exploited to control the atomic ordering of the alloy. The total number of cycles N × (n + m) determines the film thickness. chemical vapor transport (CVT), which are known to produce high-quality samples of pure TMDs [22–24], are being used to synthesize such alloys [25–27]. However, control over the growth is usually limited when using these techniques. For instance, it has not been possible to control the atomic ordering of the alloys (i.e. well-mixed or clustered) using these tech- niques [8]. Instead, the alloys grown by CVD and CVT invariably exhibit random mixing of the con- stituents, likely due to strong surface diffusion of atoms during synthesis owing to the inherently high processing temperatures of these techniques [28–30]. Figure 1. The ALD supercycle scheme used for growth of Mo1−xWxS2 alloys by alternating ALD cycles of MoS2 and WS2. 2.1.2. Chemical composition On the same set of samples, the alloy composition x and stoichiometry (defined as the S/(Mo + W) ratio) as a function of the supercycle fraction n/(n + m) Mo1−xWxS2 alloys were grown by ALD using a scheme whereby supercycles consisting of n MoS2 2 2D Mater. 9 (2022) 025016 2D Mater. 9 (2022) 025016 J J P M Schulpen et al Figure 2. Impact of the composition x (controlled through the ALD cycle fraction n/(n + m)) of the Mo1−xWxS2 films on their (a) growth per cycle measured by in-situ SE, (b) elemental composition measured by XPS, (c) vibrational spectrum measured by Raman spectroscopy, (d) electronic structure measured by optical extinction spectroscopy. The insets in (d) show the positions of the absorption peaks as a function of alloy composition, as well as the difference between the alloy spectrum and a superposed MoS2 + WS2 spectrum. Spectra in (c) and (d) have been offset vertically for legibility. Figure 2. Impact of the composition x (controlled through the ALD cycle fraction n/(n + m)) of the Mo1−xWxS2 films on their (a) growth per cycle measured by in-situ SE, (b) elemental composition measured by XPS, (c) vibrational spectrum measured by Raman spectroscopy, (d) electronic structure measured by optical extinction spectroscopy. The insets in (d) show the positions of the absorption peaks as a function of alloy composition, as well as the difference between the alloy spectrum and a superposed MoS2 + WS2 spectrum. Spectra in (c) and (d) have been offset vertically for legibility. salloy = n n + m × sMoS2 + m n + m × sWS2, were measured using x-ray photoelectron spectro- scopy (XPS) and are shown in figure 2(b). The trend in alloy composition x closely follows the rule of mix- tures given by (3) where sMoS2 and sWS2 are the stoichiometries of the pure MoS2 and WS2 films respectively. The stoi- chiometry of the alloys as a function of the cycle fraction is well described by equation (3), again con- firming the well-behaved growth of the Mo1−xWxS2 alloys over the complete composition range 0 ⩽x ⩽1. x = [Mo] [Mo] + [W] = n n+m × GPCat,Mo n n+m × GPCat,Mo + m n+m × GPCat,W , (2) (2) where the GPCat,Mo is the number of Mo atoms deposited per MoS2 ALD cycle, and GPCat,W the number of W atoms deposited per WS2 ALD cycle. 2.1.2. Chemical composition By fitting equation (2) to the data, the atomic GPC ratio between Mo and W is found to be 1.67 ± 0.03. This value is within experimental error of the GPC ratio found from the ellipsometry data, indicating that the trend in film thickness as a function of the cycle fraction can be attributed to the difference between the number of Mo and W atoms deposited per ALD cycle. As also shown in figure 2(b), the deposited alloys are approxim- ately stoichiometric (sulfur to metal ratio ≈2), with a trend towards over-stoichiometry (excess sul- fur) for tungsten-rich films. Analogously to equation (1), a rule of mixtures can be formulated for the stoichiometry: where the GPCat,Mo is the number of Mo atoms deposited per MoS2 ALD cycle, and GPCat,W the number of W atoms deposited per WS2 ALD cycle. where the GPCat,Mo is the number of Mo atoms deposited per MoS2 ALD cycle, and GPCat,W the number of W atoms deposited per WS2 ALD cycle. By fitting equation (2) to the data, the atomic GPC ratio between Mo and W is found to be 1.67 ± 0.03. This value is within experimental error of the GPC ratio found from the ellipsometry data, indicating that the trend in film thickness as a function of the cycle fraction can be attributed to the difference between the number of Mo and W atoms deposited per ALD cycle. As also shown in figure 2(b), the deposited alloys are approxim- ately stoichiometric (sulfur to metal ratio ≈2), with a trend towards over-stoichiometry (excess sul- fur) for tungsten-rich films. Analogously to equation (1), a rule of mixtures can be formulated for the stoichiometry: 2.1.3. Crystallinity and vibrational spectrum 2.1.3. Crystallinity and vibrational spectrum The impact of the alloy composition on the crystallin- ity and vibrational spectrum of Mo1−xWxS2 alloys was studied by Raman spectroscopy: the spectra are shown in figure 2(c). In the spectrum of the pure MoS2 film, two dominant peaks at 384 and 409 cm−1 are observed which can be identified as the in-plane E12g mode and out-of-plane A1g mode respectively [36]. Peaks corresponding to the same vibrational modes are observed in the spectrum of the pure WS2 films at 356 and 420 cm−1 [37]. It should be noted that the E12g peak of WS2 overlaps with its 2LA(M) mode, which dominates when using 514 nm excita- tion light on pure WS2 [38, 39]. For simplicity, we nevertheless refer to the peak complex at 356 cm−1 as the E12g peak. The E12g and A1g peaks of MoS2 and 3 3 2D Mater. 9 (2022) 025016 2D Mater. 9 (2022) 025016 J J P M Schulpen et al WS2 persist at all alloy fractions 0 < x < 1, indicat- ing a polycrystalline structure of the alloys along the complete composition range. are not independent. Since our deposition process for Mo1−xWxS2 shows no such non-ideal behavior, it is a very interesting case study into the isolated effects of supercycle length at a fixed alloy composition. Fur- thermore, since it takes approximately 15 ALD cycles to grow a closed monolayer film with our process, a short supercycle (<10 cycles) process could be used to tune the atomic ordering within the individual molecular layers of the alloy without changing their composition. As a function of the alloy fraction x, the MoS2- like and WS2-like E12g peaks remain spectrally sep- arated and their relative intensities scale with the alloy fraction, i.e. they exhibit two-mode beha- vior [40]. Similarly, though the spectral proxim- ity of the A1g modes makes their deconvolution more challenging, the asymmetric lineshape of the A1g complex at intermediate alloy fractions sug- gests two-mode behavior for this mode as well. The same behavior is observed from alloys deposited by CVT [27]. To confirm that the supercycle length only influ- ences the atomic ordering of the Mo1−xWxS2 alloys and not their composition, XPS and Rutherford backscattering spectroscopy (RBS) measurements were carried out. From these measurements, it was confirmed that the relative change in alloy fraction ∆x/x was less than 3% for supercycle lengths ranging from 2 to 48 cycles. 2.1.4. Electronic structure To study the evolution of the electronic structure of the alloys as a function of their composition, optical transmission measurements were carried out: the spectra are shown figure 2(d). In the spectrum of pure MoS2, two extinction peaks are seen around 1.86 and 2.02 eV, which can be identified as the fundamental electronic transitions of the A exciton and B exciton [41–43]. Analogous peaks are observed in the spec- trum of pure WS2 around 2.01 and 2.38 eV, which is in line with literature values for the A and B exciton energies of this material [41–43]. 2.2.1. HAADF-STEM imaging To directly study the atomic ordering of the ALD- grown Mo1−xWxS2 films, HAADF-STEM imaging was performed on two sub-monolayer samples grown using different supercycle lengths. The first was made through a single supercycle of ten ALD cycles, i.e. five MoS2 cycles followed by five WS2 cycles. The second was made through five supercycles of two cycles each, i.e. 5 × (1 MoS2 cycle + 1 WS2 cycle), again for a total of ten ALD cycles. In the atomic-resolution STEM micrographs (figures 3(a) and (b)), Mo and W atoms can be distinguished by their Z-contrast: the heavier W atoms appear brighter than the lighter Mo atoms, while S atoms are not visible in HAADF-STEM mode. The spectra of intermediate alloys each exhibit two distinct extinction peaks in the spectral region between 1.8 and 2.5 eV, analogous to the A and B excitonic peaks of pure MoS2 and WS2. Further- more, the positions of these peaks vary smoothly with the alloy composition. This behavior signi- fies good mixing of the alloy’s constituents, since any significant phase separation in the alloy would result in a spectrum that resembles a superposi- tion of the spectra of the constituents, which is not observed. Additionally, the shifts of the excitonic peaks as a function of alloy composition follow a quadratic bowing trend, in line with theoretical calculations [20]. The hexagonal crystal structure of MoS2 and WS2 is clearly visible in the STEM micrographs, dir- ectly confirming the polycrystallinity of the depos- ited alloys: the crystal (grain) sizes are approxim- ately 5–10 nm. Comparing the STEM images of the long-supercycle sample (figure 3(a)) and the short- supercycle sample (figure 3(b)) shows a clear dif- ference in atomic ordering of the alloys. The long- supercycle sample exhibits clusters of Mo atoms which are bordered by W atoms. The short-supercycle sample does not exhibit such clustering of Mo and W atoms: instead the Mo and W atoms appear to be ran- domly distributed in the alloy. 2.2. Control over atomic ordering The longer supercycle length results in clustering of Mo and W while the shorter supercycle length produces a well-mixed alloy, as also illustrated by the TEM brightness profiles (c) and (d). Results of stochastic simulations of the film growth (e) and (f) reproduce the observed differences in atomic ordering, indicating that the supercycle length together with the preferential edge growth of the material play a large role in determining the atomic ordering. in the supplementary information. The result for long supercycles is the formation of 2D core/shell-like nan- oflakes with MoS2 cores and WS2 shells. Conversely, for the short-supercycle sample, the number of con- secutive MoS2 and WS2 cycles is too low for sig- nificant clusters of MoS2 or WS2 to form. To sup- port this proposed mechanism, simulations of the initial film growth were conducted using a simpli- fied 2D growth model based on sequential precursor adsorption with preferential adsorption at grain edge sites. The results of these simulations are shown in figures 3(e) and (f), and are in qualitative agreement with the observed differences in atomic ordering as a function of supercycle length. In summary, the atomic ordering within the individual Mo1−xWxS2 molecular layers can be controlled through the super- cycle length n + m of the ALD process. Further- more, the lateral grain sizes of the deposited films can be estimated from the HAADF-STEM micro- graphs (figures 3(a), (b), S1 and S2 available online at stacks.iop.org/TDM/9/025016/mmedia) to be typ- ically 5–10 nm. supercycle length of 24 cycles, four peaks are seen at frequencies corresponding to the E12g and A1g modes of bulk MoS2 and WS2 [37, 45], indicating that the sample resembles a heterostructure as is expected for long supercycles [46]. As the supercycle period decreases, the MoS2-like E12g vibration at 383 cm−1 softens by 3.0 ± 0.5 cm−1 along with a broadening of the peak. The WS2-like E12g peak does not shift as much as the MoS2-like E12g peak, though some softening is still observed. The A1g peaks are spectrally separated at a super- cycle period of 24 cycles, while for shorter super- cycle periods the two individual peaks cannot be resolved. This merging of the two A1g phonon peaks cannot be caused purely by a broadening of these peaks, since that would broaden the total two-peak complex, which is not observed. 2.2. Control over atomic ordering The ideal rule-of-mixtures alloying behavior of the described deposition process opens up interesting new possibilities of fine-tuning the growth of the alloy. As demonstrated in the previous section, the cycle fraction of the supercycle process controls the composition of the alloy. On the other hand, it is known that the supercycle period can be used to con- trol the ordering of an alloy: short supercycle periods produce a well-mixed film while long supercycle periods produce a nanolaminate film [35]. Usually [35], non-ideal growth characteristics such as het- eronucleation delays lead to changes in composition when the supercycle length is changed, such that the effects of the cycle fraction and the supercycle length Since the long-supercycle sample was grown by performing five MoS2 cycles followed by five WS2 cycles, the distribution of Mo and W atoms in this sample is consistent with MoS2 island growth dur- ing the first five cycles followed by edge-on growth of WS2 at the edges of the MoS2 islands in the last five ALD cycles. This growth mode is consistent with earlier reports of 2D TMD growth by ALD, where nucleation is followed by preferential edge-on growth [31, 33, 44]. This growth mechanism is also discussed 4 J J P M Schulpen et al 2D Mater. 9 (2022) 025016 Figure 3. HAADF-STEM imaging of sub-monolayer samples of ALD-grown Mo1−xWxS2 with supercycle lengths of ten cycles (a) and two cycles (b), both for a total of ten ALD cycles. The scalebars indicate a size of 2 nm. The insets show local Fourier transforms, demonstrating the hexagonal crystal structure of both samples. The longer supercycle length results in clustering of Mo and W while the shorter supercycle length produces a well-mixed alloy, as also illustrated by the TEM brightness profiles (c) and (d). Results of stochastic simulations of the film growth (e) and (f) reproduce the observed differences in atomic ordering, indicating that the supercycle length together with the preferential edge growth of the material play a large role in determining the atomic ordering. Figure 3. HAADF-STEM imaging of sub-monolayer samples of ALD-grown Mo1−xWxS2 with supercycle lengths of ten cycles (a) and two cycles (b), both for a total of ten ALD cycles. The scalebars indicate a size of 2 nm. The insets show local Fourier transforms, demonstrating the hexagonal crystal structure of both samples. 2.2. Control over atomic ordering Hence, a frequency shift of one or both of the A1g peaks must be involved. Spectral deconvolution indicates the MoS2- like A1g mode stiffens by 3.5 ± 0.5 cm−1 while the WS2-like A1g mode softens by 2.0 ± 0.3 cm−1 as the supercycle length is shortened from 24 to 2 cycles. 2.2.2. Impact of atomic ordering on the vibrational structure The broadening of the two E12g peaks for short supercycle lengths can be understood as a consequence of the enhanced mixing of the alloy constituents, which reduces the crystalline order and relaxes the momentum selection rule on the Raman scattering process [47]. On the other hand, the soften- ing of the E12g modes as a function of the supercycle period cannot easily be explained on the basis of known effects. Firstly, strain [48–50] of the alloy film is not expected to be the cause of these shifts The influence of the atomic ordering of the Mo1−xWxS2 alloys on their vibrational structure was investigated by Raman spectroscopy. Few-layer (6–8 L) samples were grown using supercycle lengths of 2, 6, 10, 16 and 24 cycles at a constant MoS2 cycle fraction n/(n + m) of 0.5 and a total number of 48 ALD cycles for each sample. The Raman spectra are shown in figure 4. For the sample with the longest 5 2D Mater. 9 (2022) 025016 J J P M Schulpen et al Figure 4. Effect of atomic ordering (i.e. supercycle length) on (a) the vibrational spectrum (Raman spectroscopy) and (b) the electronic structure (optical extinction spectroscopy) of the Mo1−xWxS2 alloys. Insets in (a) show the peak positions of the Raman peaks as a function of the supercycle length. Insets in (b) show the spectral positions of the extinction peaks A and B, and an overlay of the spectrum of the longest-supercycle sample with a superposition of the pure MoS2 and WS2 spectra, indicating the heterostructure-like nature of samples grown using long supercycle lengths. Spectra are offset vertically for legibility. Figure 4. Effect of atomic ordering (i.e. supercycle length) on (a) the vibrational spectrum (Raman spectroscopy) and (b) the electronic structure (optical extinction spectroscopy) of the Mo1−xWxS2 alloys. Insets in (a) show the peak positions of the Raman peaks as a function of the supercycle length. Insets in (b) show the spectral positions of the extinction peaks A and B, and an overlay of the spectrum of the longest-supercycle sample with a superposition of the pure MoS2 and WS2 spectra, indicating the heterostructure-like nature of samples grown using long supercycle lengths. Spectra are offset vertically for legibility. indistinguishable from a superposition of the spectra of pure MoS2 and WS2 (see inset figure 4(a)). 2.2.2. Impact of atomic ordering on the vibrational structure This is expected since for very long supercycles, the depos- ited film resembles a heterostructure of MoS2 and WS2, such that the extinction spectrum contains four peaks (A and B of MoS2 and WS2). Due to their spectral broadness, not all of these four peaks can be resolved, and in the following we deconvolve the extinction spectra using a two-peak model. We will focus our attention on the interpretation of the alloy- like samples made using short supercycles (i.e. up to ten cycles) and not on the heterostructure-like samples made using longer supercycles, for which the interpretation of a two-peak deconvolution is not straightforward. since MoS2 and WS2 have identical crystal structures with in-plane lattice constants differing by only 0.2% [51]. Furthermore, opposite strain on the constitu- ents MoS2 and WS2 would lead to opposite shifts in the two E12g peaks, but parallel shifts are observed. Secondly, an increase in sulfur vacancy concentration [52] can be ruled out as the cause of the observed E12g and A1g shifts since the stoichiometry was found to remain constant at 2.0 ± 0.1 between supercycle periods of 2 and 12 cycles from RBS analysis. Addi- tionally, an increase in sulfur vacancy concentration would shift the E12g frequencies more than the A1g frequencies [52], which we do not observe. Having ruled out these alternative causes, we conclude that the E12g and A1g Raman peaks of Mo1−xWxS2 are sensitive to changes in atomic order- ing of the alloy, making Raman spectroscopy an accessible tool for probing the atomic ordering of 2D alloys. Additionally, these findings indicate that the control over atomic ordering which was demon- strated for sub-monolayer films (figure 3) extends to the few layer (6–8 L) regime. The B-peak is seen to shift to higher energy as the supercycle length is shortened (i.e. towards ran- dom atomic ordering), while the A-peak shows no significant shift. Between supercycle lengths of two and ten cycles, the B-peak has shifted by approxim- ately 20 meV. Such behavior may be related to small changes in the alloy composition (Mo/W ratio) as a function of the supercycle length [20], or instead the atomic ordering could have a direct effect on the electronic structure of the alloy. To explore the lat- ter option, DFT electronic structure calculations were carried out. For these calculations, the atomic order- ing is quantified using the order parameter J [29]: 2.2.3. 2.2.2. Impact of atomic ordering on the vibrational structure Impact of atomic ordering on electronic structure The effect of atomic ordering on the electronic structure of the Mo1−xWxS2 films was investigated by optical transmission spectroscopy. Samples were grown using supercycle lengths of 2, 6, 10, 16, 24 and 48 cycles on transparent quartz substrates. The cycle fraction n/(n + m) was again kept fixed at 0.5 (result- ing in alloy composition x = 0.6), and the total num- ber of ALD cycles was 48 for each sample. The extinc- tion spectra (figure 4(b)) show two peaks around 1.85 and 2.1 eV, labeled A and B. The spectrum of the alloy grown with the longest supercycle (48 cycles) is J = Psample Prandom = Psample x2 + (1 −x)2 , (4) (4) where Psample is the fraction of neighboring identical metal atoms (Mo–Mo or W–W) of the sample and Prandom is the fraction of neighboring identical metal atoms when the alloy is randomly mixed. The value 6 J J P M Schulpen et al 2D Mater. 9 (2022) 025016 J J P M Schulpen et al Figure 5. Calculated fundamental electronic transition energies corresponding to the A bottom and B top excitons of Mo1−xWxS2 as a function of atomic ordering. Plotted datapoints are averaged values over five unique configurations per order parameter, and the error bars denote their standard deviation. A peak than of the B peak, which was not observed experimentally. Hence, our calculations indicate that atomic ordering indeed could impact the electronic structure of Mo1−xWxS2, though further study is required to experimentally distinguish the effects of atomic ordering on the electronic structure from the effects of small changes in alloy composition. 3. Conclusions We have shown that the 2D TMD alloy Mo1−xWxS2 can be grown with excellent composition control using a supercycle-based ALD process. This well- behaved alloy growth allows for control over the ordering of the transition metal atoms within the individual molecular layers by tuning the super- cycle length of the deposition process. This control over atomic ordering was confirmed directly through HAADF-STEM imaging, and the atomic ordering was shown to tune the vibrational spectrum and elec- tronic structure of the alloys. Our results indicate that atomic ordering of 2D semiconductors alloys can be experimentally manipulated during synthesis to finely tune the opto-electronic properties of these materials for specific applications. Furthermore, our results indicate that Raman spectroscopy is sensit- ive to changes in atomic ordering of 2D TMD alloys, making it an accessible method of probing the atomic ordering of these materials. Figure 5. Calculated fundamental electronic transition energies corresponding to the A bottom and B top excitons of Mo1−xWxS2 as a function of atomic ordering. Plotted datapoints are averaged values over five unique configurations per order parameter, and the error bars denote their standard deviation. Figure 5. Calculated fundamental electronic transition energies corresponding to the A bottom and B top excitons of Mo1−xWxS2 as a function of atomic ordering. Plotted datapoints are averaged values over five unique configurations per order parameter, and the error bars denote their standard deviation. of J describes the atomic ordering of the alloy: J = 1 describes random mixing, J > 1 describes clustering of Mo and W (where larger J means larger clusters) and J < 1 describes ‘anti-clustering’, i.e. the preferential neighboring of non-identical metal atoms (Mo–W), tending towards a checkerboard pattern. 4. Methods Typical values of J for Mo1−xWxS2 alloys synthes- ized at high temperatures are around 1.0, i.e. these alloys exhibit random mixing of the transition metal atoms [29]. On the other hand, the value of J for our ALD-grown Mo1−xWxS2 using a supercycle length of ten cycles can be estimated from STEM (figure 3) to be approximately 1.8. However, since the unit cell size needed to perform DFT calculations for such large clusters would lead to prohibitively high computa- tional cost, we limit our calculations to a supercell of 5 × 5 × 1 unit cells, such that a maximum value of J of 1.41 can be simulated. The supercell configura- tions are shown in the supplementary information. The calculated electronic transition energies A and B as a function of the order parameter J are shown in figure 5. 4.1. PE-ALD process 4.1. PE-ALD process PE-ALD of Mo1−xWxS2 films was performed using an Oxford Instruments FlexAL-2 reactor equipped with a remote inductively coupled plasma source. Silicon wafers with 450 nm thermally grown oxide were used as substrates. The substrate table heater was kept at 450 ◦C during processing, resulting in a substrate temperature of 350 ◦C; a 20 min wait step was performed prior to deposition to ensure thermal equilibration. The ALD processes used for the MoS2 and WS2 cycles are described in detail in previous work [31, 33]. As precursors, Mo(NtBu)2(NMe2)2 (98%, Sigma Aldrich) and W(NtBu)2(NMe2)2 (99%, Strem Chemicals) were used for MoS2 and WS2 respectively. The precursors were kept in stainless steel canisters which were heated to 50 ◦C, and precursor deliv- ery into the reaction chamber was facilitated with a 50 sccm argon bubbling flow. Precursors were dosed for 6 s, followed by a 10 s purge of the reactor cham- ber with 100 sccm argon. A plasma of 10 sccm H2S and 40 sccm Ar was used as the coreactant in both the MoS2 and the WS2 processes. Plasma exposure was performed at a power of 100 W for 30 s at a pressure of approximately 6 mTorr. A subsequent 10 s purge of the reactor chamber with 100 sccm argon completes the ALD cycle. Since anti-clustering is not observed experiment- ally, the results for J ⩾1 are most relevant for com- parison to experiment. Between J = 1 (random mix- ing) and J = 1.41 (largest clustering), the calculated A transition energy redshifts by 11 meV, whereas the calculated B transition energy redshifts by 5 meV. Linear extrapolation to the appropriate J-value of 1.8 for Mo1−xWxS2 alloys deposited using a supercycle length of ten cycles yields redshifts of 22 and 10 meV for the A and B transitions respectively. These val- ues are of the same magnitude as the experiment- ally observed shift in the optical extinction spectrum, although the calculations predict a larger shift of the 7 2D Mater. 9 (2022) 025016 J J P M Schulpen et al 2D Mater. 9 (2022) 025016 to a residual force tolerance of 10−4 eV Å−1 using a converged plane-wave cut-off energy (400 eV) and k- space sampling (3 × 3 × 1 Monkhorst–Pack mesh). Effective electronic band structures were obtained through the unfolding procedure [59]. 4.5. Atomic ordering stochastic simulation The data that support the findings of this study are available upon reasonable request from the authors. The simulation starts with an empty hexagonal grid. In a loop, a random grid point is chosen. If this grid point is empty, there is a chance of 0.001 that a ‘MoS2 unit cell’ is placed there (simulating nucleation). If the empty grid point is next to a grid point that is already filled, this chance is 1 (simulating preferential edge growth). This loop is repeated N times (simulat- ing a full ALD cycle). By alternating such virtual ALD cycles of MoS2 and WS2, the full supercycle process of Mo1−xWxS2 alloy deposition is simulated. 4.3. Elemental composition measurement p The relative elemental composition of the Mo1−xWxS2 films was determined through XPS using a Thermo Scientific K-alpha spectrometer with an aluminum K-α (1486.6 eV) radiation source. In the experiments on atomic ordering, the obtained atomic abundances were corrected for the exponential atten- uation of the emitted photoelectrons with depth. RBS was used to determine the absolute elemental com- position of selected samples as additional validation. RBS measurements were performed with a 2 MeV 4He beam and a detector at 170◦scattering angle. 4.4. Raman and PL measurements Raman scattering spectroscopy and photolumines- cence (PL) spectroscopy measurements were per- formed with a Renishaw InVia Raman microscope equipped with a 514.5 nm laser and a CCD detector. Raman peak positions corresponding to the A1g modes were extracted by deconvolution of the spec- tra using a five-peak Voigt model, which is a strategy similar to that used in the literature [32]. Acknowledgments This work was partially funded by the Dutch Research Council (NWO) through the Gravitation program ‘Research Centre for Integrated Nanophotonics’ (No. 024.002.033). This work has also been supported by the European Research Council (Grant Agreement No. 648787-ALD of 2DTMDs). The authors acknow- ledge C O van Bommel, M G Dijstelbloem, C A A van Helvoirt, B Krishnamoorthy and J J A Zeebregts for their technical assistance, and thank S ter Huurne for performing UV-vis absorption measurements. Sol- liance and the Dutch province of Noord-Brabant are acknowledged for funding the TEM facility. 4.6. Ab-initio electronic structure calculations DFT calculations were carried out with the projector- augmented-wave [55] framework as implemented in the VASP software [56] with the exchange and correl- ation contributions to the electronic energy described semi-locally by the PBE functionals [57]. Van der Waals interactions were modeled using the DFT-D3 method of Grimme [58]. Supercells of 5 × 5 × 1 primitive cells were used with J-values between 0.64 and 1.41, which were geometrically optimized down 4.1. PE-ALD process The elec- tronic band-to-band transition energies were derived from the calculated conduction band minimum and valence band maximum at the K-point of the Bril- louin zone (where the A and B excitons are loc- ated). For each degree of atomic ordering, five unique supercells were generated and the obtained electronic transition energies were averaged over these struc- tures. The band structures were calculated at the PBE level and exciton binding was not included, such that a systematic offset is expected between the DFT-calculated band-to-band transition energies and the experimentally measured absorption peak positions. 4.2. Film thickness measurement Film growth was monitored by SE using a J.A. Wool- lam M-2000 ellipsometer in the spectral range from 1.25–4 eV. The film thickness and optical constants were determined by parametrizing the SE data using a B-spline [53]. Comparison of the optical constants obtained for pure MoS2 to literature values [54] showed good agreement, supporting the validity of the B-spline method for determining the thickness and optical constants of thin (<10 monolayers) TMD films. 4.2. Film thickness measurement Film growth was monitored by SE using a J.A. Wool- lam M-2000 ellipsometer in the spectral range from 1.25–4 eV. The film thickness and optical constants were determined by parametrizing the SE data using a B-spline [53]. Comparison of the optical constants obtained for pure MoS2 to literature values [54] showed good agreement, supporting the validity of the B-spline method for determining the thickness and optical constants of thin (<10 monolayers) TMD films. 4.7. TEM studies The sub-monolayer films have been deposited on ultra-thin (approximately 4.5 nm) Si3N4 TEM win- dows. Top view TEM studies have been performed using a probe-corrected JEOL ARM 200F, operated at 200 kV. Focusing was performed outside the area of interest. The imaged areas were only exposed to the electron beam in the single scan required of acquir- ing the image, in order to minimize beam dam- age effects. A highpass filter with a cutoff length of 100 pixels was applied to the micrographs to suppress background and allow for lossless contrast enhancement. References [23] Amani M et al 2016 High luminescence efficiency in MoS2 grown by chemical vapor deposition ACS Nano 10 6535–41 [1] Dickinson R G and Pauling L 1923 The crystal structure of molybdenite J. Am. Chem. Soc. 45 1466–71 [24] Cong C, Shang J, Wu X, Cao B, Peimyoo N, Qiu C, Sun L and Yu T 2014 Synthesis and optical properties of large-area single-crystalline 2D semiconductor WS2 monolayer from chemical vapor deposition Adv. Opt. Mater. 2 131–6 [2] Novoselov K S, Jiang D, Schedin F, Booth T J, Khotkevich V V, Morozov S V and Geim A K 2005 Two-dimensional atomic crystals Proc. Natl Acad. Sci. 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https://openalex.org/W2883413448	http://eprints.whiterose.ac.uk/134784/3/10.1007_s11606-018-4603-7.pdf	English	null	Capsule commentary on Kurtzman et al., Social incentives and gamification to promote weight loss: the LOSE IT randomized, controlled trial	Journal of general internal medicine	2,018	cc-by	685	Capsule commentary on Kurtzman et al., Social incentives and gamification to promote weight loss: the LOSE IT randomized, controlled trial Samuel G. Smith, BSc, MSc, PhD Leeds Institute of Health Sciences, University of Leeds, Leeds, UK. J Gen Intern Med DOI: 10.1007/s11606-018-4603-7 © The Author(s) 2018 outcomes. However, when such trials yield null findings we are left with unanswered questions. Did any of the intervention components have an effect on the outcome? Were there an- tagonistic interactions of the intervention components on the outcome? The multiphase optimization strategy (MOST),4 suggests such questions should be addressed earlier using an optimization trial, such as a factorial experiment or sequential multiple assignment randomized trial (SMART). When de- signing behavioral interventions, consideration should be giv- en to using MOST as a guiding framework. K urtzman et al. report a randomized controlled trial testing the effectiveness of gamification interventions on weight loss among 196 adults with obesity.1 Participants formed teams with a family member or friend prior to randomization, and were given a goal of 10,000 daily steps and a weight loss goal of 6–8% of baseline weight. Intervention participants also received a multi-component intervention involving weekly targets and entry into a team game. This involved the follow- ing: (1) a step count pledge, (2) weekly points linked with the pair’s weigh-in adherence, and (3) a scoring system that pro- moted or demoted the pair to four levels based on weight loss. One intervention group also had their weight and step data shared regularly with their primary care physician. Financial incentives were provided to participants for completing weigh-ins. K Corresponding Author: Samuel G. Smith, BSc, MSc, PhD; Leeds Institute of Health Sciences University of Leeds, Leeds, UK (e-mail: s. smith1@leeds.ac.uk). Compliance with ethical standards: Conflicts of interest: The authors declare that they do not have a conflict of interest. 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. There were within group differences in weight loss at 24 weeks among the intervention groups. However, in keeping with previous trials, 2 weight loss also occurred within the control group. There were no significant differences in weight loss between each of the intervention arms and control at 12, 24, or 36 weeks. Exploratory analysis indicated teammates living together achieved more weight loss than those not living together, particularly among the intervention conditions. This finding supports observational evidence, 3 and shows promise for the design of future behavioral interventions. Replication in a trial fully-powered for this analysis is warranted. REFERENCES 1. Kurtzman GW, Day SC, Small DS, Lynch M, Zhu J, Wang W, Rareshide CAL, Patel MS. Social incentives and gamification to promote weight loss: the LOSE IT randomized, controlled trial. J Gen Intern Med. SPI 4552 1. Kurtzman GW, Day SC, Small DS, Lynch M, Zhu J, Wang W, Rareshide CAL, Patel MS. Social incentives and gamification to promote weight loss: the LOSE IT randomized, controlled trial. J Gen Intern Med. SPI 4552 2. Johns DJ, Hartmann-Boyce J, Jebb SA, Aveyard P, and on behalf of the Behavioural Weight Management Review Group. Weight change among people randomized to minimal intervention control groups in weight loss trials. Obesity. 2016, 24(4): 772–780. 3. Jackson SE, Steptoe A, Wardle J. The influence of partner’s behavior on health behavior change: the English longitudinal study of ageing. JAMA Intern Med 2015: 175(3): 385–392. 3. Jackson SE, Steptoe A, Wardle J. The influence of partner’s behavior on health behavior change: the English longitudinal study of ageing. JAMA Intern Med 2015: 175(3): 385–392. The intervention was designed by incorporating multiple evidence-based components (e.g., gamification, incentives, targets, etc.), within a single package in the hope of maximiz- ing behavior change. This classical approach is laudable for having the ultimate end-goal in sight: improved patient 4. Collins LM. Optimization of behavioral, biobehavioral and biomedical interventions: the multiphase optimization strategy (MOST). 2018. New York, NY, Springer.
https://openalex.org/W2904050669	https://europepmc.org/articles/pmc6321037?pdf=render	English	null	S100A10 and Cancer Hallmarks: Structure, Functions, and its Emerging Role in Ovarian Cancer	International journal of molecular sciences	2,018	cc-by	11,602	Perspective S100A10 and Cancer Hallmarks: Structure, Functions, and its Emerging Role in Ovarian Cancer Tannith M. Noye 1, Noor A. Lokman 1, Martin K. Oehler 1,2 and Carmela Ricciardelli 1,* 1 Discipline of Obstetrics and Gynaecology, Adelaide Medical School, Robinson Research Institute, The University of Adelaide, Adelaide 5005, Australia; tannith.noye@adelaide.edu.au (T.M.N.); noor.lokman@adelaide.edu.au (N.A.L.); martin.oehler@adelaide.edu.au (M.K.O.) 2 Department of Gynaecological Oncology, Royal Adelaide Hospital, Adelaide 5000, Australia * Correspondence: carmela.ricciardelli@adelaide.edu.au; Tel.: +61-0883138255 Received: 5 November 2018; Accepted: 17 December 2018; Published: 19 December 2018 Received: 5 November 2018; Accepted: 17 December 2018; Published: 19 December 2018 Abstract: S100A10, which is also known as p11, is located in the plasma membrane and forms a heterotetramer with annexin A2. The heterotetramer, comprising of two subunits of annexin A2 and S100A10, activates the plasminogen activation pathway, which is involved in cellular repair of normal tissues. Increased expression of annexin A2 and S100A10 in cancer cells leads to increased levels of plasmin—which promotes the degradation of the extracellular matrix—increased angiogenesis, and the invasion of the surrounding organs. Although many studies have investigated the functional role of annexin A2 in cancer cells, including ovarian cancer, S100A10 has been less studied. We recently demonstrated that high stromal annexin A2 and high cytoplasmic S100A10 expression is associated with a 3.4-fold increased risk of progression and 7.9-fold risk of death in ovarian cancer patients. Other studies have linked S100A10 with multidrug resistance in ovarian cancer; however, no functional studies to date have been performed in ovarian cancer cells. This article reviews the current understanding of S100A10 function in cancer with a particular focus on ovarian cancer. Keywords: S100A10; annexin A2; plasmin; ovarian cancer; chemotherapy resistance International Journal of Molecular Sciences International Journal of Molecular Sciences Perspective S100A10 and Cancer Hallmarks: Structure, Functions, and its Emerging Role in Ovarian Cancer Tannith M. Noye 1, Noor A. Lokman 1, Martin K. Oehler 1,2 and Carmela Ricciardelli 1,* 1 Discipline of Obstetrics and Gynaecology, Adelaide Medical School, Robinson Research Institute, The University of Adelaide, Adelaide 5005, Australia; tannith.noye@adelaide.edu.au (T.M.N.); noor.lokman@adelaide.edu.au (N.A.L.); martin.oehler@adelaide.edu.au (M.K.O.) 2 Department of Gynaecological Oncology, Royal Adelaide Hospital, Adelaide 5000, Australia * Correspondence: carmela.ricciardelli@adelaide.edu.au; Tel.: +61-0883138255 Received: 5 November 2018; Accepted: 17 December 2018; Published: 19 December 2018 Int. J. Mol. Sci. 2018, 19, 4122; doi:10.3390/ijms19124122 2. S100A10 Structure and Function 2. S100A10 Structure and Function S100A10, which is also known as p11 or annexin A2 light chain, belongs to the calcium-binding S100 family, which is characterized by EF-hand calcium-binding motifs [17–19]. To date, at least 25 S100 proteins have been identiﬁed; the majority are clustered at the chromosome locus 1q21, which is prone to genomic alterations [18]. S100 protein interacts with multiple other proteins and exerts a broad range of cellular functions including (i) phosphorylation; (ii) maintaining cell shape and motility; (iii) calcium homeostasis; (iv) enzyme activity modulation; and (v) transduction pathway signaling [20]. S100 proteins can form both homodimeric and heterodimeric complexes with each other and undergo a conformation change following calcium binding [17]. S100A10 monomers contain four α helical domains (H-I—H-IV) (Figure 1) and are unique from other S100 protein family members as its EF-hands cannot bind to calcium [21]. S100A10 adopts a permanently open conformation comparable to the calcium-bound conformation observed with the other S100 proteins [21]. S100A10 is expressed ubiquitously in the majority of cells and plays a major role in ﬁbrinolysis, wound healing, and angiogenesis [22,23]. Recent studies have shown that S100A10 is important in regulating other physiological processes, including immune cell function [24,25], reproduction [26–28], neural cell function [29,30], and heart function [31]. S100A10, which is also known as p11 or annexin A2 light chain, belongs to the calcium-binding S100 family, which is characterized by EF-hand calcium-binding motifs [17–19]. To date, at least 25 S100 proteins have been identified; the majority are clustered at the chromosome locus 1q21, which is prone to genomic alterations [18]. S100 protein interacts with multiple other proteins and exerts a broad range of cellular functions including (i) phosphorylation; (ii) maintaining cell shape and motility; (iii) calcium homeostasis; (iv) enzyme activity modulation; and (v) transduction pathway signaling [20]. S100 proteins can form both homodimeric and heterodimeric complexes with each other and undergo a conformation change following calcium binding [17]. S100A10 monomers contain four α helical domains (H-I—H-IV) (Figure 1) and are unique from other S100 protein family members as its EF-hands cannot bind to calcium [21]. S100A10 adopts a permanently open conformation comparable to the calcium-bound conformation observed with the other S100 proteins [21]. S100A10 is expressed ubiquitously in the majority of cells and plays a major role in fibrinolysis, wound healing, and angiogenesis [22,23]. 1. Introduction Ovarian cancer is the most lethal gynecological malignancy with a 5-year survival rate of only about 46% [1]. It is estimated for 2018 that worldwide there will be about 295,414 new ovarian cancer cases and 184,799 women will die from this disease [2]. The poor survival rate can be attributed to the fact that ovarian cancer has non-speciﬁc symptoms and as a result is often diagnosed at stage 3 or 4. High recurrence rates following treatment and subsequent chemotherapy resistance is another reason [3]. Epithelial ovarian cancers are the most common ovarian malignancies and of that 70% of the subtype are high-grade serous carcinomas. High-grade serous carcinomas have high chemosensitivity following initial treatment with platinum-based therapies, but 75% of patients will relapse and ultimately die from developing chemoresistant disease [4]. Chemotherapy resistance is one of the main reasons for the fatal outcome of ovarian cancer. Thus, discovering and understanding the underlying molecular mechanisms involved in drug resistance is crucial for identifying novel and effective therapeutic targets to be able to improve survival. Resistance mechanisms identiﬁed in ovarian cancer include genetic mutations, epigenetic changes, and dysfunctional DNA repair (reviewed in [5,6]). Other identiﬁed causes for chemotherapy resistance include upregulation of ATP-binding cassette (ABC) transporters responsible for efﬂux of cancer therapies [7], activation of cancer stem cells, and epithelial to mesenchymal transition (EMT), as well as alterations to the tumor microenvironment [8,9]. www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 4122; doi:10.3390/ijms19124122 2 of 16 EMT), Int. J. Mol. Sci. 2018, 19, 4122 cancer therapies [7], acti as well as alterations to Predicting the response to drug therapies remains a major challenge in ovarian cancer. Currently, there are no predictors of response to ﬁrst-line chemotherapy in ovarian cancer, and after recurrence, the prediction of the response to second-line chemotherapy is determined empirically from the platinum-free interval (PFI) after the ﬁrst treatment [4]. Recent studies have used a chemoresponse assay (CRA) to improve patient selection for different chemotherapy treatments [10–13]. Overall, these studies found that patients with assay-sensitive tissue had improved progression-free survival (PFS) compared to patients with non-sensitive tissue. The inclusion of additional biomarkers that can predict chemotherapy response together with CRAs would enable more effective, individualized patient management and importantly would spare patients from the side effects of ineffective drugs. 1. Introduction g p g p j g Currently, there are no predictors of response to first-line chemotherapy in ovarian cancer, and after recurrence, the prediction of the response to second-line chemotherapy is determined empirically from the platinum-free interval (PFI) after the first treatment [4]. Recent studies have used a chemoresponse assay (CRA) to improve patient selection for different chemotherapy treatments [10– 13]. Overall, these studies found that patients with assay-sensitive tissue had improved progression- free survival (PFS) compared to patients with non-sensitive tissue. The inclusion of additional biomarkers that can predict chemotherapy response together with CRAs would enable more effective, individualized patient management and importantly would spare patients from the side effects of ineffective drugs. Recent studies have linked S100A10 with chemotherapy resistance and poor prognosis in serous ovarian cancer [14–16]; however, no functional studies have been performed to date. The aims of this review are to highlight the current understanding of S100A10 function in cancer cells—with a particular focus on ovarian cancer—and to discuss the potential for using S100A10 as a predictive marker and targeting S100A10 to inhibit cancer progression and treatment resistance. Recent studies have linked S100A10 with chemotherapy resistance and poor prognosis in serous ovarian cancer [14–16]; however, no functional studies have been performed to date. The aims of this review are to highlight the current understanding of S100A10 function in cancer cells—with a particular focus on ovarian cancer—and to discuss the potential for using S100A10 as a predictive marker and targeting S100A10 to inhibit cancer progression and treatment resistance. 3. S100A10 Interaction with Annexin A2 S100A10 is a plasminogen receptor and binds to the cell membrane via its cell surface receptor, annexin A2 [32,33]. Annexin A2 is a soluble monomer in the cytoplasm, and when annexin A2 is sufﬁciently expressed, it binds to S100A10, forming a stable heterotetramer. Annexin A2 is phosphorylated for conformation changes to occur and translocates the heterotetramer to the cell surface [34]. S100A10 binding to plasminogen results in the activation of plasminogen activators—tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (uPA)—and the conversion of plasminogen to plasmin (reviewed in [32]). S100A10 interacts with annexin A2 to form a heterotetramer, also known as AIIt, which consists of two subunits of annexin A2 and two subunits of S100A10 [35,36]. AIIt activates the plasminogen activation pathway to increase plasmin production in various types of cells [32,37]. Normal endothelial cells utilize S100A10 in the plasminogen activation pathway, converting plasminogen to plasmin, which is vital for ﬁbrinolysis and angiogenesis [22,32]. S100A10 has been shown to regulate up to 90% of plasmin produced in endothelial cells [32]. Plasmin can then degrade ﬁbrin and activate matrix metalloproteinases (MMPs), which in turn promotes degradation of the extracellular matrix (ECM) [38]. S100A10-null mice have been shown to exhibit increased ﬁbrin accumulation [22]. Endothelial cells from S100A10-null mice show a 40% reduction in plasminogen binding and plasmin generation in vitro compared with wild-type mice and exhibit defective angiogenesis [22]. Binding of S100A10 occurs at the annexin A2 N-terminus [39]. This interaction with the annexin A2 N-terminus is required for binding since the removal of this binding leads to the loss of the interaction with S100A10 and results in reduced plasmin production [40]. Several groups have reported that annexin A2 is required to transport S100A10 to the cell surface, which is dependent on the phosphorylation of annexin A2 [34,41–43]. S100A10 levels on the cell surface can also be regulated by IFN-γ involving annexin A2 via an exosomal secretion pathway [44]. Although earlier studies reported that annexin A2 is a plasminogen receptor [45], more recent studies support that S100A10 rather than annexin A2 is the major plasminogen receptor [46]. It has been proposed that annexin A2 in the heterotetramer plays a role in stabilizing the S100A10 protein and localizing S100A10 to the cell surface. The interaction between S100A10 and annexin A2 is, therefore, thought to protect S100A10 from degradation by the proteasome [21,46,47]. 2. S100A10 Structure and Function 2. S100A10 Structure and Function Recent studies have shown that S100A10 is important in regulating other physiological processes, including immune cell function [24,25], reproduction [26– 28], neural cell function [29,30], and heart function [31]. Figure 1 Structure of S100A10 monomer. Each monomer contains four α helical domains H-I, H-II, H-III, and H-IV. Two helical loops L1 and L2 separate H-I and H-II, and H-III and H-1V, respectively. A flexible linker or hinge region (HR1) is also located between H-II and H-III. Binding sites to annexin A2 are located in H-I, HR1, and H-IV, as indicated by the yellow boxes. S100A10 binds both tissue- type plasminogen activator (t-PA) and plasminogen (Pg) via the carboxyl-terminal lysine. Adapted from [32]. Figure 1. Structure of S100A10 monomer. Each monomer contains four α helical domains H-I, H-II, H-III, and H-IV. Two helical loops L1 and L2 separate H-I and H-II, and H-III and H-1V, respectively. A ﬂexible linker or hinge region (HR1) is also located between H-II and H-III. Binding sites to annexin A2 are located in H-I, HR1, and H-IV, as indicated by the yellow boxes. S100A10 binds both tissue-type plasminogen activator (t-PA) and plasminogen (Pg) via the carboxyl-terminal lysine. Adapted from [32]. Figure 1 Structure of S100A10 monomer. Each monomer contains four α helical domains H-I, H-II, H-III, and H-IV. Two helical loops L1 and L2 separate H-I and H-II, and H-III and H-1V, respectively. A flexible linker or hinge region (HR1) is also located between H-II and H-III. Binding sites to annexin A2 are located in H-I, HR1, and H-IV, as indicated by the yellow boxes. S100A10 binds both tissue- type plasminogen activator (t-PA) and plasminogen (Pg) via the carboxyl-terminal lysine. Adapted from [32] Figure 1. Structure of S100A10 monomer. Each monomer contains four α helical domains H-I, H-II, H-III, and H-IV. Two helical loops L1 and L2 separate H-I and H-II, and H-III and H-1V, respectively. A ﬂexible linker or hinge region (HR1) is also located between H-II and H-III. Binding sites to annexin A2 are located in H-I, HR1, and H-IV, as indicated by the yellow boxes. S100A10 binds both tissue-type plasminogen activator (t-PA) and plasminogen (Pg) via the carboxyl-terminal lysine. Adapted from [32]. Int. J. Mol. Sci. 2018, 19, 4122 3 of 16 3. S100A10 Interaction with Annexin A2 In cancer cells, increased annexin A2 and S100A10 expression result in increased plasmin production, which leads to the degradation of the ECM and activation of MMPs, thereby enabling the invasion of surrounding organs or local vasculature (Figure 2) [35–37]. Several studies have suggested that the knockdown of annexin A2 concurrently results in the loss of S100A10 [32,48–51]. Similarly, the loss of S100A10 has been shown to affect both the mRNA and protein levels of annexin A2 [23]. Therefore, it is not known whether the effects observed in many of these studies were mediated by annexin A2 or S100A10. It is likely that both proteins play reciprocal roles in mediating their function in cancer cells. 4 of 16 Int. J. Mol. Sci. 2018, 19, 4122 Figure 2. S100A10 plays a key role in regulating pro-tumorigenic processes including proliferation, adhesion, motility, invasion, metastasis, and therapy resistance. The S100A10–annexin A2 heterotetramer (AIIt) acts through the plasminogen activation pathway. AIIt on the cell surface of the plasma membrane activates plasminogen via tissue-type plasminogen activator (t-PA) and urokinase- type plasminogen activator (uPA) and increases the production of plasmin, leading subsequently to the activation of metalloproteinases (MMPs) and the degradation of the extracellular matrix (ECM) proteins, which promote tumor progression and treatment resistance. Figure 2. S100A10 plays a key role in regulating pro-tumorigenic processes including proliferation, adhesion, motility, invasion, metastasis, and therapy resistance. The S100A10–annexin A2 heterotetramer (AIIt) acts through the plasminogen activation pathway. AIIt on the cell surface of the plasma membrane activates plasminogen via tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (uPA) and increases the production of plasmin, leading subsequently to the activation of metalloproteinases (MMPs) and the degradation of the extracellular matrix (ECM) proteins, which promote tumor progression and treatment resistance. Figure 2. S100A10 plays a key role in regulating pro-tumorigenic processes including proliferation, adhesion, motility, invasion, metastasis, and therapy resistance. The S100A10–annexin A2 heterotetramer (AIIt) acts through the plasminogen activation pathway. AIIt on the cell surface of the plasma membrane activates plasminogen via tissue-type plasminogen activator (t-PA) and urokinase- type plasminogen activator (uPA) and increases the production of plasmin, leading subsequently to the activation of metalloproteinases (MMPs) and the degradation of the extracellular matrix (ECM) proteins, which promote tumor progression and treatment resistance. Figure 2. S100A10 plays a key role in regulating pro-tumorigenic processes including proliferation, adhesion, motility, invasion, metastasis, and therapy resistance. 4. Interaction of S100A10 with Other Proteins 4. Interaction of S100A10 with Other Proteins In addition to binding annexin A2, S100A10 plays a role in the trafficking of other membrane proteins including sodium channel protein Nav 1.8 [52], potassium channel protein TASK-1 [53], channel proteins TRPV5 and TRPV6 [54], the acid-sensing ion channel ASIC1a [55], and serotonin 5- HT1B receptor [56]. S100A10 is, therefore, thought to play an important role in cell surface presentation of other plasma membrane proteins in addition to annexin A2 [17]. S100A10 functionally acts as a linking protein with the ability to bind transmembrane proteins, thereby aiding the transportation of proteins to the plasma membrane [17]. Protein crystallography has also shown that AIIt interacts with AHNAK, a protein involved in wound repair [57,58], and ARCA3, which is involved in chromatin remodeling [59]. More recent studies have also shown that S100A10 is required for the organization of actin stress fibers and the formation of focal adhesions by HeLa cells via Rac1 signaling [60]. The depletion of S100A10 led to the disruption of stress fiber formation and suppression of Rac1 activation [60]. In addition to binding annexin A2, S100A10 plays a role in the trafﬁcking of other membrane proteins including sodium channel protein Nav 1.8 [52], potassium channel protein TASK-1 [53], channel proteins TRPV5 and TRPV6 [54], the acid-sensing ion channel ASIC1a [55], and serotonin 5-HT1B receptor [56]. S100A10 is, therefore, thought to play an important role in cell surface presentation of other plasma membrane proteins in addition to annexin A2 [17]. S100A10 functionally acts as a linking protein with the ability to bind transmembrane proteins, thereby aiding the transportation of proteins to the plasma membrane [17]. Protein crystallography has also shown that AIIt interacts with AHNAK, a protein involved in wound repair [57,58], and ARCA3, which is involved in chromatin remodeling [59]. More recent studies have also shown that S100A10 is required for the organization of actin stress ﬁbers and the formation of focal adhesions by HeLa cells via Rac1 signaling [60]. The depletion of S100A10 led to the disruption of stress ﬁber formation and suppression of Rac1 activation [60]. 3. S100A10 Interaction with Annexin A2 The S100A10–annexin A2 heterotetramer (AIIt) acts through the plasminogen activation pathway. AIIt on the cell surface of the plasma membrane activates plasminogen via tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (uPA) and increases the production of plasmin, leading subsequently to the activation of metalloproteinases (MMPs) and the degradation of the extracellular matrix (ECM) proteins, which promote tumor progression and treatment resistance. 5. S100A10 Expression in Cancers 5. S100A10 Expression in Cancers The expression of S100A10 in cancer has been widely studied and is summarized in Table 1. The role of S100A10 in cancer was first identified in acute promyelocytic leukemia (APL), a subtype characterized by the expression of a fusion protein formed by the fusion of retinoic acid receptor alpha with promyelocytic leukemia (PML-RARα) genes [61]. Patients with APL experience severe bleeding that results from increased plasmin production [61,62]. Increased plasmin levels are due to the upregulation of both annexin A2 and S100A10 in APL cells due to the PML-RARα oncoprotein [46,63]. S100A10 mRNA and protein expression are also upregulated in patients with acute lymphoblastic leukemia (ALL) [64]. S100A10 expression is increased in many other cancers [65–70] The expression of S100A10 in cancer has been widely studied and is summarized in Table 1. The role of S100A10 in cancer was ﬁrst identiﬁed in acute promyelocytic leukemia (APL), a subtype characterized by the expression of a fusion protein formed by the fusion of retinoic acid receptor alpha with promyelocytic leukemia (PML-RARα) genes [61]. Patients with APL experience severe bleeding that results from increased plasmin production [61,62]. Increased plasmin levels are due to the upregulation of both annexin A2 and S100A10 in APL cells due to the PML-RARα oncoprotein [46,63]. S100A10 mRNA and protein expression are also upregulated in patients with acute lymphoblastic leukemia (ALL) [64]. S100A10 expression is increased in many other cancers [65–70] and is generally 5 of 16 Int. J. Mol. Sci. 2018, 19, 4122 associated with a poor prognosis (Table 1). In kidney cancers, S100A10 expression is 2.5-fold higher than in normal kidney tissue [71]. Non-cancerous resections adjacent to kidney tumors show no expression of S100A10; however, S100A10 is expressed in renal cell carcinoma lesions [72]. S100A10 expression is increased in melanoma in comparison to levels in normal skin [69]. The expression of S100A10 is also upregulated in basal-type breast cancers [70]. S100A10 is overexpressed in gastric cancers [65,73], precancerous lesions in the stomach [67], and high S100A10 expression is associated with gastric cancer metastasis to the lymph nodes [67]. Overexpression of S100A10 has also been observed in anaplastic large cell lymphoma [68]. However, reduced S100A10 expression has also been reported in prostate cancer [74] and thyroid carcinoma [75]. Chetcuti et al. reported S100A10 mRNA expression in prostate cancer tissues but surprisingly found that S100A10 protein is not expressed [74], signifying potential post-translational modiﬁcations. 5. S100A10 Expression in Cancers 5. S100A10 Expression in Cancers S100A10 protein is present in the normal follicular thyroid tissues, but S100A10 expression is reduced in follicular adenoma and follicular thyroid carcinomas [75]. However, increased levels of S100A10 are present in all anaplastic thyroid carcinomas, which is the most aggressive form of thyroid malignancy, suggesting that S100A10 plays a role in the progression of thyroid carcinomas [76]. Tan et al. reported that high cytoplasmic S100A10 expression in advanced stage gallbladder carcinoma is associated with poor prognosis [77]. Overexpression of S100A10 is also associated with poor prognosis in lung carcinoma [78,79] and pancreatic cancer [80]. A recent study identiﬁed S100A10 as one of a three-gene expression signature to independently predict survival of lung adenocarcinoma patients [81]. Similarly, in colorectal carcinomas, increased S100A10 protein is associated with a poor prognosis and reduced overall survival (OS) [82]. Increased S100A10 expression is independently associated with recurrence in colorectal cancer patients [83]. Table 1. S100A10 gene and protein expression in cancers. Cancer S100A10 Expression Ref. Blood S100A10 expression is increased on the surface of leukemia cells [46,63] S100A10 mRNA and protein is upregulated in B-cell acute lymphoblastic leukemia [64] Breast S100A10 expression is upregulated in basal-type breast cancer [70] Colorectal S100A10 is increased in hereditary polyposis colorectal cancer [66] Increased S100A10 expression is associated with poor prognosis and reduced overall survival in colorectal cancer [82] S100A10 gene expression is associated with tumor recurrence in colon cancer [83] Gallbladder High cytoplasmic S100A10 expression is associated with poor prognosis [77] Kidney S100A10 expression is 2.5-fold higher in renal cell carcinoma compared with normal kidney tissue [71] S100A10 is expressed in renal cell carcinoma and absent in non-cancerous renal tumors [72] Lung Overexpression of S100A10 is associated with poor prognosis [78,79,84] Lymphatic S100A10 is overexpressed in anaplastic large cell lymphoma [68] Pancreas S100A10 mRNA and protein is overexpressed in pancreatic cancer and predicts patient outcome [80] Prostate S100A10 expression is lost in prostate cancer tissues [74] Skin S100A10 expression is increased in melanoma compared with normal skin [69] Stomach S100A10 is overexpressed in gastric cancer [65,73] S100A10 expression is upregulated in pre-cancerous lesions and associated with gastric cancer metastasis to the lymph node [67] Thyroid S100A10 is overexpressed in anaplastic thyroid carcinomas compared with normal tissues [76] S100A10 expression is decreased in follicular adenomas and thyroid carcinomas [75] Table 1. S100A10 gene and protein expression in cancers. 6. Functional Role of S100A10 in Cancer S100A10 plays a pro-tumorigenic role by regulating proliferation, cell adhesion, migration, invasion, metastasis, and therapy resistance in various malignancies (summarized in Table 2 and Figure 2). Collectively, these studies establish a pro-tumorigenic role for S100A10 as a key contributor in plasmin regulation, tumor progression, and metastasis. 6 of 16 Int. J. Mol. Sci. 2018, 19, 4122 Table 2. The functional roles of S100A10 in cancer cells. Table 2. The functional roles of S100A10 in cancer cells. Table 2. The functional roles of S100A10 in cancer cells. Function Observation Ref. 6. Functional Role of S100A10 in Cancer Proliferation S100A10 is upregulated by p53 activation in breast cancer cells [85] Lewis Lung carcinoma and T241 ﬁbrosarcoma proliferation is inhibited in S100A10 knockout mice [86] S100A10 is downregulated by a knockdown of annexin A2, which decreases the proliferation of breast cancer cell lines [50] The displacement of S100A10 from annexin A2 attenuates plasminogen activation, impairing colony formation and growth of lung cancer cells [87] Cell growth inhibition by the microRNA miR-590-5P in hepatic carcinoma cells is mediated via S100A10 [88] Annexin A2/S100A10 activates the ERK1/2 and AKT pathways in multiple myeloma cells to enhance cell growth [89] S100A10 knockdown reduces proliferation of colon cancer cells [82] S100A10 knockdown inhibits growth of pancreatic cancer cells PANC-1 in immunocompromised NOD/SCID mice [80] Adhesion Annexin A2/S100A10 regulates adhesion of leukemia cells and prostate cancer cells to osteoblasts [64,90] Cell surface S100A10 expression promotes adhesion of breast and prostate cancer cells to endothelial cells in vitro [90,91] Migration S100A10 expression is associated with the recruitment and migration of macrophages [24,70,86] The displacement of S100A10 from annexin A2 attenuates plasminogen activation and impairs the migration of A549 lung cancer cells [87] S100A10 knockdown reduces the migration of colon cancer cells [82] Invasion S100A10 in colon cancer cells is crucial for promoting plasmin production and cell invasiveness [92] S100A10 antibodies inhibit the invasion of acute promyelocytic leukemia cells [63] S100A10 expression in ﬁbrosarcoma cells increases plasmin production and cell invasiveness [93] The displacement of S100A10 from annexin A2 attenuates plasminogen activation and impairs invasion of lung cancer cells [87] Macrophages from S100A10 knockout mice have reduced plasmin-dependent invasion [24] S100A10 depletion in RAS-transformed cell lines (HEK293, NIH-3T3) results in a loss of plasmin production and reduced cell invasiveness [94] siRNA S100A10 reduces invasion of HCT-116 and DLD-1 colon cancer cell lines [82] Angiogenesis S100A10-null mice have reduced blood vessel density compared to wild-type mice [86] Metastasis Loss of S100A10 reduces metastatic burden in the HT1080 ﬁbrosarcoma mouse model [93] Overexpression of S100A10 increases the metastatic burden in the HT1080 ﬁbrosarcoma mouse model [93] S100A10 is one of 170 genes activated during the process of intravasation in breast cancer cells [95] Treatment resistance Disruption of both annexin A2 and S100A10 interactions sensitize leukemia cells to chemotherapy [64] Overexpression of S100A10 reduces the sensitivity of colorectal cancer cells to oxaliplatin [51] Knockdown of S100A10 inhibits annexin A2 phosphorylation and increases sensitivity of acute lymphoblastic leukemia cells to prednisolone [96] S100A10 protein expression is increased in tamoxifen-resistant MCF-7 breast cancer cells and breast cancer tissues [97,98] 6.4. Invasion Several studies have shown that S100A10 plays a role in promoting the invasion of cancer cells. Transfection of human HT1080 ﬁbrosarcoma cells with S100A10 antisense oligonucleotides result in a loss of S100A10 protein from the cell surface, decreased plasmin production, and reduced cell invasion [93]. Knockdown of S100A10 by siRNA signiﬁcantly reduces the invasion capacity of HCT-116 and DLD-1 colorectal cancer cell lines [82]. S100A10 alone in the absence of annexin A2 is crucial for promoting plasmin production and the invasiveness of CCL-22 colorectal cancer cells [92]. A study by Phipps et al. (2011) has also demonstrated that LLC and T241 cells are unable to grow and invade in S100A10-null mice due to the inability to recruit macrophages to the tumor site [86]. The macrophages from S100A10 knockout mice exhibit reduced plasmin-dependent invasion [24]. The displacement of S100A10 from annexin A2 by DLC1, a Rho GTPase-activating protein (RhoGAP) that functions as a tumor suppressor, results in the attenuation of plasminogen activation and impaired invasion of A549 lung cancer cells [87]. The depletion of S100A10 in the kidney (HEK293) and ﬁbroblast (NIH-3T3) cell lines also result in the loss of plasmin production and reduced cell invasiveness [94]. Moreover, a recent study has shown that U937/PR9 and NB4 leukemic cell invasion can be blocked by either annexin A2 or S100A10 antibodies in vitro [63]. 6.2. Adhesion Adhesion of cancer cells is an important phase in the progression of disease. Myrvang and coworkers (2013) were able to show that cell surface S100A10 promotes the adhesion of breast cancer cells to endothelial cells in vitro [91]. S100A10 together with annexin A2 has been shown to regulate the adhesion of leukemia cells [64] and prostate cancer cells to osteoblasts [90]. These ﬁndings suggest that S100A10 and annexin A2 may aid the metastatic process by allowing cancer cells to reach the bone marrow. 6.1. Proliferation S100A10 has been linked to play a key role in proliferation in many different types of cancers. Increased cell proliferation of basal-type breast cancer cells is associated with upregulation of S100A10 expression [70]. The knockdown of annexin A2 and concurrent loss of S100A10 expression decreases the cell proliferation of invasive MDA-MB-435S breast cancer cells [50]. The growth of Lewis Lung carcinoma (LLC) and T241 ﬁbrosarcomas is greatly reduced in S100A10 knockout mice compared with wild-type mice [86]. The displacement of S100A10 from annexin A2 attenuates plasminogen activation, impairing colony formation and growth of A549 lung cancer cells [87]. S100A10 has also been shown to bind to Bcl-2-associated death promoter (BAD) protein and adversely affects BAD-induced apoptosis in Chinese hamster ovary (CHO) cells [99]. S100A10 is upregulated by p53 activation, which regulates pro-survival functions in MCF-7 breast cancer cells [85]. Inhibition of hepatic carcinoma, HepG2 cell growth by the microRNA, miR-590-5P is mediated via S100A10 expression [88]. Together annexin A2/S100A10 has also been shown to activate the ERK1/2 and AKT pathways in MM.1S multiple myeloma cells to enhance cell growth [89]. Knockdown of S100A10 by siRNA signiﬁcantly reduces the proliferation of both HCT-116 and DLD-1 colon cancer cells [82]. A more recent study found that knockdown of S100A10 inhibits the growth of pancreatic cancer cells PANC-1 in immunocompromised NOD/SCID mice [80]. Int. J. Mol. Sci. 2018, 19, 4122 7 of 16 7 of 16 6.3. Migration S100A10 plays a role in promoting the migration of cancer cells. S100A10 expression promotes the migration of non-small cell lung cancer (NSCLC) A549 cancer cells in vitro [87]. Knockdown of S100A10 by siRNA signiﬁcantly reduces the migration capacity of two colorectal cancer cell lines, HCT-116 and DLD-1 [82]. Several studies also suggest a direct link between S100A10 expression with the recruitment and migration of macrophages [24,70,86]. S100A10 also plays a critical role in the migration of macrophages to tumor sites and is reported to be a rate-limiting step that controls tumor progression [86]. 6.5. Angiogenesis The process of forming new blood vessels also depends on the presence of S100A10 [86]. Phipps et al. (2011) showed using the S100A10-null mouse model that the density of blood vessels is decreased by over 50% compared with wild-type [86]. S100A10-null macrophages are not able to stimulate angiogenesis and LLC tumor growth in the S100A10-null mice. 7. Role of S100A10 in Ovarian Cancer and Chemotherapy Resistance To date, there have been only three studies that have investigated the expression of S100A10 in ovarian cancer. The study by Gillet et al. (2012), which included 80 serous ovarian cancer patients treated with carboplatin and paclitaxel, found S100A10 to be one of the 11 signature genes whose expression is involved in multidrug resistance [100]. Another study by Nymoen et al. (2015) found that S100A10 protein expression in ovarian cancer tissues is related to poor chemotherapy response and associated with shorter overall and progression-free survival [15]. In the third study, Lokman et al. (2016) used 13 publicly available ovarian cancer microarray datasets including 722 serous ovarian cancer patients who had received single platinum treatment and 468 patients with combined platinum–taxane treatment [16]. They showed that high mRNA levels of S100A10 predict reduced OS and that high cytoplasmic S100A10 expression is signiﬁcantly associated with reduced OS in serous ovarian cancer patients [16]. Moreover, high stromal annexin A2 and high cytoplasmic S100A10 expression in serous ovarian cancer tissues are associated with a 3.4-fold increased risk of progression and a 7.9-fold risk of ovarian cancer death [16]. Our preliminary studies investigating the mechanisms of chemotherapy resistance in ovarian cancer have shown increased S100A10 expression in chemotherapy-resistant disease compared to expression observed in the same patient tissue at diagnosis (Figure 3). Together these ﬁndings suggest that S100A10 plays an important role in the progression of serous ovarian cancer and chemotherapy resistance. Future studies are required to investigate further the functional role of S100A10 in ovarian cancer, its usefulness in predicting chemotherapy response, and as a therapeutic target to overcome chemoresistance. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 16 cancer patients who had received single platinum treatment and 468 patients with combined platinum–taxane treatment [16]. They showed that high mRNA levels of S100A10 predict reduced OS and that high cytoplasmic S100A10 expression is significantly associated with reduced OS in serous ovarian cancer patients [16]. Moreover, high stromal annexin A2 and high cytoplasmic S100A10 expression in serous ovarian cancer tissues are associated with a 3.4-fold increased risk of progression and a 7.9-fold risk of ovarian cancer death [16]. Our preliminary studies investigating the mechanisms of chemotherapy resistance in ovarian cancer have shown increased S100A10 expression in chemotherapy-resistant disease compared to expression observed in the same patient tissue at diagnosis (Figure 3). 6.6. Metastasis As the hallmark of disease progression, metastasis has been shown to be promoted by the presence of S100A10. S100A10 plays an important role in this process, as overexpression of S100A10 in HT1080 ﬁbrosarcoma was shown to increase the lung metastatic burden in mice by 16-fold while the loss of S100A10 reduced the metastatic burden by 3-fold [93]. A more recent study analyzing circulating tumor cells from breast cancer patients reported that S100A10 is one of the 170 genes activated during intravasation, an important process in the initial stages of metastasis [95]. Int. J. Mol. Sci. 2018, 19, 4122 8 of 16 6.7. Therapy Resistance Increased S100A10 has also been linked to therapy resistance. Treatment with small molecules that inhibit the S100A10–annexin A2 interaction, antibodies against annexin A2 and S100A10, or the knockdown of S100A10 could all increase the sensitivity of NTPL-20 leukemia cells to the chemotherapy drug vincristine [64]. Spijkers-Hagelstein et al. (2013) were able to show that to improve the treatment success of glucocorticoid therapy in ALL, phosphorylation of annexin A2 is required; additionally, this phosphorylation requires S100A10 expression and the absence of both annexin A2 and S100A10 reduces the resistance to treatment in the ALL SEM cell line [96]. COLO-320 colorectal cancer cells that overexpress S100A10 also show reduced sensitivity to oxaplatin [51]. Increased S100A10 has been associated with tamoxifen resistance in MCF-7 breast cancer cells [97] and breast cancer tissues [98]. The mechanisms whereby S100A10 regulates therapy resistance is poorly understood and requires further investigation. 8. Strategies to Target S100A10 in Cancer Cells Different therapeutic strategies have been used to target S100A10 including annexin A2 peptides, S100A10 neutralizing antibodies, small molecule inhibitors, and the vitamin A metabolite all-trans retinoic acid (ATRA). An annexin A2 peptide containing the S100A10 binding site prevents the binding of prostate cancer cells [90] and multiple myeloma cells to osteoblasts [89]. S100A10 antibodies are effective in reducing leukemia cell invasion in vitro [63] and homing of leukemia cells to the bone marrow in vivo [64]. Current studies in our laboratory are investigating the ability of S100A10 antibodies to block serous ovarian cancer motility and invasion. Using both, a receptor-guided and ligand-guided virtual screening approach has led to the identification of a number of small molecules that inhibit the interaction between annexin A2 and S100A10 [101–104]. One of these inhibitors 5-benzyl-4-methyl-2-(toluene-4-sulfonylamino)-thiophene-3-carboxylic acid amide that could block the interaction between annexin A2 and S100A10 has recently been shown to inhibit the adhesion of leukemic cells to osteoblasts in vivo and increase the sensitivity of leukemic cells to drugs such as dexamethasone and vincristine in vitro [64]. Further in vitro and in vivo studies are required to determine the effectiveness of these small molecule inhibitors in blocking pro-tumorigenic behavior in a wide range of cancer cells, including ovarian cancer. ATRA is currently used as a primary treatment for patients with APL [61,62,105]. ATRA has been shown to be beneﬁcial in inducing differentiation and promoting apoptosis of leukemic cells and improving bleeding symptoms by inhibiting plasmin production and decreasing annexin A2 and S100A10 expression [46,106–108]. Treatment of APL leukemic cell lines with ATRA causes the rapid loss of both cell surface annexin A2 and S100A10 protein [46,63,109]. Gladwin et al. (2000) also showed that ATRA reduces S100A10 protein levels but not S100A10 mRNA levels in bronchial epithelial cells (BEAS-2B) [110]. These ﬁndings suggest that ATRA exerts its effects to inhibit S100A10 protein levels via a post-translational mechanism. Recent studies further investigating the mechanism whereby ATRA inhibits S100A10 protein levels suggest that ATRA promotes the proteasomal degradation of S100A10 in a ubiquitin-independent manner [111]. ATRA treatment in MCF-7 breast cancer cells reduce S100A10 but not annexin A2 transcript and protein levels, indicating that ATRA can regulate S100A10 levels independently of PML/RARα and annexin A2 [111]. 7. Role of S100A10 in Ovarian Cancer and Chemotherapy Resistance Together these findings suggest that S100A10 plays an important role in the progression of serous ovarian cancer and chemotherapy resistance. Future studies are required to investigate further the functional role of S100A10 in ovarian cancer, its usefulness in predicting chemotherapy response, and as a therapeutic target to overcome chemoresistance. Figure 3. S100A10 immunostaining in matching tissues from a serous ovarian cancer patient at diagnosis (a) and recurrence with chemotherapy-resistant disease (b). S100A10 expression is increased in tumor tissue at relapse with chemotherapy-resistant disease compared to tumor tissue at diagnosis. S100A10 antibody using citrate buffer retrieval (1/1000, BD Biosciences) [16]. Scale bar = 100µm. Figure 3. S100A10 immunostaining in matching tissues from a serous ovarian cancer patient at diagnosis (a) and recurrence with chemotherapy-resistant disease (b). S100A10 expression is increased in tumor tissue at relapse with chemotherapy-resistant disease compared to tumor tissue at diagnosis. S100A10 antibody using citrate buffer retrieval (1/1000, BD Biosciences) [16]. Scale bar = 100 µm. Figure 3. S100A10 immunostaining in matching tissues from a serous ovarian cancer patient at diagnosis (a) and recurrence with chemotherapy-resistant disease (b). S100A10 expression is increased in tumor tissue at relapse with chemotherapy-resistant disease compared to tumor tissue at diagnosis. S100A10 antibody using citrate buffer retrieval (1/1000, BD Biosciences) [16]. Scale bar = 100µm Figure 3. S100A10 immunostaining in matching tissues from a serous ovarian cancer patient at diagnosis (a) and recurrence with chemotherapy-resistant disease (b). S100A10 expression is increased in tumor tissue at relapse with chemotherapy-resistant disease compared to tumor tissue at diagnosis. S100A10 antibody using citrate buffer retrieval (1/1000, BD Biosciences) [16]. Scale bar = 100 µm. Int. J. Mol. Sci. 2018, 19, 4122 9 of 16 8. Strategies to Target S100A10 in Cancer Cells While the effects of ATRA on ovarian cancer cell proliferation and apoptosis have been previously investigated [112], to date no data has been reported on the effects of ATRA on both annexin A2 and S100A10 expression in ovarian cancer cells. Ongoing studies in our laboratory are investigating the ability of ATRA to inhibit cell proliferation and S100A10 expression in a range of serous ovarian cancer cell lines and serous ovarian cancer tissues using an ex-vivo explant assay [113]. A potential risk of using anti-S100A10 or anti-annexin A2 therapies is the increased risk of thromboembolic events. Previous studies have found increased thrombosis in patients receiving ATRA [114,115] while in other studies the incidence of thrombosis was reported to be lower [116,117]. It will be important to monitor patients treated with anti-S100A10 therapies and consider antithrombotic prophylaxis if they have a high risk of thromboembolic events [118]. 9. Summary and Conclusions In conclusion, S100A10 has been shown to play an important role in promoting pro-tumorigenic behavior in many cancers. Emerging evidence shows an important role of the AIIt heterotetramer in the plasminogen activator system in cancer cells and a key role in macrophage migration. The interaction between annexin A2, S100A10, and t-PA mediates the conversion of plasminogen to plasmin, which facilitates the ECM degradation, MMP activation, and angiogenesis, leading to increased cancer cell migration, invasion, and metastasis. S100A10 also plays a signiﬁcant role in the development of treatment resistance; however, the mechanisms involved are poorly understood and warrant further investigation. A greater understanding of the functional role of S100A10 in ovarian cancer cells could lead to the development of effective strategies to target S100A10 and annexin A2, inhibit progression, and overcome chemotherapy resistance in ovarian cancer patients. Int. J. Mol. Sci. 2018, 19, 4122 10 of 16 10 of 16 Author Contributions: Conceptualization, T.M.N, C.R. and N.A.L.; writing—original draft preparation writing—review and editing, T.M.N., C.R., N.A.L and M.K.O. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. Abbreviations AIIt S100A10-annexin A2 heterotetramer ALL acute lymphoblastic leukemia APL acute promyelotic leukemia ABC ATP-binding cassette ATRA all-trans retinoic acid BAD Bcl-2-associated death promoter BEAS-2B bronchial epithelial cells CHO Chinese hamster ovary CRA Chemoresponse assay DLCI data-link connection identiﬁer ECM extracellular matrix LLC Lewis Lung Carcinoma MMP matrix metalloproteinases NSCLC non-small cell lung cancer PFI platinum-free interval PFS progression-free survival Pg plasminogen PML promyelocytic leukemia OS overall survival RhoGAP Rho GTPase-activating protein t-PA tissue-type plasminogen activator uPA urokinase-type plasminogen activator References 1. Miller, K.D.; Siegel, R.L.; Lin, C.C.; Mariotto, A.B.; Kramer, J.L.; Rowland, J.H.; Stein, K.D.; Alteri, R.; Cancer treatment and survivorship statistics, 2016. CA Cancer J. Clin. 2016, 66, 271–289. [CrossRef] [P 2. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. 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https://openalex.org/W2412072712	https://europepmc.org/articles/pmc4633164?pdf=render	English	null	TNF-R1, an immune checkpoint in melanoma?	Genes & cancer	2,015	cc-by	1,107	Genes & Cancer, Vol. 6 (9-10), September 2015 Genes & Cancer, Vol. 6 (9-10), September 2015 www.impactjournals.com/Genes & Cancer/ TNF-R1, an immune checkpoint in melanoma? Florie Bertrand, Céline Colacios and Bruno Ségui We and others have documented that the factor associated with neutral sphingomyelinase activation (FAN), which is an adaptor protein of the TNF-R1 [2], enhances TNF-induced apoptosis [3], leukocyte migration [4] and antibody immune response towards a thymo- dependent antigen [5]. In addition, downregulation of FAN in B16 melanoma cells decreases TNF-induced B16 melanoma motility and invasion [6]. We recently investigated the consequences of host FAN deficiency on the tumor growth of B16 melanoma cells. In sharp contrast to what we observed in mice lacking either TNF or TNF-R1, the tumor growth of B16K1 cells was not impaired in FAN-deficient mice as compared to their wild- type counterparts (Montfort and Ségui, unpublished data). The latter observation suggests that the host TNF-R1 signaling, which likely represents an immune checkpoint facilitating B16K1 melanoma growth, does not critically involve the FAN-dependent neutral sphingomyelinase activation. The role of FAN may thus be restricted to Through binding to and activation of two receptors, TNF-R1 and TNF-R2, Tumor Necrosis Factor α (TNF) modulates various biological processes in the immune system. A growing body of evidence in the literature indicates that TNF may behave as a tumorigenic cytokine, facilitating cancer cell immune escape and tumor progression. We have recently provided evidence that host TNF signaling impairs CD8+ T cell-dependent immune response against melanoma. TNF blockade (with Etanercept) or deficiency (TNF knock out) facilitates the accumulation of CD8+ Tumor-Infiltrating Lymphocytes (TILs), thereby limiting the growth of melanoma cell lines, which express Major Histocompatibility Class 1 molecules (MHCI) at high levels. Similar findings were observed in mice lacking TNF-R1, but not TNF-R2, indicating that host TNF-R1 plays a critical role in limiting the establishment of such a CD8+ T cell- dependent immune response against melanoma under our experimental conditions [1]. Figure 1: TNF triggers TNF-R1-dependent cell death of activated CD8+ T cells, limiting their tumor accumulation in melanoma. In contrast to naive CD8+ T cells, activated CD8+ T cells express TNF-R1, which transduces cell death in response to TNF and likely constitutes an immune checkpoint in melanoma. TNF-R1 deficiency impairs TNF-induced CD8+ T cell death. Inhibiting TNF or TNF-R1 may enhance CD8+ T cell tumor infiltration, thereby limiting melanoma progression. TNF-R1, an immune checkpoint in melanoma? Florie Bertrand, Céline Colacios and Bruno Ségui Naive CD8+ T cells Activated CD8+ T cells Activated CD8+ T cells TNF-R1-/- TNF-induced cell death Resist TNF- induced cell death Resist TNF- induced cell death TNF-R1 TNF-R2 TNF TNF-R1+/+ CD8+ T cell accumulation in melanoma Naive CD8+ T cells Resist TNF- induced cell death CD8+ T cell accumulation in melanoma Figure 1: TNF triggers TNF-R1-dependent cell death of activated CD8+ T cells, limiting their tumor accumulation in melanoma. In contrast to naive CD8+ T cells, activated CD8+ T cells express TNF-R1, which transduces cell death in response to TNF and likely constitutes an immune checkpoint in melanoma. TNF-R1 deficiency impairs TNF-induced CD8+ T cell death. Inhibiting TNF or TNF-R1 may enhance CD8+ T cell tumor infiltration, thereby limiting melanoma progression. www.impactjournals.com/Genes & Cancer 369 Genes & Cancer Our recent findings demonstrate that TNF-R1- dependent TNF signaling impairs CD8+ TIL content and likely constitutes a potent immune checkpoint in melanoma, facilitating melanoma progression. Targeting either TNF or TNF-R1 may reduce the death of activated CD8+ TILs in patients affected with melanoma and thus may constitute an emerging immunotherapy to enhance CD8+ T cell-dependent immune response against melanoma. Clinical trials are necessary to evaluate the therapeutic value of TNF blockade strategy in melanoma patients and define the eligibility criteria such as (i) TNF expression in melanoma biopsies, (ii) pre-existing CD8+ T cell infiltration, (iii) MHCI expression by melanoma cells. some, but not all, TNF-dependent immune responses. Accordingly, the TNF-dependent anti-infectious immune response towards Listeria monocytogenes is not compromised in mice lacking FAN [5]. One of the main mechanisms that likely accounts for the TNF-dependent immunosuppression in our experimental mouse melanoma model is the capacity of TNF to trigger cell death of activated CD8+ T cells. Whereas TNF potently induced cell death of activated CD8+ T cells as documented by others, it remained unclear which TNF receptor is required for this process. On the one hand, activated CD8+ T cells expressed TNF-R2, which mediated activation-induced cell death [7]. On the other hand, exogenous TNF elicited cell death in concanavalin-A activated CD8+ T cells in a TNF- R1-dependent manner [8]. Of note, the latter study was carried out in the presence of cycloheximide to artificially sensitize lymphocytes to TNF. The use of cycloheximide may be a source of artefact and is unlikely appropriate to evaluate the physiological role of TNF and its receptors in CD8+ T cell death. TNF-R1, an immune checkpoint in melanoma? Florie Bertrand, Céline Colacios and Bruno Ségui Under our experimental conditions in the absence of cycloheximide, TNF-induced cell death of activated CD8+ T cells occurred mainly through a TNF- R1-dependent process [1]. This conclusion is supported by the following findings: (i) naive CD8+ T cells, which expressed TNF-R2 but not TNF-R1, resisted TNF- induced cell death; (ii) activated CD8+ T cells, which significantly expressed both TNF-R1 and TNF-R2, were sensitive to exogenous TNF; (iii) TNF-R1-deficient CD8+ T cells were fully resistant to TNF-induced cell death; (iv) TNF-R2 deficiency minimally affected CD8+ T cell death in response to TNF. The role of TNF-R1 as an immune checkpoint in melanoma gets further credence as illustrated by our data showing that the accumulation of activated CD8+ T cells into melanoma was facilitated by TNF-R1 deficiency in an adoptive transfer experiment performed in CD8-deficient hosts [1] (Figure 1). The authors declare no conflict of interest. The authors declare no conflict of interest. Bruno Ségui: INSERM UMR 1037, CRCT, Toulouse, France Correspondence: Bruno Ségui, email bruno.segui@inserm.fr Correspondence: Bruno Ségui, email bruno.segui@inserm.fr Keywords: TNF, CD8+ T lymphocytes, apoptosis, tumor-infil trating lymphocytes Received: September 08, 2015 Published: October 05, 2015 Keywords: TNF, CD8+ T lymphocytes, apoptosis, tumor-infil trating lymphocytes Received: September 08, 2015 Published: October 05, 2015 Published: October 05, 2015 REFERENCES 1. Bertrand F, et al. Cancer Res. 2015; 75: 2619-2628. 2. Montfort A, et al. J Leukoc Biol. 2010; 88: 897-903. 3. Segui B, et al. J Clin Invest. 2001; 108: 143-151. 4. Boecke A, et al. J Immunol. 2012; 189: 1559-1566. 5. Montfort A, et al. J Immunol. 2009; 183: 5369-5378. 6. Boecke A, et al. Br J Cancer. 2013; 109: 422-432. 7. Zheng L, et al. Nature. 1995; 377: 348-351. 8. Teh HS, et al. J Immunol. 2000; 165: 4814-4821. 370 Genes & Cancer
https://openalex.org/W4306828717	https://diagnprognres.biomedcentral.com/counter/pdf/10.1186/s41512-022-00133-x	English	null	Development and validation of personalised risk prediction models for early detection and diagnosis of primary liver cancer among the English primary care population using the QResearch® database: research protocol and statistical analysis plan	Diagnostic and prognostic research	2,022	cc-by	9,707	Abstract Background and research aim: The incidence and mortality of liver cancer have been increasing in the UK in recent years. However, liver cancer is still under-studied. The Early Detection of Hepatocellular Liver Cancer (DeLIVER-QRe‑ search) project aims to address the research gap and generate new knowledge to improve early detection and diag‑ nosis of primary liver cancer from general practice and at the population level. There are three research objectives: (1) to understand the current epidemiology of primary liver cancer in England, (2) to identify and quantify the symptoms and comorbidities associated with liver cancer, and (3) to develop and validate prediction models for early detection of liver cancer suitable for implementation in clinical settings. Methods: This population-based study uses the QResearch® database (version 46) and includes adult patients aged 25–84 years old and without a diagnosis of liver cancer at the cohort entry (study period: 1 January 2008–30 June 2021). The team conducted a literature review (with additional clinical input) to inform the inclusion of variables for data extraction from the QResearch database. A wide range of statistical techniques will be used for the three research objectives, including descriptive statistics, multiple imputation for missing data, conditional logistic regres‑ sion to investigate the association between the clinical features (symptoms and comorbidities) and the outcome, fractional polynomial terms to explore the non-linear relationship between continuous variables and the outcome, and Cox/competing risk regression for the prediction model. We have a specific focus on the 1-year, 5-year, and 10-year absolute risks of developing liver cancer, as risks at different time points have different clinical implications. The internal–external cross-validation approach will be used, and the discrimination and calibration of the prediction model will be evaluated. Correspondence: weiqi.liao@phc.ox.ac.uk 1 Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK 1 Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK © 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. Development and validation of personalised risk prediction models for early detection and diagnosis of primary liver cancer among the English primary care population using the QResearch® database: research protocol and statistical analysis plan Weiqi Liao1 , Peter Jepsen2 , Carol Coupland1,3, Hamish Innes4, Philippa C. Matthews5,6,7 , Cori Campbell7, Eleanor Barnes7 , Julia Hippisley‑Cox1 and on behalf of the DeLIVER consortium Weiqi Liao1* , Peter Jepsen2 , Carol Coupland1,3, Hamish Innes4, Philippa C. Matthews5,6,7 , Cori Campbell7, Eleanor Barnes7 , Julia Hippisley‑Cox1 and on behalf of the DeLIVER consortium © 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/. Liao et al. Diagnostic and Prognostic Research (2022) 6:21 https://doi.org/10.1186/s41512-022-00133-x Liao et al. Diagnostic and Prognostic Research (2022) 6:21 https://doi.org/10.1186/s41512-022-00133-x (2022) 6:21 Diagnostic and Prognostic Research Open Access Introduction and research background The QResearch team has conducted previous work on developing the QCancer prediction models to estimate future risk of lung, colorectal, gastro-oesophageal, pan- creas, blood and renal tract cancers for both men and women, breast, cervical, and ovarian cancers for women, and prostate and testicular cancers for men [8]. The QCan- cer algorithms have been implemented in the NHS com- puter system as decision support tools for GPs. An online risk calculator is also available at https://www.qcancer.org/, free for public use. However, liver cancer is still not part of the QCancer model family yet. Therefore, we will develop a prediction model for liver cancer in this project. According to the most recent statistics from Cancer Research UK, liver cancer represents the 18th most common cancer in the UK, accounting for 2% of all new cancer cases in 2017. However, it is the 8th most common cause of cancer death, accounting for 3% of all cancer deaths in 2018 [1]. Deaths due to liver can- cer have increased by around 50% in the last decade [2]. The age-standardised incidence and incidence-based mortality rates of primary liver cancer increased during 1997–2017, particularly notable in hepatocellular carci- noma (HCC) [3]. The rapid increase in incidence and mortality rates is a public health concern, and a burden to health and social care. Compared with other cancers, the prognosis of liver cancer is poor, with respective 1- and 5-year survival estimates of only 38.1% and 12.7% in England during 2013–2017 [1]. Early diagnosis is associated with better survival. When it is diagnosed at its earliest stage, 78% of people can survive for 1 year or longer, compared with 20% when diagnosed at the lat- est stage [1, 4, 5]. Abstract 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/. Page 2 of 11 Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Discussion: The DeLIVER-QResearch project uses large-scale representative population-based data to address the most relevant research questions for early detection and diagnosis of primary liver cancer in England. This project has great potential to inform the national cancer strategic plan and yield substantial public and societal benefits. Keywords: Liver cancer, Hepatocellular carcinoma (HCC), Cholangiocarcinoma, Risk prediction model, Early detection, Diagnosis, Symptom, Comorbidity Research aim and objectivesh This study aims to generate new knowledge for early detec- tion and diagnosis of primary liver cancer from the English population, with a special interest in HCC. There are three research objectives in the DeLIVER-QResearch project: 1. To understand the current epidemiology of people diagnosed with liver cancer in England, including the incidence, route to diagnosis, cancer stage and his- tology, treatments, survival duration, and the main causes of death; g There are two general approaches for early detection and diagnosis of (ED&D) cancer (World Health Organi- zation) [6]. Early diagnosis aims to detect symptomatic patients as early as possible. For individuals at high risk but without symptoms, using reliable tests in screening can detect cancer at early stages. Therefore, the study design in this project is around these two approaches. A better understanding of the clinical features that indicate the development and progression of liver cancer would improve prompt referrals for investigation and interven- tion. Risk prediction models could estimate individual patients’ risk of developing liver cancer and identify those at high risk from the primary care population (risk strati- fication) for active surveillance (screening) using liver ultrasonography and serum alpha-fetoprotein (AFP) test [7]. Both approaches could help to shift the diagnosis of liver cancer towards earlier stages, when patients have a greater chance of curative treatments and better survival outcomes. All of these are the rationale and motivations to conduct the DeLIVER-QResearch project, which is part of an initiative funded by Cancer Research UK to early detect hepatocellular liver cancer (DeLIVER), pro- ject website: www.deliver.cancer.ox.ac.uk. 2. To explore and characterise the symptom and comor- bidity profile for patients diagnosed with liver cancer, compared with those without liver cancer. The find- ings can help both patients and GP to achieve early diagnosis through symptomatic presentation in gen- eral practice. p 3. To develop and validate personalised prediction models for estimating the risk of patients getting a diagnosis of liver cancer in the next 5 or 10 years using primary care electronic health records (EHRs), which could be used to identify patients at the high- est risk who will benefit the most from active surveil- lance and early clinical intervention. Study setting and populationh Data source the QResearch database Routinely collected EHRs linked to the QResearch database (version 46) will be the data source for this study. QResearch is a large consolidated database with anonymised EHRs of over 35 million patients from 1800 + general practices using the Egton Medical Infor- mation Systems (EMIS) spread across England. The database includes patients who are currently registered with the practices as well as historical patients who may have left or died. Historical records date back to 1989 with linked data on all practices since 1998. Patients’ primary care records are linked with other national datasets, such as the Hospital Episode Statistics (HES, secondary care data, including inpatient, outpatient, accident and emergency (A&E), and critical care), death registration data (up to 15 causes of death) from the Office for National Statistics (ONS), and cancer registry from Public Health England (PHE). This is a population-based study using the QResearch database. We use similar inclusion and exclusion cri- teria as those in previous studies [8–10] to develop and validate the other QCancer models. The study popula- tion will be adult patients aged between 25 and 84 years old and without a diagnosis of liver cancer before the date of cohort entry. Patients diagnosed with cancer aged 16–24 years old are classified as Teenage and Young Adult (TYA) cancer [11]. The age range covers the major- ity of patients, who are more likely to benefit from active surveillance and screening for early diagnosis of liver cancer. The included patients need to have been regis- tered in the general practices for at least 12 months, and these practices need to have contributed to the QRe- search database for a minimum of 12 months before the cohort entry date. This is to ensure complete data before cohort entry. Figure 1 shows the timeline of the dynamic patient cohort for the DeLIVER-QResearch project. g The team conducted a rapid literature review (includ- ing the NICE guidelines) and had clinical input from physicians (hepatology, primary care, infectious dis- eases) to inform the inclusion of variables and prepare code lists to extract data from the QResearch data- base. Two patient representatives reviewed the lists of symptoms and comorbidities and shared their experi- ences and disease trajectories with the lead researcher. Identification of liver cancer cases Incident primary liver cancer cases during 2008–2018 will be identified and followed up to 30 June 2021. The most recent available data from the cancer registry was 31 December 2018 when the data were extracted in August 2021. However, the primary care records, HES, and the death registry only had a time lag of sev- eral months. Therefore, we could know whether patients received treatments or not after diagnosis of liver cancer and the outcomes (e.g. death, left cohort, or still alive) during the follow-up period. Study setting and populationh We prepared Read/SNOMED-CT code lists to extract events from the GP records, ICD-10 code lists for diag- nosed diseases in the HES, cancer registry and death records, and OPCS code lists for interventions and procedures conducted in NHS hospitals. Tables 1 and 2 summarise the variables we requested for the three research objectives. Some variables may not be signifi- cantly associated with the outcome after the analysis. However, we do not want to miss any potential asso- ciation. Therefore, the variables are included as broadly and comprehensively as possible in the project set-up phase. Study designs An open cohort study will be used for research objec- tives 1 and 3, and a nested case–control study (within the same cohort) for research objective 2. Page 3 of 11 Page 3 of 11 Page 3 of 11 Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Table 2 Symptoms, comorbidities, and clinical characteristics relevant to liver cancer Change in taste, loss of taste, dry mouth/thirst, dysphagia, loss of appetite or feeling full after eating small amounts, heartburn/gastro-oesophageal reflux (GOR), indigestion, nausea, vomiting, hemate‑ mesis, stomach pain/discomfort/cramp Abdominal pain (right upper quadrant), abdominal lump on the right side, abdominal distension, abdominal mass, swollen abdomen/flatulence/bloating, ascites, change in bowel habits, diarrhoea, constipation, melaena Weight loss, fatigue/tiredness/lethargy (lack of energy), nausea, confusion (may suggest hepatic encephalopathy), fever, shivering, jaundice (yellow skin), pruritis (itchy skin), rash, dark urine, pale stools, night sweats, right shoulder pain, back pain, peripheral oedema, unexplained bruising Cirrhosis, hepatitis B infection, hepatitis C infection, non-alcoholic fatty liver disease (NAFLD), alco‑ holic liver disease, autoimmune hepatitis, liver adenoma, any other chronic liver diseases Hypertension, coronary heart disease, heart failure, chronic obstructive pulmonary disease (COPD), venous thromboembolism Type 1 and type 2 diabetes mellitus, electrolyte disorders, dyslipidaemia, obesity, gout Chronic pancreatitis, hepatic cyst, renal cyst, pancreatic cyst, hemochromatosis, cholangitis, gall‑ bladder stones, gastrointestinal haemorrhage, peptic ulcer disease, anaemia, thrombocytopenia Heroin use or other drug addiction, alcoholism, HIV/AIDS Haemoglobin, HBV (antibody/antigen/DNA), ALT, AST, PLT, BR, GGT, ESR, CRP Haemoglobin, HBV (antibody/antigen/DNA), ALT, AST, PLT, BR, GGT, ESR, CRP Furosemide, spironolactone, propranolol, carvedilol, lactulose, rifaximin, tenofovir, entecavir, lamivu‑ dine, antiretroviral therapy; statins, aspirin and metformin Medications Furosemide, spironolactone, propranolol, carvedilol, lactulose, rifaximin, tenofovir, entecavir, lamivu‑ dine, antiretroviral therapy; statins, aspirin and metformin HES hospital episode statistics, HBV hepatitis B virus, HCV hepatitis C virus, HEV hepatitis E virus, ALT alanine transaminase, AST aspartate aminotransferase, PLT platelet count, BR bilirubin, GGT gamma-glutamyl transferase, ESR erythrocyte sedimentation rate, CRP C-reactive protein HES hospital episode statistics, HBV hepatitis B virus, HCV hepatitis C virus, HEV hepatitis E virus, ALT alanine transaminase, AST aspartate aminotransferase, PLT platelet count, BR bilirubin, GGT gamma-glutamyl transferase, ESR erythrocyte sedimentation rate, CRP C-reactive protein Fig. 1 Timeline of the dynamic patient cohort for the DeLIVER-QResearch (version 46) project. Data from primary care, secondary care, and death records are available for liver cancer cases during the follow-up period, up till 30 June 2021 at diagnosis (likely to convert into a binary variable, i.e. early vs late stage). be different from those in the general primary care pop- ulation, and may differ by geographical region and over time. Therefore, up to 10 controls will be matched with each cancer case by age, sex, general practice (regions), and calendar year (time) wherever possible using inci- dence density sampling [13]. Table 2 Symptoms, comorbidities, and clinical characteristics relevant to liver cancer With such matching vari- ables, we can better infer statistically what symptoms and comorbidities are associated with liver cancer. Each control will be allocated an index date which is the date of cancer diagnosis in their matched case. We will investigate the number of patients with and with- out cirrhosis in liver cancer cases. If the sample size is big enough, we will stratify liver cancer cases by cirrho- sis in subgroup analysis. Study outcomesh The primary outcome is the incident diagnosis of pri- mary liver cancer. The date of cancer diagnosis was the earliest date recorded in the four linked data sources (pri- mary care, hospital admission, cancer registration, and death registry), which will be the index date in the nested case–control study. The secondary outcome is the stage Table 1 General variables for this study HES hospital episode statistics, ONS Office for National Statistics, PHE Public Health England Data source Categories Variables GP record Demographics Age, sex, ethnicity, socioeconomic deprivation (Townsend quintile), geographical regions in England Lifestyle Body mass index (BMI, continuous variable), smoking and drinking status and intensity (with units)—longitudinal data available for all variables HES Diagnoses and treatments in the outpatient appointments and hospital admissions Cancer registry (PHE) Date of liver cancer diagnosis, route to diagnosis, stage at diagnosis, cancer grade and histology HES Treatments Surgical resection of liver, liver transplantation, ablation of liver, transarterial chemoembolization (TACE), transarterial radioembolization (TARE)—OPCS codes ONS death Date of death, all causes of death (up to 15) Page 4 of 11 Liao et al. Study outcomesh Diagnostic and Prognostic Research (2022) 6:21 Table 2 Symptoms, comorbidities, and clinical characteristics relevant to liver cancer HES hospital episode statistics, HBV hepatitis B virus, HCV hepatitis C virus, HEV hepatitis E virus, ALT alanine transaminase, AST aspartate aminotransferase, PLT platelet count, BR bilirubin, GGT gamma-glutamyl transferase, ESR erythrocyte sedimentation rate, CRP C-reactive protein Symptoms (GP records) Appetite and upper gastrointestinal symptoms Change in taste, loss of taste, dry mouth/thirst, dysphagia, loss of appetite or feeling full after eating small amounts, heartburn/gastro-oesophageal reflux (GOR), indigestion, nausea, vomiting, hemate‑ mesis, stomach pain/discomfort/cramp Abdominal and bowel symptoms Abdominal pain (right upper quadrant), abdominal lump on the right side, abdominal distension, abdominal mass, swollen abdomen/flatulence/bloating, ascites, change in bowel habits, diarrhoea, constipation, melaena Non-specific and systemic symptoms Weight loss, fatigue/tiredness/lethargy (lack of energy), nausea, confusion (may suggest hepatic encephalopathy), fever, shivering, jaundice (yellow skin), pruritis (itchy skin), rash, dark urine, pale stools, night sweats, right shoulder pain, back pain, peripheral oedema, unexplained bruising Comorbidities (GP and HES records) Liver diseases Cirrhosis, hepatitis B infection, hepatitis C infection, non-alcoholic fatty liver disease (NAFLD), alco‑ holic liver disease, autoimmune hepatitis, liver adenoma, any other chronic liver diseases Cardiorespiratory diseases Hypertension, coronary heart disease, heart failure, chronic obstructive pulmonary disease (COPD), venous thromboembolism Metabolic disorders Type 1 and type 2 diabetes mellitus, electrolyte disorders, dyslipidaemia, obesity, gout Other GI and systemic diseases Chronic pancreatitis, hepatic cyst, renal cyst, pancreatic cyst, hemochromatosis, cholangitis, gall‑ bladder stones, gastrointestinal haemorrhage, peptic ulcer disease, anaemia, thrombocytopenia Others Heroin use or other drug addiction, alcoholism, HIV/AIDS Clinical and family history (GP records) Family history Liver cancer, (any) gastrointestinal cancers (gastric, bowel, liver, pancreas) Personal history Previous cancers Investigation and patient management (logged) in primary care records Blood tests Haemoglobin, HBV (antibody/antigen/DNA), ALT, AST, PLT, BR, GGT, ESR, CRP Medications Furosemide, spironolactone, propranolol, carvedilol, lactulose, rifaximin, tenofovir, entecavir, lamivu‑ dine, antiretroviral therapy; statins, aspirin and metformin Explore the clinical features and red‑flag symptoms associated with liver cancer (research objective 2) The analyses for the nested case–control study In the nested case–control study design, the cases and con- trols have the same length of time exposure in the risk set. The index date was the date of cancer diagnosis in cases or the equivalent date in the matched controls. In this way, it is easier to date back the exposure of symp- toms (e.g. 3 months, 6 months, 1 year,… before diagnosis) and investigates the association between symptoms and the outcome at different periods to inform early diagno- sis of liver cancer through symptomatic presentation. We will explore the symptoms and comorbidities in patients’ EHRs in different timeframes, e.g. up to 3 months, 4–6 months, 7–12 months, 1–2 years, 2–3 years, and 3–5 years, before the date of diagnosis in cases, or the equivalent date in controls. Multivariable conditional logistic regression will be used for the matched case– control design, adjusting for confounders (e.g. patient’s sociodemographic characteristics and lifestyle factors) and considering the interaction terms. This will deter- mine which ‘red flag’ symptoms remain significant when other factors are taken into account in the model. After handling the missing data (subsection above), the results from the imputed datasets will be the primary estimates, but these will be compared with the estimates from two sets of sensitivity analyses, which are complete case anal- ysis, and analyses restricted to cases and controls that have more than 3 years of EHRs before the index date. The analyses will be carried out separately for the two control groups (the general primary care population and patients diagnosed with cirrhosis), and the findings from the two control groups will be compared. After imputing cancer staging, we will conduct further analyses to iden- tify clinical features that can help diagnose liver cancer earlier (likely to divide as binary, early vs late stages). In summary, the analyses in the nested case–control studies are more exploratory. We may develop a diagnostic pre- diction model in the future (depending on the findings and subjective to further funding), but not in this project. The reporting of the case–control study will follow the recommendations of the STROBE statement [20]. Handling missing data For symptoms, comorbidities, and medication use, the absence of information in the EHRs will be assumed that the patient did not have the health conditions or was not prescribed the medications. Control groups in the nested case–control study Around 80–90% of patients have cirrhosis before being diagnosed with HCC [12]. Cirrhosis could be seen as one possible precursor and on the disease trajectory of developing HCC. Therefore, to better understand the clinical features and early signs of liver cancer, we will select two control groups (the general primary care population and patients diagnosed with cirrhosis) in the nested case–control study. The demographic and clinical characteristics of liver cancer cases are likely to Page 5 of 11 Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Statistical analysis plan General statistical methods used in multiple research objectives presentation, late stage at diagnosis, patients received curative treatments or any treatment, and survival dura- tion, with a specific interest in the effects of sex, socio- economic deprivation and ethnicity. Considering the importance of cirrhosis on the disease trajectory of liver cancer, we will use similar methods to describe the epi- demiology (e.g. incidence, prevalence, the trend over the years, and distribution in the English primary care popu- lation) of cirrhosis using EHRs. Descriptive statistics Descriptive statistics will be used to describe the sociodemographic (e.g. age, sex, ethnic- ity, Townsend quintile) and clinical characteristics (e.g. symptoms, comorbidities, cancer stage, grade, histol- ogy) of the study population, using means and standard deviations, medians and interquartile ranges (IQR), and proportions, for different types of data as appropriate. For the nested case–control study, characteristics will be described separately for cases and the two sets of con- trols. We will describe the temporal changes in incidence, routes to diagnosis, stage at diagnosis and treatments for liver cancer by year during the study period. Methodology for prediction models to estimate individual risk of developing liver cancer (research objective 3) Sample size considerations We used the criteria by Riley et al. [21] and the ‘pmsampsize’ package in R to calculate the minimum required sample size for developing a clini- cal prediction model. The parameters for sample size esti- mation for time-to-event outcome were set or assumed as follows. The previous QCancer prognostic models have around 30 predictors, we assume 50 predictors in the new models to allow more flexibility. The median duration from cohort entry to the incident diagnosis of liver cancer is about 10 years (QResearch has linked data on all practices since 1998, see the “Data source—the QResearch® database” section above), which is suitable for the predictive period (up to 10 years). The age-stand- ardised incidence rate (event rate) of liver cancer in the UK during 2016–2018 was 14.5 (95% CI 14.3–14.8) per 100,000 population for men and 6.2 (95% CI 6.0–6.3) for women (statistics from Cancer Research UK [22]). A con- servative R2 Cox−Snell (15% of the maximum R2 Cox−Snell ) was used as recommended [21]. Based on the above param- eters, the minimum sample size required for developing a new model is 149,750 for men and 299,750 for women. Hence, the derivation dataset needs a minimum sample of about 450,000 men and women. Considering the application of our prediction model in clinical practice, the baseline values (evaluated at cohort entry) will be used in the model, as this is a similar clini- cal situation that physicians can use the current informa- tion to predict patient’s future risks when they see the patient. Cox proportional hazards model will be used as the main method to develop risk prediction models, using robust variance estimates to allow for clustering of patients within general practices, also accounting for cen- soring (due to death, loss of follow-up, or the end of the observation period). The proportional hazards assump- tions for Cox regression will be checked. The time ori- gin is the date of entry into the study cohort. The risk period of interest is from the time origin up to the date of incident diagnosis of liver cancer. Patients who died from other causes will be considered censored in the Cox regression. We will also consider using competing risk regression as an alternative. In such a case, death other than liver cancer before diagnosis will be competing risks. Explore the clinical features and red‑flag symptoms associated with liver cancer (research objective 2) Then a MICE model will be fitted in the development dataset to impute missing values and will include all predictors along with age inter- action terms, the Nelson-Aalen estimator of the baseline cumulative hazard, and the outcome (incident liver can- cer). Separate models will be fitted for men and women. Model development We will use similar established ana- lytical strategies to develop and evaluate the risk predic- tion equations in this study that were used in the other QResearch studies [8, 25–29]. The internal–external cross-validation approach [30, 31] will be used to quan- tify the heterogeneity of predictor effects in different geo- graphical regions in England, rather than splitting data by general practice into development and validation data- sets. Eight geographical regions will be used to develop the model and the remaining two regions to validate the model. This process will be performed five times, leaving out two different regions at each time. The indicators of model performance (subsection below) from the inter- nal–external cross-validation approach will be pooled using random-effects meta-analysis [30, 32]. The final model will be used to derive risk estimates for each year of follow-up, with a specific focus on 1-year (short-term), 5-year (medium-term) and 10-year (long-term) risk esti- mates. Separate models will be developed and evaluated for men and women, as the coefficients for the risk fac- tors may be different between sexes. Explore the clinical features and red‑flag symptoms associated with liver cancer (research objective 2) There may be missing data in some variables, as they were not collected and recorded in the EHRs. We will use multiple imputation with chained equations (MICE) to replace missing values for ethnicity, Townsend quintile, BMI, smoking status, alcohol intake and cancer staging with the assumption of data missing at random (MAR) [14–17]. We will first investigate the proportion of miss- ing values in each variable. At least five imputations will be conducted, as the TRIPOD statement suggests that 5 or 10 imputations be sufficient [18]. We will conduct ten imputations if computationally feasible for the whole cohort (around 9 million patients). We can conduct more imputations in the case–control study, as the sample size is much smaller. Rubin’s rules will be used to combine the model parameter estimates across the imputed datasets [19]. Epidemiology of primary liver cancer (research objective 1) The overall incidence rate of liver cancer and by age groups, sex, ethnicity, socioeconomic deprivation (Townsend quintiles), the 10 geographical regions in England, and liver cancer subtypes (HCC, cholangiocar- cinoma, other specified or unspecified liver cancer) per 100,000 person-years will be calculated. Poisson regres- sion will be used to investigate how patient characteristics are associated with the trend over time and the variation in the incidence of liver cancer and HCC throughout the study period, where interactions between age, sex, eth- nicity and Townsend quintile will be explored. In addi- tion, we will explore factors associated with emergency Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Page 6 of 11 Liao et al. Diagnostic and Prognostic Research (2022) 6:21 The most common symptoms and the combina‑ tions Patients may present to their GP with a series of different symptoms. Besides each individual symptom, the frequency of the most common symptom combi- nations will be summarised. For individual significant symptoms and symptom combinations, we will calculate the sensitivity, specificity, and positive predictive values. Such additional information would be helpful for GPs to manage patients based on symptomatic presentation and decide whether to refer the patients to a specialist or not, especially when patients present with several non-spe- cific symptoms. for the non-linear relationships. Methodology for prediction models to estimate individual risk of developing liver cancer (research objective 3) 10%) to conduct competing risk regression as a sensitivity analysis, and compare the results between the two approaches. The main analyses will be multivariable models includ- ing various predictors and interaction terms. The regres- sion coefficients for each variable in the final model will be used as weights. From which, we will use a formula to derive the absolute risk estimates by combining them with the baseline survivor function evaluated for each year of follow-up, with a maximum of 10 years [33]. We will compare our developed model with the other vali- dated risk prediction models in this field [34]. Variable selection and considerations As Sauerbrei et al. pointed out [35], despite as an important topic, there is not yet enough evidence to recommend the selection of variables and functional forms in multivariable analysis. We aim to develop a parsimonious and clinically rel- evant prediction model. We will start with a full model by including all the variables based on clinical knowledge and research evidence from the literature. We will then remove variables that might not be clinically significant with the following criteria. We will retain binary/cate‑ gorical/ordinal variables having hazard ratios (HR) < 0.91 or > 1.10, as HR closer to 1 may be less clinically signifi‑ cant for such variables. But the thresholds of HRs will not be used for continuous variables, as they are usually small for each incremental value. We apply a statisti- cal significance level of 0.05 (two-tailed). To simplify the models, we will combine similar variables with compa- rable HRs where possible. If some variables do not have enough events to obtain point estimates and standard errors, we will combine some of these if clinically simi- lar in nature. The variables in our prediction models are routinely recorded in the EHRs. Therefore, we can update the model periodically without burdening the healthcare professionals to collect data besides their routine work. f We will assess the calibration of the prediction model by comparing the predicted risks at 10 years with the observed risks, and present it in a smooth calibration plot. The observed risks for men and women will be obtained by using the Kaplan–Meier estimates or cumu- lative incidence functions to account for competing risks. We will also evaluate these performance measures in six pre-specified age groups (25–39, 40–49, 50–59, 60–69, 70–79, 80 +) and by sex. Methodology for prediction models to estimate individual risk of developing liver cancer (research objective 3) As we will use multiple imputation and fractional polynomials in such a big sample size (about 9 million patients), together with the internal–external cross-vali- dation approach, it may not be computationally feasible to use competing risk regression. We will explore the With about 9 million patients in the open cohort and an estimated 7000 incident liver cancer cases during 2008– 2018 in the QResearch database, there is sufficient data for the development and validation datasets. We will use all the eligible patients in the database to maximise the power. Exploration of non‑linear relationships Before imputa- tion, a complete-case analysis will be fitted using a model containing only the continuous variables (e.g. age, BMI) within the development dataset to derive the fractional polynomial terms (up to two polynomial terms) [23, 24] Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Page 7 of 11 statistic [40] (Wolber’s C statistic [41] if using a compet- ing risk model), and time-dependent ROC [42] at 1, 5 and 10 years and combine these across the imputed datasets using Rubin’s rules. R2 is the explained variation, where a higher value indicates a greater proportion of variation in survival time is explained by the model [37]. The D statistic is a measure of discrimination, which quantifies the separation in survival between patients with differ- ent levels of predicted risk, where higher values indicate better discrimination [38]. The Brier score is an aggregate measure of disagreement (the average squared error dif- ference) between the observed and the predicted out- comes [39]. The Harrell’s C statistic [40] is a measure of discrimination that quantifies the extent to which those with earlier events have higher risk scores. Higher values of Harrell’s C indicate better performance of the model for predicting the relevant outcome. A value of 1 indi- cates that the model has perfect discrimination. A value of 0.5 indicates that the model discrimination is no better than chance, which is the same interpretation for time- dependent ROC. The 95% confidence intervals for the performance statistics will be calculated to allow com- parisons with alternative models for the same outcome and across different subgroups [43]. compatibility of multiple advanced statistical techniques used together and make pragmatic decisions based on the available computing power and resources. We may use Cox regression as the main method, and draw a smaller random sample (e.g. Methodology for prediction models to estimate individual risk of developing liver cancer (research objective 3) Decision curve analysis [44] will be used to evaluate the net benefit of the prediction model (clinical usefulness). We will follow the recom- mendations from the TRIPOD guideline [18] to report the multivariable prognostic model. Risk stratification Risk stratification allows patients with a high predicted risk to be identified electronically from primary care records for tailored advice, active monitoring of the disease progression, and screening. Since there is no widely accepted threshold for classifying a high risk of liver cancer, we will examine the distribu- tion of the predicted risks and calculate a series of cen- tile values in the model. We will evaluate the sensitivity and specificity of various risk thresholds of the predicted absolute risk at the population level in the 10-year pre- dictive horizon. We will also report how many partici- pants with scores at least equal to each threshold, how many cancer cases will be potentially detectable, and how many cancer cases will be missed at each threshold. Such classification statistics would be useful for policymakers, Model validation—evaluate the model performance An imputation model (MICE) will be fitted for missing val- ues in the validation datasets with five imputations (same as in the deviation dataset) using the methods described in the earlier subsection. We will apply the risk equa- tions for men and women derived from the previous step to the validation data and calculate measures of model performance. As in previous studies [36], we will calculate the R2 [37], the D statistic [38], the Brier score [39], Harrell’s C Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Page 8 of 11 Page 8 of 11 Liao et al. Diagnostic and Prognostic Research (2022) 6:21 [28], QCancer (10-year risk) [8] and other algorithms. UK primary care records have high levels of accuracy and completeness of clinical diagnoses and prescribed medications. This study has good face validity, as it is conducted in the same setting where most patients are clinically assessed, managed, and followed up in England. Thanks to the expansion and upgrade of the QResearch database in recent years, we now have information on cancer staging, grade, and histology, although there are still missing data in these variables. The rich data source makes in-depth exploration possible. Summary: relevant guidelines used in this project • NICE NG12 (Suspected cancer: recognition and referral) [45]h • The European Association for the Study of the Liver (EASL) clinical practice guidelines (management of HCC) [46]h • The STROBE statement (reporting guideline for observational studies) [20]h Due to the available resources and the funding, we will use the internal–external approach to validate the devel- oped model using data from the same database. QRe- search uses data from the EMIS, which is the computer system used by 55% of the GP surgeries in the UK. Our study population is based in England and representa- tive of the whole English primary care population. Some previous studies [48–50] independently examined other risk equations developed by the QResearch team and concluded that using external data gave a similar perfor- mance as the internal validation approach using the QRe- search database, which is reassuring. It may be possible to externally validate the developed model by our collab- orators in Scotland or Denmark using datasets outside of England in future studies. • The TRIPOD statement (reporting guideline for mul- tivariable prediction model) [18, 47] The strengths and limitations of this project The strengths and limitations of this project To our best knowledge, this study will be the largest of its kind for early detection and diagnosis of liver cancer from the English primary care population. It has sig- nificant potential to address several important research gaps and gain a deeper understanding of the presenta- tion, characteristics, and outcomes of liver cancer. The key strengths of this population-based study include prospective recording of predictors and outcomes, good ascertainment of outcomes through linkage of multiple national databases, and a large sample size from an estab- lished validated database (QResearch) that has been used to develop many risk prediction tools, such as QRisk3 Methodology for prediction models to estimate individual risk of developing liver cancer (research objective 3) This study also minimises the most common biases in epidemiological studies, such as selection bias, recall bias, and respond- ent bias. We will also use relevant clinical and statisti- cal guidelines to follow the best research practice and to guide the analysis and report the findings for transparent and reproducible research. All of these strengths make the study findings more robust and more likely to gener- alise to the wider UK population. if a cut-off is needed to identify patients who may benefit from surveillance and make recommendations for liver cancer screening based on risk stratification in the future. However, we will not give recommendations on which risk threshold is suitable for screening, as it needs a full cost-effectiveness analysis, which is out of the scope of the current study. Dissemination and implementation plan of the prediction model The developed algorithm will be published in peer-reviewed journals and presented at academic con- ferences. A web-based program can implement the new risk algorithm similar to the QCancer tool (https://www. qcancer.org/), subject to funding and the Medicines and Healthcare Products Regulatory Agency (MHRA) medi- cal device compliance. It will also be possible to imple- ment the risk algorithm in the EHR systems, using exist- ing data to calculate individual risks for the primary care population. These implementation intentions will be subject to the terms and conditions of QResearch, the University of Oxford, the Cancer Research UK grant, and the agreement of all parties. It will be covered by another implementation protocol, which is out of the scope of this research protocol. The limitations of this study may include potential information bias and missing data. Based on our experi- ences of using primary care data, some lifestyle factors such as BMI, smoking and alcohol status may not always track the true values in real-time, and often lack consist- ency. In addition, the recording of family history of can- cer in primary care records may be sparse. Although the completeness of cancer staging is improving year by year, we are concerned that incomplete cancer staging limits further exploration of the factors influencing early/late diagnosis of liver cancer. However, we may overcome this limitation by imputing cancer stage, as we have rich clini- cal data, treatments, and survival outcomes linked to the QResearch database, which can be used in the imputa- tion model. Acknowledgements We are grateful that two named patient representatives (both diagnosed with HCC) reviewed our lay summary for ethical approval and provided very helpful feedback. They also commented on the list of potential symptoms and comorbidities of liver cancer that we provided and shared their disease trajectories and personal lived experiences of HCC with us. We also thank the former project manager, Dr. Katja Pfafferott, for coordinating the patient and public involvement (PPI) process and the project setup. We appreciate the constructive comments from the three reviewers for the statistical analysis plan in this project. Clinical implications and research impacth Individuals at the highest risk of developing liver cancer are most likely to benefit from active surveillance using liver ultrasonog- raphy and serum alpha-fetoprotein test. Applying per- sonalised risk prediction models to select individuals at high risk from the population for screening could be a cost-effective approach to improve early diagnosis of liver cancer and patient outcomes, without unduly burdening the overstretched NHS, and avoiding harm to patients at low risk. The implementation of the prediction model will allow the public to calculate individual risks of devel- oping liver cancer for free, like the other QCancer tools, which is a way to engage the public to be more aware of their health status and initiate help-seeking when neces- sary. The UK government and NHS England have com- mitted to improving early diagnosis and cancer outcomes [51]. This project has great potential of making contribu- tions to the national early detection and diagnosis cancer strategic plan, with associated public and societal ben- efits, such as reducing premature death due to liver can- cer, reducing care costs for the NHS, and reducing cancer burden to the society. National Statistics. The Hospital Episode Statistics data used in this study are re-used with permission from NHS Digital, who retains the copyright of the data. None of the acknowledged organisations or funding bodies has been involved in any research process, including study design, data specification, statistical analysis, interpretation of results, preparing manuscripts, or the decision to publish. Study and authors’ information DeLIVER website: www.deliver.cancer.ox.ac.uk. A full list of co-investigators and members of the DeLIVER consortium is in the appended Additional file 1. DeLIVER website: www.deliver.cancer.ox.ac.uk. A full list of co-investigators and members of the DeLIVER consortium is in the appended Additional file 1. Funding The Early Detection of Hepatocellular Liver Cancer (DeLIVER) project is funded by Cancer Research UK (Early Detection Programme Award, grant reference: C30358/A29725). QResearch received funding from the NIHR Biomedi‑ cal Research Centre, Oxford, grants from John Fell Oxford University Press Research Fund, grants from Cancer Research UK (Grant number C5255/ A18085) through the Cancer Research UK Oxford Centre, grants from the Oxford Wellcome Institutional Strategic Support Fund (204826/Z/16/Z). EB acknowledges the support of the NIHR as an NIHR Senior Investigator. The views expressed in this manuscript are those of the authors and not the NIHR or NHS. PCM is supported by a Wellcome Trust intermediate clinical fellow‑ ship (Ref. 110110/Z/15/Z). This research is funded in whole, or in part, by the Wellcome Trust. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any author accepted manuscript version arising from this submission. Ethics approval and consent to participate Additional file 1. Co-investigators and members in the DeLIVER consortium. This study has been approved by the QResearch Scientific Committee on 8 July 2021. QResearch is a Research Ethics Approved Research Database, confirmed by the East Midlands – Derby Research Ethics Committee (research ethics reference: 18/EM/0400, project reference: OX30 DeLIVER). A dedicated webpage for this project has been created on the QResearch website https:// www.qresearch.org/research/approved-research-programs-and-projects/ development-and-validation-of-personalised-risk-prediction-models-for-early- detection-and-diagnosis-of-hepatocellular-carcinoma-hcc-among-the-engli sh-population-from-primary-care/. A lay summary of this project for the public audience is available on the webpage. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s41512-022-00133-x. The online version contains supplementary material available at https://doi. org/10.1186/s41512-022-00133-x. Authors’ contributions EB and JH-C secured the funding. EB is the chief investigator of the DeLIVER project, and JH-C is the package lead and the guarantor of this study. All authors contributed to the study conceptualisation. WL specified the data, led on the ethical approval, and is the lead statistician for the DeLIVER-QResearch project. WL designed the statistical analysis plan, with methodological input from JH-C, C. Coupland, PJ and HI, clinical input from PJ, JH-C, EB and PM, and contextual input from JH-C, PJ, EB, PM, HI and C. Campbell. WL drafted the whole research protocol. All authors read and commented on the earlier drafts, contributed to the revision of the manuscript, and approved the final version of the manuscript for submission. Availability of data and materials Due to the sensitive nature of anonymised patient-level health records and the agreement with the data provider, the data are only accessible to the named researchers of the team, stored and analysed in a data safe haven (the QResearch server). Clinical implications and research impacth There are three key components in the DeLIVER-QRe- search project: reporting the epidemiology of liver cancer Liao et al. Diagnostic and Prognostic Research (2022) 6:21 Page 9 of 11 in England, identifying clinical features (symptoms and comorbidities) to support earlier diagnosis of liver cancer from primary care, and developing prognostic models to identify patients at high risk but without any symptoms yet. We prioritise these research objectives as they could be linked to and contribute to the broader DeLIVER pro- ject. These studies will generate new knowledge that will allow us to gain a deeper understanding of the current situation of liver cancer in England, and inform health policy on early detection, diagnosis, and management of liver cancer at the population level. Individuals at the highest risk of developing liver cancer are most likely to benefit from active surveillance using liver ultrasonog- raphy and serum alpha-fetoprotein test. Applying per- sonalised risk prediction models to select individuals at high risk from the population for screening could be a cost-effective approach to improve early diagnosis of liver cancer and patient outcomes, without unduly burdening the overstretched NHS, and avoiding harm to patients at low risk. The implementation of the prediction model will allow the public to calculate individual risks of devel- oping liver cancer for free, like the other QCancer tools, which is a way to engage the public to be more aware of their health status and initiate help-seeking when neces- sary. The UK government and NHS England have com- mitted to improving early diagnosis and cancer outcomes [51]. This project has great potential of making contribu- tions to the national early detection and diagnosis cancer strategic plan, with associated public and societal ben- efits, such as reducing premature death due to liver can- cer, reducing care costs for the NHS, and reducing cancer burden to the society. in England, identifying clinical features (symptoms and comorbidities) to support earlier diagnosis of liver cancer from primary care, and developing prognostic models to identify patients at high risk but without any symptoms yet. We prioritise these research objectives as they could be linked to and contribute to the broader DeLIVER pro- ject. These studies will generate new knowledge that will allow us to gain a deeper understanding of the current situation of liver cancer in England, and inform health policy on early detection, diagnosis, and management of liver cancer at the population level. References New advances in the diagnosis and management of hepatocellular carcinoma. 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Competing interests This project involves anonymised data from patient-level information col‑ lected by the NHS, as part of the care and support of patients. We acknowl‑ edge the patients and general practices contributing to the EMIS (Egton Medical Information Systems) clinical computer system and the QResearch database and the Universities of Nottingham and Oxford for the expertise in establishing, developing, and supporting the QResearch database. The cancer registration data used in this study are supplied by Public Health England. The mortality registration data are provided by the Office for JH-C is an unpaid director of QResearch, a not-for-profit organisation in a part‑ nership between the University of Oxford and EMIS Health, who supply the QResearch database for this work. JH-C is a founder and shareholder of Clin‑ Risk Ltd. and was its medical director until 31 May 2019. ClinRisk Ltd. produces open and closed source software to implement clinical risk algorithms into clinical computer systems including the original QCancer algorithms referred to above. Other authors have no interests to declare for this submitted work. Page 10 of 11 Liao et al. Diagnostic and Prognostic Research (2022) 6:21 22. Cancer Research UK. Liver cancer incidence. 2021. Available from: https:// www.cancerresearchuk.org/health-professional/cancer-statistics/stati stics-by-cancer-type/liver-cancer/incidence. Received: 26 January 2022 Accepted: 16 August 2022 25. Hippisley-Cox J, et al. Predicting risk of type 2 diabetes in England and Wales: prospective derivation and validation of QDScore. BMJ. 2009;338:b880-. 26. Hippisley-Cox J, Coupland C. Predicting risk of osteoporotic fracture in men and women in England and Wales: prospective derivation and validation of QFractureScores. BMJ. 2009;339:b4229. References 1. Cancer Research UK. Liver cancer statistics. 2021. 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Author details 1fi 1 Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK. 2 Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark. 3 Division of Primary Care, School of Medicine, University of Nottingham, Nottingham, UK. 4 School of Health and Life Sciences, Glasgow Caledonian University, Glasgow, UK. 5 The Francis Crick Institute, London, UK. 6 University College London, London, UK. 7 Nuffield Department of Medicine, University of Oxford, Oxford, UK. 23. Royston P, Altman DG. Regression using fractional polynomials of continuous covariates: parsimonious parametric modelling. Appl Stat. 1994;43(3):429–67. https://doi.org/10.2307/2986270. https://www.jstor. org/stable/2986270?origin=crossref#metadata_info_tab_contents. 24. Royston P, Ambler G, Sauerbrei W. The use of fractional polynomials to model continuous risk variables in epidemiology. Int J Epidemiol. 1999;28(5):964–74. 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https://openalex.org/W2136434605	https://imafungus.biomedcentral.com/counter/pdf/10.5598/imafungus.2013.04.02.16	English	null	MycoBank gearing up for new horizons	IMA fungus	2,013	cc-by	7,139	MycoBank gearing up for new horizons China 3Campus Miguel de Unamuno, Universidad de Salamanca, 37007, Salamanca, Spain 4\| / ` x x@/##@ " ~ ~ #+ Pathumthani 12120, Thailand 4\| / ` x x@/##@ " ~ ~ #+ Pathumthani 12120, Thailand 5 / x " 4 \| "~x6KJJJ" ; ½"?#JPJK" Sweden 5 / x " 4 \| "~x6KJJJ" ; ½"?#JPJK" Sweden / x \| + 4 \| x 4NJ'$N_"; 7Research Laboratory, Center for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School-Westmead Hospital, The University of Sydney, Westmead Millennium Institute and Westmead Hospital, Sydney, Australia 'x ; _ ~ x NJ`PJ#N#~ / x \| + 4 \| x 4NJ'$N_"; 7Research Laboratory, Center for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School-Westmead Hospital, The University of Sydney, Westmead Millennium Institute and Westmead Hospital, Sydney, Australia 'x ; _ ~ x NJ`PJ#N#~ 9 x^ D < < _ / 4 ~ G / 4 N'JPJ" > + " @ \| 4 / + " Kx _> 4 " x 9 x^ D < < _ / 4 ~ G / 4 N'JPJ" > + " @ \| 4 / +"Kx_> 4" x ` " @$ " _ 10Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA Abstract: 4x NJJP 6 6 < < Names. IMA FUNGUS · VOLUME 4 · NO 2: 371–379 IMA FUNGUS · VOLUME 4 · NO 2: 371–379 I#JKK$'L NJ#JPJN# MycoBank gearing up for new horizons Since January 2013, registration of fungal names is a mandatory requirement for valid publication under the International Code of Nomenclature for algae, fungi and plants (ICN). This 6 4x@ for lecto, epi- and neotypes), the multi-lingual database interface, the nomenclature discussion forum, annotation system, and web services with links to third parties. MycoBank has also introduced novel \|; ;4x 6 6 \|; formal system for naming fungi known from DNA sequence data only. To further improve the quality of MycoBank data, remote access will now allow registered mycologists to act as MycoBank curators, / 6 Key words: MycoBank _x+ Forum Fungi International Nucleotide Sequence Database Collaboration \| 6` " Nomenclature Registration Repositories @ Article info:" I NJ#>; I#J NJ#>~ I# NJ#* Article info:" I NJ#>; I#J NJ#>~ I# NJ#* © 2013 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: Non-commercial: No derivative works: For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. MycoBank gearing up for new horizons ARTICLE Vincent Robert1, Duong Vu1, Ammar Ben Hadj Amor1, Nathalie van de Wiele1, Carlo Brouwer1, Bernard Jabas1, Szaniszlo Szoke1, Ahmed Dridi1, Maher Triki1, Samy ben Daoud1, Oussema Chouchen1, Lea Vaas1, Arthur de Cock1, Joost A. Stalpers1, Dora Stalpers1` ¤4D 14 G ` 1, Felipe Borges dos Santos1` " 1, Wei Li2, Linhuan Wu2, Run Zhang2, Juncai Ma2, Miaomiao Zhou1 " ~] G ` 3, Lily Eurwilaichitr4, Supawadee Ingsriswang4, Karen Hansen5, Conrad Schoch, Barbara Robbertse, Laszlo Irinyi7, Wieland Meyer7` / ', David L. Hawksworth9, John W. Taylor10, and Pedro W. Crous Vincent Robert1, Duong Vu1, Ammar Ben Hadj Amor1, Nathalie van de Wiele1, Carlo Brouwer1, Bernard Jabas1, Szaniszlo Szoke1, Ahmed Dridi1, Maher Triki1, Samy ben Daoud1, Oussema Chouchen1, Lea Vaas1, Arthur de Cock1, Joost A. Stalpers1, Dora Stalpers1` ¤4D 14 G ` 1, Felipe Borges dos Santos1` " 1, Wei Li2, Linhuan Wu2, Run Zhang2, Juncai Ma2, Miaomiao Zhou1 " ~] G ` 3, Lily Eurwilaichitr4, Supawadee Ingsriswang4, Karen Hansen5, Conrad Schoch, Barbara Robbertse, Laszlo Irinyi7, Wieland Meyer7` / ', David L. Hawksworth9, John W. Taylor10, and Pedro W. Crous 1/x"?\|;< x / _ 'K'P/@_ @ \| > ? I 2Institute of Microbiology, Chinese Academy of Sciences, NO.1 West Beichen Road, Chaoyang District, Beijing 100101, P. R. INTRODUCTION Registration ? > 6 the MB numbers in the protolog, and upon publication, 4x 6 be released to the community with date, volume and page numbers. (software, databases and website) has been transferred to a professional datacentre where power supply, Internet connections and backups are guaranteed. ARTICLE In order to keep MycoBank users aware of the latest news and improvements related to the database and software, a \| www. Lx4/"\| 6. Since the MycoBank website offers a large number of < ; are now available, providing a number of answers and videos associated with commonly asked questions (these features are available under the Help button on the main menu). So popular was the system with mycologists, that proposals to make the deposit of the key elements mandatory ranks, were prepared (Hawksworth et al. 2010), and debated at the 9th International Mycological Congress in Edinburgh in 2010. These were put before the Nomenclature Section #' x / 4 in July 2011, and incorporated into the International Code of Nomenclature for algae, fungi, and plants (McNeill et al. 2012). Type registration During the nomenclature discussion sessions at the 9th International Mycological Congress in Edinburgh (IMC9), the 6 4x < different authors might easily designate lectotypes, epitypes or neotypes the same name, which would be unfortunate and could lead to the same name being applied in different senses. We strongly support this suggestion, and anticipate mandatory will be made to the Nomenclature Section of the 19th International Botanical Congress in 2017. Queries The new software interface was created in order to improve 8 6 x Lx 6Ú@ £4 ) and advanced queries (www. Lx 6Ú@ £4 Ñ; ) ; 6x queries by combining AND, OR and NOT together with brackets. This makes it possible to ask a question such as Candida species published after 1990 by Kurtzman < @ I@ 6 Candida) AND (Publication date is after 1990) AND (Authors contains Kurtzman) AND NOT (Authors contains Fell). 6 4"?6 4"? 6 also added a bibliographic query system (www.mycobank. Lx 6Ú@ £4 NJ ) as well as a thesaurus of terms commonly used in mycology (www. Lx 6Ú@ £@ ). Although MycoBank was initially set up by CBS- \|; 6< 2009 it was decided that the ownership of the MycoBank system, database and website should be transferred to the International Mycological Association (IMA). In 2010 a new version of the MycoBank website was launched, based on the BioloMICS software (Robert et al. 2011). The advantage of the latter software is that the structure of the database ? users and the curators of MycoBank. The new BioloMICS- based version of MycoBank has been regularly updated and improved since then. In this article, we present the major developments achieved during the past four years, as well as some usage statistics of the MycoBank system. In the last 8 evolving in coming years. INTRODUCTION because they were published in obscure sources. Due to the large number of names published each year in a range of publications, MycoBank curators were not always able to verify and include all of them in the database. To address this issue, we approached a large number of journal editors 6 request authors deposit nomenclatural data, descriptions and illustrations in MycoBank, as good practice. This equates 4x ` x database for genotypic data. Authors would receive a unique 4x NJJP repository with the primary aim to register all fungal 6 combinations), and make this available in an open access database to the mycological community (Crous et al. 2004). 6 that many newly published fungal names were not accessible to researchers in developing countries or simply overlooked, © 2013 International Mycological Association V O L U M E 4 · N O . 2 Robert et al. ` x simultaneously be assured that no homonyms were published, as the search engine would inform authors if the name was already occupied (www.mycobank.org). Registration ? > 6 the MB numbers in the protolog, and upon publication, 4x 6 be released to the community with date, volume and page numbers. ` x simultaneously be assured that no homonyms were published, as the search engine would inform authors if the name was already occupied (www.mycobank.org). INTRODUCTION MycoBank can do much more than complete the basic requirements of the Code, but the only mandatory elements I > > > > 6 #¤ NJ#N + > replaced name for new combinations, names at new ranks, > 6 details of the name-bearing type and the institution or other place in which it is permanently preserved. Name registration @ 6 (Fig. 1). Popup windows are presented to depositors in order 6 6 It is recommended in the Code (McNeill et al. 2012: Rec. 42A.1) that registration numbers are obtained only “after a @ as during review it sometimes becomes necessary to change the chosen names. Infrastructure The latest version of the MycoBank software was released in ; NJ#N according to the natural evolution of their increasing needs and the one of the end-users, without the intervention of any software developers. This is essential when new types of data and the associated analytical tools will be incorporated into the system. In order to ensure a high level of security and availability of the MycoBank website, the whole MycoBank system NJ# 372 I M A F U N G U S I M A F U N G U S MycoBank gearing up for new horizons Fig. 1. Main form for the dep of a new name. Depositors usually entering data relate 6 P average per entry. ARTICLE Fig. 1. Main form for the deposit of a new name. Depositors are usually entering data related to 6 P average per entry. 4x@ sections of papers as follows: system was thus added to MycoBank. Mycologists can now log in to the system, and choose to register a type specimen 6 6 delivers MB numbers). It means that mycologists can now 4x@ 4x @ designation of lectotypes, epitypes, and neotypes. However, 4x can directly indicate during the registration process of a on an epitype, neotype, or holotype specimen. Type. Italy: Padua, on withering leaves of Hedera helix, July #'KL. Ranger (L–lectotype designated here4x@#NPK> Sardegna, Cologne near Oleina, leaf litter of H. helix, 31 Aug. 1970, I. Hulk /x" ?#$$N ? epitype designated here 4x@#NPP 6? /x"$J created and a large number of topics and discussions were initiated (Fig. 3). 6 < allow multiple languages to be displayed, and we contacted ; / < ` ~ " @ 6 (Fig. 2). Additional languages will be added as required by the community. Japanese and Russian will be added in 2014. Annotations and remote curation Like many working databases, MycoBank is incomplete and contains errors and omissions that requires continuous updates by curators. However, it is virtually impossible for the small team of MycoBank curators to sustain such a huge task. The annotation system was therefore created to allow users (after a registration open to anyone) to post comments, suggest corrections or propose new data associated with 6 / or simply leave the comments as pending (Fig. 4). It is not, / 6 Multi-lingual system A major complaint of some users of the earlier version of MycoBank was that it was only available in English thus 373 V O L U M E 4 · N O . 2 Robert et al. Fig. 2. MycoBank is now multi- lingual and offers the possibil- ity to access the information in several major languages. Myco- Bank homepage displayed here in Arabic. ARTICLE Fig. 2. MycoBank is now multi- lingual and offers the possibil- ity to access the information in several major languages. Myco- Bank homepage displayed here in Arabic. Fig. 2. MycoBank is now multi- lingual and offers the possibil- ity to access the information in several major languages. Myco- Bank homepage displayed here in Arabic. created and a large number of topics and discussions were initiated (Fig. 3). Forum Since the Amsterdam declaration on fungal nomenclature (Hawksworth et al. 2011), and the introduction of the new Code (McNeill et al. 2012), mycologists have several new challenges to face reaching consensus with regard to the NJ## et al. 2012). Two years ago, when discussions were initiated, we felt that there was a need to create a discussion 6 name that should be retained. Hence, the Forum option was The same reasons that led us to include an open annotation system, led to a request for help from additional Curators. In April 2014 and to achieve this goal, remote 374 I M A F U N G U S I M A F U N G U S MycoBank gearing up for new horizons Fig. 3. MycoBank Forum. MycoBank gearing up for new horizons ARTICLE Fig. 3. MycoBank Forum. V O L U M E 4 · N O . 2 g y < Fig. 4. The MycoBank annotation system. V O L U M E 4 · N O . 2 < Fig. 4. The MycoBank annotation system. Fig. 4. The MycoBank annotation system. 375 V O L U M E 4 · N O . 2 Robert et al. Table 1. Top 10 countries using MycoBank. Rank Country Percentage of total users 1 USA #K 2 France # 3 ` J* 4 Spain P' 5 Italy 4.2 Brazil ' 7 Russia 3.4 ' India 3.32 9 Canada 3.22 10 China 3.03 Table 1. Top 10 countries using MycoBank. / 6 ; specialists (approved as curators by the MycoBank Advisory Board) to manage sections of the database related to their @ / /x"_ " N; NJ#P further session planned at the International Mycological Congress (IMC10) in Bangkok. ARTICLE Links to third parties Many other websites are rich resources that can be associated with fungal names available in the MycoBank system. Structural links to the following websites have been created: Catalogue of Life (CoL), Encyclopedia of + + ` x < `x< 6 < < @ 6 " @"` " ~4 ` Wikipedia, Wikispecies, BOLD Systems, EMBL, NCBI, All Russian Collection of Microorganisms (VKM), and CBS collection and StrainInfo. More ad hoc links are also available 6 @ 4x ? I#K users are located in the USA, but people from 205 countries have used MycoBank since April 2012. Table 1 lists the top 10 countries using of MycoBank around the world. MycoBank, interested in forum discussions or willing to 6 4x ~ K'J 4x NJJP #$'K NJ#N'J# 6 @ #'L @ KJ NN$ @ #JJ N# @ #JJJ P* J # and 5 new species only. One hundred and seventeen authors published more than 100 new species and during this period. Statistics In total 254,120 unique visitors have visited the English version of MycoBank between April 2012 and 3 December 2013. In December 2012 we launched several language versions of the website, French (3992 unique visitors), Arabic NP / #$K* #J$ ` #'N' Portuguese (2141) and Spanish (2207). Recently, a Thai language version has been introduced. #'N MycoBank portals, while the average visit duration is between #J Web services and central system for registration of fungal names Many users and websites are interested to obtain data in batches and incorporate this in their own databases. Since MycoBank is a public database used by many other repositories, it was important to provide a number of web services that can be consumed by third party machines. We therefore created several dynamic web services that can easily be changed or adapted if needed. wanted reference databases at the same time or separately and results are gathered centrally and proposed as a unique matching list. 6 registration website was established (Fungal Names - FN in China at ILL L L html 6 Fungorum website, which also provides the option for 6< ?< _ www. 6 ). The International Commission on the @ 6</@< \| / for Fungi (NCF) suggested that the three registrars should synchronize their data and MycoBank was asked to create a central web service that would provide unique numbers to the 6 @ was released in June 2013, and IF and FN are currently implementing the needed changes to their system in order to have a fully synchronized system. possible using a combination of morphological, physiological L L 6Ú~ £ ). { MycoBank is not only a repository of data associated with fungal names and vouchers, but also offers unique online www.mycobank. L 6) against curated databases ? www.q-bank.eu), CBS collections websites (www.cbs.knaw.nl, Fusarium, dermatophytes, indoor fungi, Calonectria, Yeasts, etc), Fungal Barcoding (www. fungalbarcoding.org), EUBOLD system (www.eubold.org), ISHAM ITS Database for Human and Animal Pathogenic Fungi (www.mycologylab.org) or UNITE (ILL ). \|/xL` pairwise sequence alignments. Users interested in identifying unknown sequences can compare them against all the The evolution of the number of newly described species between 1759 and 2009 can be seen in Fig. 5. The number 6 6 This is likely due to new technologies allowing mycologists to better distinguish specimens and cultures and therefore separate species, and new techniques permitting them to process and handle larger numbers of specimens. Between 376 I M A F U N G U S MycoBank gearing up for new horizons MycoBank gearing up for new horizons Fig. 5. Decennial evolution of the number of described species between 1759 and 2009. < < < "< < < < < "< < "< < < < < < < < < < "< < < < < < < < < < "< < < < < ARTICLE Fig. 5. Decennial evolution of the number of described species between 1759 and 2009. < < "< < < < < < < < < < "< < < < < < < < < < "< < < < < Fig. 5. Decennial evolution of the number of described species between 1759 and 2009. Fig. 5. Decennial evolution of the number of described species between 1759 and 2009. material and barcoding sequences. { Some projects are dealing with the establishment of reference databases in \|; pathogenic fungi as the basis for the development of novel G 4 D !"/ ; \|!4/ @ " WDCM databases are gathering strain data from culture collections and are providing links to INSDC databases, but their scope is limited to cultivable strains and it is now commonly accepted that most of the diversity is not present in culture collections or museums but is simply unknown (Hawksworth 2001, Kirk et al.NJJ'x NJ##4 et al. 2011). Projects to digitize herbaria will provide additional 6 4 by the US National Science Foundation. There still remain many research collections around the world with useful information of unique strains or specimens, but these are often unavailable to third parties. MycoBank also maintains 6? species descriptions, which is in the process of being linking to INSDC-based sequences, in order to objectify or at least to provide a molecular background to species circumscriptions. Hence, the co-authors of this paper as well as other prominent mycologists and institutions are actively working on this matter and are preparing workshops, guidelines and tools to better to solve this problem is to suggest MycoBank depositors of new species to provide molecular data, in addition to strains or specimens, or links to these data during the registration process. It is common knowledge that the voluntary deposit of additional data is a burden to many researchers, but it must be remembered that it is not mandatory, even though 6 screens. { On the other hand reliable, openly available data from databases and associated websites is a cornerstone of @ 2003 and 2012, the number of newly described species #$NNJJKKP#NJ#NNPNPKJ#$N #''NN#N$#NNPN#KKNP KP# A more detailed analysis of changing patterns over time in the description of new fungal species will be presented elsewhere. ACKNOWLEDGEMENTS @ as already mentioned, this approach is only partly successful. The second one is by incitements such as funding, increased citations and improved visibility facilitated by providing researchers free, useful software and database related tools. The third one is by enforcement and using new rules to be International Code of Nomenclature for algae, fungi, and plants, by journals or reference databases. A combination of the three options 6 based on molecular data linked to accessible strains and specimens, and not only on phenotypic criteria. ARTICLE @ 4x 6 such, the authors would like to thank them for their contribution. Paul 4 6< 4x is thankfully acknowledged for his huge contribution. The MycoBank software developments were partially made within 6 + P+ e-Infrastructure project co-funded by the European Commission’s Seventh Framework Program for Research and Technological Development. Some data entries were made possible thanks to the EMbaRC project also supported by the European Commission’s Seventh Framework Program for Research and Technological Development Linking species data via molecular data using strains and specimens is important, but will not solve all problems or opportunities induced by the usage of modern technologies. \| 6 ` " \|`" throughput screening technologies already allow us to obtain large datasets that would not be accessible using traditional sampling, isolation and collecting methods. New species are traditionally based on the isolation of one, or ideally several specimens that are studied and deposited in reference \|`" sequences from a single soil sample in a few hours and get 6 is also possible and relatively easy to monitor the changes in ecosystems or hosts over time. ACKNOWLEDGEMENTS The known diversity constitutes only a small fraction of the real fungal biodiversity (Hawksworth 2001, Kirk et al. NJJ' x NJ## 4 et al.NJ##` 6 6 a near complete idea of the scope of microbial diversity. @ \|`" 6 Currently, there are no mechanisms allowing researchers 6 such new technologies, other than the recently proposed system of UNITE (Kõljalg et al. 20131). Although there are a number of issues associated with these new technologies in terms of data quality, reproducibility and quantity, there is 4x collaboration with INSDC, UNITE and other DNA Barcoding and tools to record non-specimen based descriptions for candidates species (Taylor 2011). We are currently working on tools for the semi-automated curation of large datasets, for fast and accurate assignments to species or candidate ` analyzed, new technologies need to be developed. This can only be accomplished through the collaboration of several 6 6 molecular researchers, bioinformaticians, informaticians, mathematicians, database specialists to technologists focused in molecular or information technologies and /~_`~_ <~`; Parts of the developments were done in the framework of the \|/x < " <" 4 Culture and Science (OCW). ACKNOWLEDGEMENTS Parts of the developments were also done in the framework of the “DNA barcoding of pathogenic fungi as the basis for the G ; \|!4/ grant #APP1031952 to WM and VR. CLS acknowledges support from the Intramural Research Program of the NIH, National Library of Medicine. CLS acknowledges support from the Intramural Research Program of the NIH, National Library of Medicine. CLS acknowledges support from the Intramural Research Program of the NIH, National Library of Medicine. Future 4x names now required by the Code. While it is increasingly becoming a rich source of knowledge at species, genus, family, and higher levels, the databases of the International Nucleotide Sequence Database Collaboration (INSDC), a consortium consisting of NCBI, EMBL and DDBJ, serves as the international repository for molecular sequences. The task of linking MycoBank entries based on reference material (specimens and strains) to INSDC sequences, often only known from environmental sequences, is a real challenge. 6 many species described and circumscribed on the basis of non-molecular criteria (morphology, physiology, ecology, etc.). Voucher data annotated consistently in all databases will possibly remains the most effective way to link species names and their associated molecular data. The Darwin / ILL LL proposed and is one way to standardize the formulation of such data. The links between species (and subsequently to 6 via strains and specimens. The biological repositories of fungal voucher data, culture collections and herbaria (listed 6 reference material, information and strains (Durães Sette et al. 2013). Other initiatives such as the barcoding of life (BOLD systems, EUBOLD, China BOLD, etc.) or the UNITE database are providing useful links between reference 377 V O L U M E 4 · N O . 2 Robert et al. 4¤ x }" x x` ¤} Lombard L, Crous PW (2012) One fungus, one name promotes progressive plant pathology. Molecular Plant Pathology 13IJP # International Code of Nomenclature for algae, fungi, and plants (Melbourne Code) adopted by the Eighteenth International Botanical Congress Melbourne, Australia, July 2011. [Regnum D \|#KP¹¼ I G" x REFERENCES Blackwell M (2011) The Fungi: 1, 2, 3… 5.1 million species? American Journal of Botany 98IPNP' / ~ ` " ¤; D " ` NJJP MycoBank: an online initiative to launch mycology into the 21st century. Studies in Mycology 50: 19–22. Durães Sette L, Pagnocca FC, Rodrigues A (2013) Microbial culture collections as pillars for promoting fungal diversity, conservation 6 Fungal Genetics and Biology 60IN' 6 Fungal Genetics and Biology 60IN' Hawksworth DL (2001) The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycological Research 105: 1422–1432. Hawksworth DL (2011) A new dawn for the naming of fungi: impacts of decisions made in Melbourne in July 2011 on the future publication and regulation of fungal names. MycoKeys 1: 7–20. IMA Fungus 2I#KK#N Hawksworth DL, Cooper JA, Crous PW, Hyde KD, Iturriaga T, et al. (2010) Proposals to make the pre-publication deposit of key nomenclatural information in a recognized repository a requirement for valid publication of organisms treated as fungi under the Code. Taxon 59IJN>Mycotaxon 111: 514–519. Hawksworth DL, Crous PW, Redhead SA, Reynolds DR, Samson RA, " ;@ ¤ 4¤et al. (2011) The Amsterdam Declaration on Fungal Nomenclature. IMA Fungus 2: 105–112. ~4/ ~<4 " ¤;NJJ' Ainsworth & Bisby's Dictionary of the Fungi, 10th edn. Wallingford, CABI Publishing. Kõljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS [and 37 ¹NJ#@ ? Kõljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS [and 37 ¹NJ#*@ ? Molecular Ecology 22: 5271–5277. Molecular Ecology 22: 5271–5277. 4\| ¤x <x D` DL, Herendeen PS, Knapp S, Marhold K, Prado J, Prud’homme <"` ¤@ \|¤ NJ#N 1See IMA Fungus 4: (33), 2013. 378 I M A F U N G U S MycoBank gearing up for new horizons Taylor JW (2011) One Fungus = One Name: DNA and fungal nomenclature twenty years after PCR. IMA Fungus 2: 113–120. REFERENCES Taylor JW (2011) One Fungus = One Name: DNA and fungal nomenclature twenty years after PCR. IMA Fungus 2: 113–120. International Code of Nomenclature for algae, fungi, and plants (Melbourne Code) adopted by the Eighteenth International Botanical Congress Melbourne, Australia, July 2011. [Regnum D \|#KP¹¼ I G" x ARTICLE 4 /@ ~;"";` xNJ## species are there on Earth and in the ocean? PLoS Biology 9(8): e1001127. Robert V, Szoke S, Jabas J, Vu D, Chouchen O, Blom E, Cardinali ` NJ## x4/" x 4 / " The Open Applied Informatics Journal 5I'$' 379 V O L U M E 4 · N O . 2
https://openalex.org/W2034459215	https://opus.bibliothek.uni-augsburg.de/opus4/files/59335/journal.pone.0071486.PDF	English	null	Cognitive Problem Solving Patterns of Medical Students Correlate with Success in Diagnostic Case Solutions	PloS one	2,013	cc-by	8,133	Abstract The fu design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: Funds for this project were provided by Dr. med. Hildegard Hampp Trust administered by LMU Munich, Germany. T design, data collection and analysis, decision to publish, or preparation of the manuscript. project were provided by Dr. med. Hildegard Hampp Trust administered by LMU Munich, Germany. The funders had no role in study nd analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jan.kiesewetter@med.lmu.de Jan Kiesewetter1*, Rene´ Ebersbach1, Anja Go¨ rlitz1, Matthias Holzer1, Martin R. Fischer1, Ralf Schmidmaier2 1 Lehrstuhl fu¨r Didaktik und Ausbildungsforschung in der Medizin am Klinikum der Universita¨t Mu¨nchen, Ludwig-Maximilians-University, Munich, Germany, 2 Medizinische Klinik und Poliklinik IV, Klinikum der Universita¨t Mu¨nchen, Ludwig-Maximilians-University, Munich, Germany Abstract Context: Problem-solving in terms of clinical reasoning is regarded as a key competence of medical doctors. Little is known about the general cognitive actions underlying the strategies of problem-solving among medical students. In this study, a theory-based model was used and adapted in order to investigate the cognitive actions in which medical students are engaged when dealing with a case and how patterns of these actions are related to the correct solution. Methods: Twenty-three medical students worked on three cases on clinical nephrology using the think-aloud method. The transcribed recordings were coded using a theory-based model consisting of eight different cognitive actions. The coded data was analysed using time sequences in a graphical representation software. Furthermore the relationship between the coded data and accuracy of diagnosis was investigated with inferential statistical methods. Results: The observation of all main actions in a case elaboration, including evaluation, representation and integration, was considered a complete model and was found in the majority of cases (56%). This pattern significantly related to the accuracy of the case solution (w = 0.55; p,.001). Extent of prior knowledge was neither related to the complete model nor to the correct solution. Conclusions: The proposed model is suitable to empirically verify the cognitive actions of problem-solving of medical students. The cognitive actions evaluation, representation and integration are crucial for the complete model and therefore for the accuracy of the solution. The educational implication which may be drawn from this study is to foster students reasoning by focusing on higher level reasoning. Citation: Kiesewetter J, Ebersbach R, Go¨rlitz A, Holzer M, Fischer MR, et al. (2013) Cognitive Problem Solving Patterns of Medical Students Correlate with Success in Diagnostic Case Solutions. PLoS ONE 8(8): e71486. doi:10.1371/journal.pone.0071486 Editor: Alessandro Antonietti, Catholic University of Sacro Cuore, Italy Received February 13, 2013; Accepted June 28, 2013; Published August 12, 2013 ewetter 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. Copyright: 2013 Kiesewetter et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funds for this project were provided by Dr. med. Hildegard Hampp Trust administered by LMU Munich, Germany. Participants When confronted with a problem, humans tend to take the same cognitive actions regardless of the content of the problem [17]. Cognitive actions could be defined as follows: the retrieval of the problem, the processing of the information, a formulation of the plan to tackle the task, carrying out the plan and an evaluation of the results. These cognitive actions have been thoroughly researched and are found in abundance known as action theoretic approaches in cognitive psychology [17,18], mathematics [19], pedagogy [20], in medicine [21] and many other fields [22]. A medical problem-solving process including the underlying cogni- tive actions could be exemplified as follows: When a patient sees a doctor, the doctor recognizes or finds out about the symptoms of the patient (i.e. she complains about red urine), analyses these symptoms and generates differential diagnostic ideas (i.e. urinary tract infection). In order to get more information the physician asks further questions and performs further investigations (i.e. by examining the patient and carrying out a urine sample and a blood test). When presenting the patient to another physician, the doctor would summarize what he or she has learned so far from an inner representation of the patient (i.e. 57 year old female patient, hematuria since three days, no signs of an infection). This inner representation includes positive and negative findings and might as well contain differential diagnostic ideas (i.e. malignant tumour or glomerulonephritis). After an evaluation of the differential diagnoses, decisions about further steps would be reached and communicated to the patient. All models include the above mentioned cognitive actions with varying emphasis [17]. These cognitive actions serve as the foundation of the strategies of problem-solving within a field including medicine. A more adaptable and faster learning of clinical reasoning founding on the empirical verification of cognitive actions has been stipulated very recently [23,24]. The model using typified objects (MOT- model) comprehensively describes cognitive clinical reasoning process as suggested by experts. On the top-level of this hierarchically built model the experts agreed on the following processes: Identify early cues, determine the objectives of the encounter, categorize for the purpose of action, implement purposeful action and evaluate the results. All processes are interlinked and receive specific inputs and produce certain outputs thus representing the dynamic nature of the problem-solving process of experts. However, cognitive actions were not examined empirically among medical students. Operationalization of the Model It has been criticized that action theoretic models might be useful for instructional purposes, but are not suitable to describe the real-life problem-solving processes [22]. To conduct empirical research, an analysis model was needed to concretize the task, most likely applicable to medical students and detailed enough not to miss fundamental cognitive actions. After a thorough literature review and comprehensive expert discussions the empirically tested model from Schoenfeld [25] was chosen as a starting point as it represents the widely used action theoretic models, with the following cognitive actions: read, analyse, explore, plan, implement and verify. Schoenfeld’s model was especially formulated for simple problem-solving dealing with a single problem, but not for complex problems [25]. Problems can be considered as complex where diverse and volatile goals have to be considered [17]. Medical problem-solving is complex problem-solving [17]. Thus, more cognitive actions needed to be defined to gain a compre- hensive view. Therefore, the original Schoenfeld model was modified in the following way. The doctor needs an inner representation to cope with the complexity of the problems, the development of which is another cognitive action within the analysis model. With this inner representation of the problems, the doctor evaluates the different actions taken and integrates the results to finally come to a solution. This decision for a working diagnosis or for the final solution is another cognitive action in the analysis model. The here presented ‘‘modified Schoenfeld model for complex problem-solving’’ (further referred to as ‘‘modified Schoenfeld model’’) consists of eight selective cognitive actions, dealing with the problems given: Denomination, Analysis, Exploration, Plan, Implementation, Evaluation, Representation, Integration (see table 1). This ‘‘modified Schoenfeld model’’ was used for the case sessions of a pilot study. The detailed subactions and contents of each cognitive action were observed, summarized and defined using qualitative research methods (qualitative content analysis, induc- tive category development, open coding process [26]). After several test codings, a fixed coding scheme was defined and applied to the whole sample of cases. The aim of this study was to empirically examine how medical students think clinically with the following objectives: (1) can the process of clinical problem-solving be described using the proposed cognitive actions; (2) can a specific pattern in case-based problem-solving be extracted using the relation of the proposed cognitive actions to each other; (3) is this pattern correlated with the diagnostic accuracy? Course of the Study Figure 1 shows that the study consisted of a controlled knowledge training, a subsequent knowledge test, and the paper- based clinical case-scenarios. Participants solved three cases in clinical nephrology with the think-aloud-method after three hours practising a standardized learning unit in the field of clinical nephrology. Recordings were transcribed and coded according to the ‘‘modified Schoenfeld model’’. Codings were analysed for Introduction has identified a spectrum of four consecutive strategies for problem-solving in medicine: guessing, hypothetical-deductive reasoning, scheme induction and pattern recognition [6]. With increasing knowledge and experience, medical students derive hypotheses from the patient’s information and try to verify them purposefully. These strategies of generating and testing of hypotheses have successfully been observed empirically[7–9] and described in detail[6,10–12]. In the last decade there has been a tendency towards case-based learning as an instructional approach for students to learn medical problem-solving [13,14]. To foster the development of expertise early in medical careers learning from authentic patient cases has been stipulated [15]. The key to successful learning of medical students seems to lie in the consequent process character of the cases [16]. Despite this empirical basis it remains hard to assess the verification if, when and how to foster medical students’ problem-solving skills. Even more, there is currently no established model in medical education to accurately describe the cognitive process of clinical problem solving. In order to The physician’s profession demands a number of competen- cies. One of these is the ability to reason clinically. Clinical reasoning focuses on the signs and symptoms of a patient and the subsequent identification of relevant questions on the patients history, further the physical examination, the correct interpretation of those results and information, as well as procedures required to reach the correct diagnosis in an efficient manner [1]. The actual reasoning process involves medical decision-making on the one hand and problem-solving on the other hand [2]. This study focuses on medical problem- solving. There is a broad base of knowledge on expertise of physicians and their decision-making (cf. [3]), but only little is known about cognitive actions of medical students. This lack of knowledge exacerbates attempts of medical educators to foster problem-solving adapted to their students’ needs. This study focuses therefore only on medical students. Prior knowledge is essential for successful problem-solving as shown by various studies regarding ‘‘content specificity’’ [4,5]. Previous research August 2013 \| Volume 8 \| Issue 8 \| e71486 August 2013 \| Volume 8 \| Issue 8 \| e71486 1 PLOS ONE \| www.plosone.org Cognitive Problem Solving Patterns subcomponents of thinking. Paper-based cases with basic patient information and further on test-results were given to the subjects. educate with a resource-oriented instructional approach it is a prerequisite to first investigate the actual process of medical student’s problem-solving. Participants This is especially surprising as the development of medical students’ problem-solving skills could be fostered using knowledge about an optimum relation of cognitive actions. Furthermore, so far there is no evidence available that using certain cognitive action models predict successful case solutions. Twenty-three medical students in their 4th or 5th year (female = 11) of two medical faculties volunteered (M = 23.9 years; range 20–34) to take part in the study. These years of the medical curriculum were chosen because the participants should have enough prior knowledge to solve clinical problems, but should not have experienced their final 6th clinical year of full time electives to focus on the the problem-solving of the student. Furthermore these participants had finished their internal medicine curriculum. Written informed consent was obtained from all participants. This study was approved by the Ethical Committee of the Medical Faculty of LMU Munich. Participants received a small monetary compensation for their expenses. Methods Operationalization of the Research Questions PLOS ONE \| www.plosone.org Operationalization of the Research Questions The stated research questions were investigated in a laboratory setting with a controlled set of clinical content. A think-aloud method was used to be able to identify patterns and certain August 2013 \| Volume 8 \| Issue 8 \| e71486 August 2013 \| Volume 8 \| Issue 8 \| e71486 PLOS ONE \| www.plosone.org 2 Cognitive Problem Solving Patterns Table 1. Illustration and operationalized definition of the ‘‘modified Schoenfeld model for complex problem solving’’. Cognitive Action Operationalized definition Denomination Retrieve information; read Analysis Analyse information; generate differential diagnostic ideas Exploration Associate, compare, vaguely propose strategies how to understand the problem Plan Generate plans, weigh up these plans against each other, decide on a plan Implementation State and justify one definite plan; request certain additional information and/or examinations Evaluation Verify or dismiss hypotheses with regard to new information or examination results; evaluative thinking Representation Inner representation of the case; statement of the situation as far as it is summarized in the mind of the student Integration Decision for one working diagnosis, differential diagnoses and/or therapy doi:10.1371/journal.pone.0071486.t001 Table 1. Illustration and operationalized definition of the ‘‘modified Schoenfeld model for complex problem solving’’. Cognitive Action Operationalized definition Denomination Retrieve information; read Analysis Analyse information; generate differential diagnostic ideas Exploration Associate, compare, vaguely propose strategies how to understand the problem Plan Generate plans, weigh up these plans against each other, decide on a plan Implementation State and justify one definite plan; request certain additional information and/or examinations Evaluation Verify or dismiss hypotheses with regard to new information or examination results; evaluative thinking Representation Inner representation of the case; statement of the situation as far as it is summarized in the mind of the student Integration Decision for one working diagnosis, differential diagnoses and/or therapy doi:10.1371/journal.pone.0071486.t001 Table 1. Illustration and operationalized definition of the ‘‘modified Schoenfeld model for complex problem solving’’. Table 1. Illustration and operationalized definition of the ‘‘modified Schoenfeld model for complex problem solving’’. doi:10.1371/journal.pone.0071486.t001 completion, the students’ retention of content specific medical knowledge was tested [13,16,28]. accuracy of the diagnosis. Learner characteristics were obtained by questionnaires. g [ ] Clinical case scenarios. The three paper-based case scenarios with diagnoses within the field of clinical nephrology were real cases of the department of internal medicine adapted from experts with anonymized real supplemental material (i.e. lab values). Operationalization of the Research Questions After the transformation into paper-based scenarios, authenticity was additionally ensured through review by two content experts and one didactic expert. All cases were structured the same way, containing two or three pages describing the patients complaints and medical history. The results of the physical examination, blood tests, urine sample, ECG and ultrasound scan were each described on separate pages. The first case described a patient with hematuria due to glomerulonephritis. The second case concerned a patient with both the symptoms of acute renal failure as well as depression. The third case was on a patient with hypertensive crisis due to renal arterial stenosis. Students were not allowed to use secondary aids such as books or computers. Pre-study questionnaire. All participants filled out a questionnaire containing items about their socio-demographic data, gender and age as possible confounders. The reliability of this multiple-choice exam is very high (Cronbachs a = .957) [27]. The performance of participants in this exam was used as an indicator for general prior knowledge in medicine. The results of the questionnaire and all other obtained data were anonymized. Knowledge training and test. Although all participants were in the advanced part of medical school and had all passed the internal medicine curriculum a pre-learning phase was established. The pre-learning phase involved an extensive 3-hour computer- based tutorial on clinical nephrology to account for content specificity [4]. This was to help ensure that all students were able to show their problem-solving strategy and ability because they had the knowledge needed for application of strategies. Upon In a short practice exercise participants were instructed on the think-aloud method [29]. The students’ task was to work on each case to show their problem-solving abilities with no other instructions being given than ‘‘please work on this case’’. They were not explicitly asked to state a diagnosis. Only one single student and the test instructor were present in the room during the case elaboration. The test instructor sat behind the participant to avoid any diversion of thought [29]. The only interaction between the participant and instructor was when the instructor provided the next page of a case. Every case was interrupted after ten minutes, independent of whether the case was solved or not. While participants were working on the cases using the think-aloud method, they were audiorecorded. Figure 1. Overview on the course of the study. doi:10.1371/journal.pone.0071486.g001 PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e71486 action Denomi-nation Analysis Exploration Plan Implemen-tation Evalu-ation Represen- tation Integra-tion All cases ve %) 360.5 min (36.76%) 281.5 min (28.71%) 40.4 min (4.12%) 53.8 min (5.49%) 75.4 min 7.69%) 85.5 min (8.76%) 45.5 min (4.65%) 37.3 min (3.81%) 980.4 (100%) of elative %) 474 (32.09%) 330 (22.34%) 62 (4.2%) 77 (5.21%) 237 (16.04%) 159 (10.76%) 79 (5.35%) 59 (3.99%) 1.477 (100%) The cognitive actions Evaluation, Representation and Integration were not present at the beginning and emerged during the case elaboration in this order. This pattern evolved for each of the three cases in a similar way (compare Fig. 2a–c). Elucidation of a ‘‘Complete Model Pattern’’ In most individual case elaborations, two or three cognitive actions took place at the same time. Mostly this was Analysing or Evaluating while Denominating (44% of coded categories). To identify patterns in the case elaborations, the time-line graphs of the single cases were analysed. The analysis revealed a typical reproduced sequence how the participants traversed through the cognitive actions: they mostly started with Denomination, progressed through Analysis (or sometimes Exploration) to Implementation (or more rarely Plan). The obtained new information, due to the requests of the cognitive action Implementation, are then read and denominated, and another loop starts from the beginning of this sequence again. We Table 2. Time-on-task: Distribution of the cognitive actions (all cases, n = 66). Cognitive action Denomi-nation Analysis Exploration Plan Implemen-tation Evalu-ation Represen- tation Integra-tion All cases time (relative %) 360.5 min (36.76%) 281.5 min (28.71%) 40.4 min (4.12%) 53.8 min (5.49%) 75.4 min 7.69%) 85.5 min (8.76%) 45.5 min (4.65%) 37.3 min (3.81%) 980.4 (100%) Frequency of episodes (relative %) 474 (32.09%) 330 (22.34%) 62 (4.2%) 77 (5.21%) 237 (16.04%) 159 (10.76%) 79 (5.35%) 59 (3.99%) 1.477 (100%) at the same time. Subsequently, the codings were marked as time- sections in the transcription software ‘‘f4’’ (f4 2011, Dr. T. Dresing, http://www.audiotranskription.de) and then exported to Microsoft Excel 2010 (Microsoft, 2010). For further analysis the statistical environment ‘‘R’’ was used (http://www.r-project.org/). A predefined alpha level set at p,0.05 was used for all tests of significance. Graphical illustrations were processed as the percentage of time spent on one action relative to the overall time. Although the cognitive actions of the model were described qualitatively, this was the basis for a quantitative analysis and graphical illustration of the results. As quantitative dependent variables the frequencies of cognitive actions were analysed, as well as the length of the episodes. The accuracy of diagnosis was established in a binary form (correct or not correct) as a dependent variable. Chi-squared tests were used to verify the relationship of dependent variables to all dichotomous participant variables, while Pearson correlation was used for all continuous dependent variables to correlate them to previously obtained participant data. action Denomi-nation Analysis Exploration Plan Implemen-tation Evalu-ation Represen- tation Integra-tion All cases ve %) 360.5 min (36.76%) 281.5 min (28.71%) 40.4 min (4.12%) 53.8 min (5.49%) 75.4 min 7.69%) 85.5 min (8.76%) 45.5 min (4.65%) 37.3 min (3.81%) 980.4 (100%) of elative %) 474 (32.09%) 330 (22.34%) 62 (4.2%) 77 (5.21%) 237 (16.04%) 159 (10.76%) 79 (5.35%) 59 (3.99%) 1.477 (100%) Chi-squared tests were processed in SPSS 20.0 with a predefined alpha level set at p,0.05. One investigator (R. E.) coded all transcripts. A second rater coded more than 10% of the transcripts. Based on the coded time, the interrater coefficient analysed with Cohens kappa was k = .935. Based on the coded text, the interrater coefficient was k = .884. Results The ‘‘modified Schoenfeld model for complex problem-solving’’ in medicine enables us to describe the cognitive actions of medical students. The times-on-task participants spent overall on each of the eight cognitive actions are shown in table 1. Most time was spent on the cognitive actions Denomination and Analysis. The frequencies of the episodes overall showed a similar distribution with minor distinctions. Action Denomination and Analysis have mainly long episodes (MDenomination = 45sec 61.74, MAnalysis = 51- sec 62.34). Action Implementation often consists of short episodes (MImplementation = 19.10sec 61.11), so the percentage in terms of frequencies is higher than the percentage in terms of session-time (as illustrated in table 2). Figure 2 shows how the cognitive actions were distributed over time. All elaborations are presented separately for each of the three cases (Fig. 2a–c) and aggregated for all three cases (Fig. 2d). The case elaborations of all participants were mapped onto each other. As the figure shows, Denomination and Analysis were spread over the entire case elaboration, equally Plan and Implementation. The cognitive actions Evaluation, Representation and Integration were not present at the beginning and emerged during the case elaboration in this order. This pattern evolved for each of the three cases in a similar way (compare Fig. 2a–c). August 2013 \| Volume 8 \| Issue 8 \| e71486 Data Analysis All audio recordings (total time of 13:05 hours) were transcribed and coded using the model described above. For technical reasons, three tapes were not completely evaluable and 66 of 69 cases were analyzed. The standard qualitative content analysis by Mayring [26] was used as method to assess, code and analyse the process of thought, as it also yields very detailed quantitative data in consecutive analysis. It uses models with several categories for the coding of a text. In this study, the cognitive actions were used as categories. A section of text matching a particular cognitive action was determined as an episode. One text section could be coded as more than one episode, when different cognitive actions took place Figure 1. Overview on the course of the study. doi:10.1371/journal.pone.0071486.g001 August 2013 \| Volume 8 \| Issue 8 \| e71486 August 2013 \| Volume 8 \| Issue 8 \| e71486 PLOS ONE \| www.plosone.org 3 Cognitive Problem Solving Patterns ly, the codings were marked as time- n software ‘‘f4’’ (f4 2011, Dr. T. anskription.de) and then exported to soft, 2010). For further analysis the was used (http://www.r-project.org/). at p,0.05 was used for all tests of strations were processed as the n one action relative to the overall actions of the model were described asis for a quantitative analysis and esults. variables the frequencies of cognitive l as the length of the episodes. s was established in a binary form ependent variable. Chi-squared tests onship of dependent variables to all ables, while Pearson correlation was ndent variables to correlate them to pant data. Chi-squared tests were th a predefined alpha level set at oded all transcripts. A second rater transcripts. Based on the coded time, nalysed with Cohens kappa was d text, the interrater coefficient was model for complex problem-solving’’ cribe the cognitive actions of medical participants spent overall on each of re shown in table 1. Most time was ons Denomination and Analysis. The overall showed a similar distribution tion Denomination and Analysis have mination = 45sec 61.74, MAnalysis = 51- ation often consists of short episodes 1.11), so the percentage in terms of e percentage in terms of session-time gnitive actions were distributed over resented separately for each of the gregated for all three cases (Fig. 2d). participants were mapped onto each Denomination and Analysis were spread ion, equally Plan and Implementation. Data Analysis ion, Representation and Integration were ng and emerged during the case pattern evolved for each of the three are Fig. 2a–c). te Model Pattern’’ laborations, two or three cognitive me time. Mostly this was Analysing or 44% of coded categories). To identify ons, the time-line graphs of the single alysis revealed a typical reproduced nts traversed through the cognitive with Denomination, progressed through tion) to Implementation (or more rarely ormation, due to the requests of the n, are then read and denominated, and beginning of this sequence again. We Table 2. Time-on-task: Distribution of the cognitive actions (all cases, n = 66). Cognitive action Denomi-nation Analysis Exploration Plan Implemen-tation Evalu-ation Represen- tation Integra-tion All cases time (relative %) 360.5 min (36.76%) 281.5 min (28.71%) 40.4 min (4.12%) 53.8 min (5.49%) 75.4 min 7.69%) 85.5 min (8.76%) 45.5 min (4.65%) 37.3 min (3.81%) 980.4 (100%) Frequency of episodes (relative %) 474 (32.09%) 330 (22.34%) 62 (4.2%) 77 (5.21%) 237 (16.04%) 159 (10.76%) 79 (5.35%) 59 (3.99%) 1.477 (100%) action Denomi-nation Analysis Exploration Plan Implemen-tation Evalu-ation Represen- tation Integra-tion All cases ve %) 360.5 min (36.76%) 281.5 min (28.71%) 40.4 min (4.12%) 53.8 min (5.49%) 75.4 min 7.69%) 85.5 min (8.76%) 45.5 min (4.65%) 37.3 min (3.81%) 980.4 (100%) of elative %) 474 (32.09%) 330 (22.34%) 62 (4.2%) 77 (5.21%) 237 (16.04%) 159 (10.76%) 79 (5.35%) 59 (3.99%) 1.477 (100%) at the same time. Subsequently, the codings were marked as time- sections in the transcription software ‘‘f4’’ (f4 2011, Dr. T. Dresing, http://www.audiotranskription.de) and then exported to Microsoft Excel 2010 (Microsoft, 2010). For further analysis the statistical environment ‘‘R’’ was used (http://www.r-project.org/). A predefined alpha level set at p,0.05 was used for all tests of significance. Graphical illustrations were processed as the percentage of time spent on one action relative to the overall time. Although the cognitive actions of the model were described qualitatively, this was the basis for a quantitative analysis and graphical illustration of the results. As quantitative dependent variables the frequencies of cognitive actions were analysed, as well as the length of the episodes. The accuracy of diagnosis was established in a binary form (correct or not correct) as a dependent variable. Chi-squared tests were used to verify the relationship of dependent variables to all dichotomous participant variables, while Pearson correlation was used for all continuous dependent variables to correlate them to previously obtained participant data. Chi-squared tests were processed in SPSS 20.0 with a predefined alpha level set at p,0.05. One investigator (R. E.) coded all transcripts. A second rater coded more than 10% of the transcripts. Based on the coded time, the interrater coefficient analysed with Cohens kappa was k = .935. Based on the coded text, the interrater coefficient was k = .884. Results The ‘‘modified Schoenfeld model for complex problem-solving’’ in medicine enables us to describe the cognitive actions of medical students. The times-on-task participants spent overall on each of the eight cognitive actions are shown in table 1. Most time was spent on the cognitive actions Denomination and Analysis. The frequencies of the episodes overall showed a similar distribution with minor distinctions. Action Denomination and Analysis have mainly long episodes (MDenomination = 45sec 61.74, MAnalysis = 51- sec 62.34). Action Implementation often consists of short episodes (MImplementation = 19.10sec 61.11), so the percentage in terms of frequencies is higher than the percentage in terms of session-time (as illustrated in table 2). Figure 2 shows how the cognitive actions were distributed over time. All elaborations are presented separately for each of the three cases (Fig. 2a–c) and aggregated for all three cases (Fig. 2d). The case elaborations of all participants were mapped onto each other. As the figure shows, Denomination and Analysis were spread over the entire case elaboration, equally Plan and Implementation. Elucidation of a ‘‘Complete Model Pattern’’ In most individual case elaborations, two or three cognitive actions took place at the same time. Mostly this was Analysing or Evaluating while Denominating (44% of coded categories). To identify patterns in the case elaborations, the time-line graphs of the single cases were analysed. The analysis revealed a typical reproduced sequence how the participants traversed through the cognitive actions: they mostly started with Denomination, progressed through Analysis (or sometimes Exploration) to Implementation (or more rarely Plan). The obtained new information, due to the requests of the cognitive action Implementation, are then read and denominated, and another loop starts from the beginning of this sequence again. We PLOS ONE \| www.plosone.org 4 Cognitive Problem Solving Patterns Figure 2. Time-line graphs of all participants of each clinical case, and time-line graph of all clinical cases (Figure 2 a to d from upper left to lower right corner). It shows the distribution of cognitive actions over time. The darker the blue is presented, the more case elaborations are containing this action at this part of the process. doi:10.1371/journal.pone.0071486.g002 Figure 2. Time-line graphs of all participants of each clinical case, and time-line graph of all clinical cases (Figure 2 a to d from upper left to lower right corner). It shows the distribution of cognitive actions over time. The darker the blue is presented, the more case elaborations are containing this action at this part of the process. doi:10.1371/journal.pone.0071486.g002 keyed this sequence, which was found in every case elaboration, a ‘‘lower loop’’ (Mloop = 3.18 loops/case 61.46). The most widely used sequence of cognitive actions in the lower loops was Denomination, Analysis, Implementation, Denomination (116 of 210 loops; 55%). The actions Evaluation, Representation and Integration did also show a typical sequence in more than half of the case elaborations (37/66; 56%). This sequence was called ‘‘higher loop’’. The sequence began with Evaluation and optionally Representation, followed or closed by Integration. As only explicitly stated representations were coded, Representation was considered to be optional. When the case elaboration included both, the lower loops as well as higher loops of the actions Evaluation, Representation and Integration these case elaborations were labelled a ‘‘complete model’’ (37/66; 56%). If the actions Evaluation, Representation and Integration were in another order or only single actions were coded, the case elaboration was labelled ‘‘incomplete’’ (29/66; 44%). August 2013 \| Volume 8 \| Issue 8 \| e71486 Elucidation of a ‘‘Complete Model Pattern’’ The complete model was equally distributed over the three given paper- based cases, with a lower frequency in the third case (first case: 14/ keyed this sequence, which was found in every case elaboration, a ‘‘lower loop’’ (Mloop = 3.18 loops/case 61.46). The most widely used sequence of cognitive actions in the lower loops was Denomination, Analysis, Implementation, Denomination (116 of 210 loops; 55%). The actions Evaluation, Representation and Integration did also show a typical sequence in more than half of the case elaborations (37/66; 56%). This sequence was called ‘‘higher loop’’. The sequence began with Evaluation and optionally Representation, followed or closed by Integration. As only explicitly stated August 2013 \| Volume 8 \| Issue 8 \| e71486 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Cognitive Problem Solving Patterns Figure 3. Time-line graphs of case elaborations with incomplete (3a) and complete model (3b). When the case elaboration included also the higher loops of the actions Evaluation, Representation and Integration these case elaborations were labelled a ‘‘complete model’’. If the actions Evaluation, Representation and Integration were in another order or only single actions were coded, the case elaboration was labelled ‘‘incomplete’’. doi:10.1371/journal.pone.0071486.g003 Figure 3. Time-line graphs of case elaborations with incomplete (3a) and complete model (3b). When the case elaboration included also the higher loops of the actions Evaluation, Representation and Integration these case elaborations were labelled a ‘‘complete model’’. If the actions Evaluation, Representation and Integration were in another order or only single actions were coded, the case elaboration was labelled ‘‘incomplete’’. doi:10.1371/journal.pone.0071486.g003 23; 61%, second case: 13/22; 59%, third case: 10/21; 48%). Figure 3 shows representative case examples each for a complete and an incomplete model. possible to describe the process of problem-solving using this model. More specifically, it was found that all medical students used the following cognitive actions: Denomination, Analysis, and Implementation. When dealing with the cases, the medical student participants spent 73% of the session time with these relatively basic cognitive actions. Further, the results yield that the students spend less time on the actions Exploration and Plan. Furthermore, the cognitive actions of Evaluation, Representation and Integration were found only in a subset of the students. On average, students spent only 17% of the total session time on these higher cognitive actions. The Complete Model Pattern is Significantly Correlated with the Correct Diagnostic Case Solution Neither socio-demographic data of the participants (age, year of studies), nor prior knowledge (grades of PME as general prior knowledge, assessment of the learning phase in the field of clinical nephrology) were related to the completion of the model, analysed with Pearson correlation. As well, the dichotomous variables of sex and practical experience were not related to the completion of the model, analysed with Chi square test. Previous knowledge is not correlated with the complete model or for the correct solution in this setting with this level of knowledge in clinical nephrology. The second objective of this study was to assess whether certain patterns can be extracted in the distribution of the actions over the duration of the case sessions. In our analysis, certain repeating patterns were found. Among all students the pattern of Denomi- nation to Analysis and to Implementation could be found and was called a lower loop. This finding is consistent with the loops in the problem-solving process of medical doctors as described by Barrows and Tamblyn [21]. The higher cognitive actions (higher loops) could be coded in 56% of all cases. Solving a case with both, the lower loops and the higher loops was defined as the complete model pattern. The overall process of the case elaboration revealed a dynamic and complex sequence of actions with various lengths and often rapid switching between the different actions. The non- sequential workflow observed in the case elaboration in this study can be assumed to be necessary to cope with the complexity of the problems (as described in action theoretic approaches [17]). The correct solution was obtained in 27 of all cases (27/66; 41%), the incorrect solution or no solution in the majority of the case elaborations (39/66; 59%), respectively. Out of the 37 cases with the complete model, the correct solution was reached in 24 cases (24/37; 64%). In contrast, out of the 29 cases with the incomplete model, the correct solution was reached in 3 cases only (3/29; 10%) (see table 3). The complete model was a strongly correlated with the correct solution. (Chi-squared test, p,.0001; phi coefficient [mean square contingency coefficient] w = 0.55). August 2013 \| Volume 8 \| Issue 8 \| e71486 1. Borleffs JCC, Custers EJFM, van Gijn J, ten Cate OTJ (2003) ‘‘Clinical Reasoning Theater’’: A New Approach to Clinical Reasoning Education. Academic Medicine 78: 322. Cognitive Problem Solving Patterns With the model it is now possible to evaluate instructional strategies regarding their underlying cognitive actions. However, before the model should be used in this way it is important to understand why the students chose certain cognitive actions and did not choose others. Future studies on this subject could be stipulated by selection strategy research (i.e. [40]). Conclusions The model used in this study investigates the complex and dynamic nature of the medical problem-solving process. We have investigated and validated a first model to describe the cognitive actions during problem-solving of clinical medical students. This provides the platform for further research especially for the evaluation of novel instructional methods that intend to foster clinical reasoning. Potential Applications for Medical Education There is an abundance of educational models using sequential steps [17,35]. For clinical reasoning, the most common models are problem-based learning [21,36,37] or worked examples [13,16,38]. These models were designed for instructional purposes of core curriculum knowledge but have been criticized to be unsuitable for a description of realistic free individual medical problem-solving as happens in daily clinical work [22]. The findings in this study demonstrate that the proposed model is well- suited to describe realistic free individual medical problem-solving of medical students. The value of the model consists in its capacity to enable one to trace back the cognitive steps students take during the medical problem-solving process, independent of the correct solution. This is different from current educational strategies where the focus lies on the correct solution rather than the process towards the correct solution (cf. van Gog [16]). One educational application which can be drawn from this study is the necessity to foster higher level reasoning (evaluation, representation and integration) during case elaboration. This could for example be applied by supporting students to express a verbal representation during their individual problem-solving process. Furthermore, training students to present their patients also may foster higher level thinking; research is needed to verify how this might work. 2. Elstein AS, Schwarz A (2002) Clinical problem solving and diagnostic decision making: selective review of the cognitive literature. Bmj 324: 729–732. Limitations of the Study The qualitative design, the data preparation, as well as the analysis made it necessary to include a limited number of participants and a limited number of cases and domains per participant, respectively. On the other hand, qualitative research chooses to rather focus on carefully constructed valid measures (over thirteen hours of transcribed, coded and analysed material) than on less meaningful yet reliable measures, and for a qualitative study, the sample is relatively large. The composition of participants in the study was selected by stratification in groups regarding to their years of study, age and sex. However, the findings support that the completion of the model and solution of the cases were not linked to the participants at all. A natural limitation created by the think-aloud method is that only what is expressed verbally can be analysed, coded and interpreted. Furthermore, the model is rather complex and not easy to code. The eight cognitive actions were chosen in order not to miss a cognitive action. For further investigations, it could be useful to work with a simplified model by fusing both the cognitive action of Analysis and Exploration as well as Plan and Implementation. The implementation of the model into a cognitive architecture (i.e. ACT-R; adaptive control of thought–rational) would be interesting. Cognitive architectures have also been used to model the problem-solving processes of mathematicians and then implemented to foster the mathematical problem-solving of high-school students [33]. Although medical problem-solving is different from mathematical problem-solving a transfer of this application seems highly desirable. Additionally the model could be used as a tool for expertise research in medical problem solving and for research on specific biases of decision making of physicians [34]. Our model represents one way of approaching the cognitive processes behind clinical reasoning. Our model was drawn inductively from various models and pilot study data. Certainly other existing models have been proposed that could also fit. Recently elaborated and extensive modelling did find steps similar to our proposed model [23]. Nonetheless, to our knowledge our study represents the first empirical verification of a model to describe the process of individual medical problem-solving among medical students and it strongly suggests a link between higher cognitive actions and successful case solutions. Author Contributions Conceived and designed the experiments: RE AG MH MRF RS. Performed the experiments: RE. Analyzed the data: JK RE MH RS. Contributed reagents/materials/analysis tools: JK RE AG MH RS MRF. Wrote the paper: JK RE AG MH RS MRF. Conceived and designed the experiments: RE AG MH MRF RS. Performed the experiments: RE. Analyzed the data: JK RE MH RS. Contributed reagents/materials/analysis tools: JK RE AG MH RS MRF. Wrote the paper: JK RE AG MH RS MRF. Cognitive Problem Solving Patterns Cognitive Problem Solving Patterns time. Therefore, the ability to build an inner representation from the case information and its evaluation enabled the students to reach the correct solution. Surprisingly, the extent of general prior medical knowledge (PME) was neither related to the complete model pattern nor to the correct solution of the case. Therefore, this result suggests that the completion of the model is independent from the person. The question remains whether the higher cognitive actions are a predictor for diagnostic accuracy or rather a prerequisite. Furthermore, the fulfilment of the model could not simply be attributed to students with higher grades. According to content specificity, knowledge in a certain field is a prerequisite for the strategies applied. Although content specificity was controlled through the learning phase, the subjects did not consistently use or not use the complete model nor did the grades of the assessment after the learning phase relate to the use of the complete model. This result indicates that the cognitive actions described could be indeed fundamental abstractions, that they are not completely based on content specificity. Further research should clarify the counterintuitive finding regarding general prior knowledge (as tested with the PME). For example, the relation of knowledge types (factual knowledge, conceptual knowledge and procedural knowledge [30], [16]) and meta cognitive knowledge and regulation [31,32] to the cognitive actions, the completion of the model and the solution of the case should be investigated. This study showed that the majority of the students were already able to think on the higher-level. Therefore, instruction and encouragement alone could be a resource-oriented approach [39]. In case-based learning, worked examples could advance students’ learning to higher-level thinking as especially Integration could be fostered. With the model it is now possible to evaluate instructional strategies regarding their underlying cognitive actions. However, before the model should be used in this way it is important to understand why the students chose certain cognitive actions and did not choose others. Future studies on this subject could be stipulated by selection strategy research (i.e. [40]). This study showed that the majority of the students were already able to think on the higher-level. Therefore, instruction and encouragement alone could be a resource-oriented approach [39]. In case-based learning, worked examples could advance students’ learning to higher-level thinking as especially Integration could be fostered. Discussion The aim of the study was to empirically verify the process of complex problem-solving among medical students. The first objective was to determine whether the process of problem-solving can be described using the cognitive actions in the proposed ‘‘modified Schoenfeld model’’. The results indicate that it is The third objective was to reveal whether the identified pattern is associated with the solution of the case. The complete model pattern was significantly correlated with a higher frequency of the correct solution (w = 0.55). It appeared that the higher cognitive actions Evaluation, Representation and Integration were crucial for successful problem-solving. A reason for this finding might be that these cognitive actions exceed the other five cognitive actions with regard to their cognitive complexity needed to execute these actions as they require the ability for abstract thinking. For problem-solving of complex medical cases by medical students the quality of process was strongly associated with the quality of product in our study (cf. van Gog [16]). Furthermore, this finding can be explained through the attributes of complex problem- solving [17]. Here, working on a case does not happen in a sequential order but rather in a dynamic and complex process where transitions from one action to another back and forth are necessary due to multiple problems and aims which change over Table 3. Frequencies of the incorrect and correct solution relative to the completion of the model. Table 3. Frequencies of the incorrect and correct solution relative to the completion of the model. Incorrect solution Correct solution Incomplete model 26/29; 90% 3/29; 10% 29 cases; 44% Complete model 13/37; 35% 24/37; 64% 37 cases; 56% 39/66; 59% 27/66; 41% doi:10.1371/journal.pone.0071486.t003 doi:10.1371/journal.pone.0071486.t003 August 2013 \| Volume 8 \| Issue 8 \| e71486 PLOS ONE \| www.plosone.org 6 Cognitive Problem Solving Patterns Fletcher L, Carruthers P (2012) Metacognition and Reasoning 32. Fletcher L, Carruthers P (2012) Metacognition and Reasoning. Philosophical Transactions of the Royal Society B: Biological Sciences 369. New England Journal of Medicine 355: 2251–225 12. Kassirer JP, Kopelman RI, Wong JB (1991) Learning clinical reasoning: Williams & Wilkins Baltimore. 33. Anderson JR, Fincham JM, Qin Y, Stocco A (2008) A central circuit of the mind. Trends in cognitive sciences 12: 136–143. 13. Kopp V, Stark R, Fischer MR (2008) Fostering diagnostic knowledge through computer-supported, case-based worked examples: effects of erroneous examples and feedback. Medical Education 42: 823–829. 34. Gigerenzer G, Hertwig R, Pachur T (2011) How are heuristics selected? Heuristics 1: 241–242. 14. Fischer M (2000) CASUS-An authoring and learning tool supporting diagnostic reasoning. Zeitschrift fu¨r Hochschuldidaktik 1: 87–98. 35. Bugdahl V, Bugdahl B (1995) Kreatives Problemloslen im Unterricht: Cornelsen Scriptor. 14. Fischer M (2000) CASUS-An authoring and learning t reasoning. Zeitschrift fu¨r Hochschuldidaktik 1: 87–98 g 15. Charlin B, Boshuizen HPA, Custers EJ, Feltovich PJ (2007) Scripts and clinical reasoning. Medical Education 41: 1178–1184. g 15. Charlin B, Boshuizen HPA, Custers EJ, Feltovich reasoning. Medical Education 41: 1178–1184. 15. Charlin B, Boshuizen HPA, Custers EJ, Feltovich PJ (2007) Scripts and clinical reasoning. Medical Education 41: 1178–1184. 15. Charlin B, Boshuizen HPA, Custers EJ, Feltovich PJ (2007) S 36. Wood DF (2003) Problem based learning. Bmj 326: 328. reasoning. Medical Education 41: 1178–1184. 37. Davis MH (1999) AMEE Medical Education Guide No. 15: Problem-based learning: a practical guide. Medical Teacher 21: 130–140. 37. Davis MH (1999) AMEE Medical Education Guide No 16. Van Gog T, Paas F, Van Merrie¨nboer JJG (2004) Process-oriented worked examples: Improving transfer performance through enhanced understanding. Instructional Science 32: 83–98. learning: a practical guide. Medical Teacher 21: 130–140. 38. Paas F, Van Gog T (2006) Optimising worked example instruction: Different ways to increase germane cognitive load. Learning and Instruction 16: 87–91. 17. Funke J (2003) Problemlo¨sendes Denken: Hogrefe & Huber. 17. Funke J (2003) Problemlo¨sendes Denken: H 39. Rogers Carl R, Lyon Harold CJ, Tausch R (2013) On becoming an effective teacher - Person-Centered Teaching, Psychology, Philosophy, and Dialogues with Carl R. Rogers. New York: Routledge. 18. Dewey J (1997) How we think: Dover Pubns. 19. Polya G (1945) How to solve it. Princeton University Press. y ( ) y 20. Weidenmann B, Krapp A (2006) Pa¨dagogische Psychologie. 40. References August 2013 \| Volume 8 \| Issue 8 \| e71486 PLOS ONE \| www.plosone.org 7 Cognitive Problem Solving Patterns 3. Gigerenzer G (2011) Better doctors, better patients, better decisions: Envisioning health care 2020: MIT Press. 22. Frensch PA, Funke J (1995) Complex problem solving: The European perspective: Lawrence Erlbaum. 4. Elstein A, Shulman L, Sprafka S (1978) Medical Problem Solving–An Analysis of Clinical Reasoning. 23. Charlin B, Lubarsky S, Millette B, Crevier F, Aude´tat MC, et al. (2012) Clinical reasoning processes: unravelling complexity through graphical representation. Medical Education 46: 454–463. 5. Wimmers PF, Splinter TAW, Hancock GR, Schmidt HG (2007) Clinical competence: General ability or case-specific? Advances in health sciences education 12: 299–314. 24. Groves M (2012) Understanding clinical reasoning: the next step in wo how it really works. Medical Education 46: 444–446. 24. Groves M (2012) Understanding clinical reasoning: the n M (2012) Understanding clinical reasoning: the next step in working o how it really works. Medical Education 46: 444–446. 6. Harasym PH, Tsai TC, Hemmati P (2008) Current trends in developing medical students’ critical thinking abilities. The Kaohsiung journal of medical sciences 24: 341–355. 25. Schoenfeld A (1985) Mathematical problem solving. 26. Mayring P (2004) Qualitative content analysis. A companion to qualitative research. Thousand Oaks: SAGE. 266–269. 7. Eva KW, Hatala RM, LeBlanc VR, Brooks LR (2007) Teaching from the clinical reasoning literature: combined reasoning strategies help novice diagnosticians overcome misleading information. Medical Education 41: 1152–1158. 27. Fischer MR, Herrmann S, Kopp V (2005) Answering multiple-choice questions in high-stakes medical examinations. Medical Education 39: 890–894. 28. Schmidmaier R, Ebersbach R, Schiller M, Hege I, Holzer M, et al. (2011) Using electronic flashcards to promote learning in medical students: retesting versus restudying. Medical Education 45: 1101–1110. 8. Gra¨sel C, Mandl H (1993) Fo¨rderung des Erwerbs diagnostischer Strategien in fallbasierten Lernumgebungen. 29. Ericsson K, Simon H (1993) Protocol analysis: verbal reports as data. 1993. Rev ed Bradford, Cambridge, MA. 9. Neufeld V, Norman G, Feightner J, Barrows H (1981) Clinical problem-solving by medical students: a cross-sectional and longitudinal analysis. Medical Education 15: 315–322. 30. Krathwohl DR (2002) A revision of Bloom’s taxonomy: An overview. Theory into practice 41: 212–218. 10. Eva KW (2005) What every teacher needs to know about clinical reasoning. Medical Education 39: 98–106. p 31. Flavell JH (1979) Metacognition and cognitive monitoring: A new area of cognitive–developmental inquiry. American psychologist 34: 906. 11. Norman G (2006) Building on experience–the development of clinical reasoning. New England Journal of Medicine 355: 2251–2252. 32. Cognitive Problem Solving Patterns Marewski JN, Schooler LJ (2011) Cognitive niches: An ecological model of strategy selection. Psychological Review 118: 393. 21. Barrows HS, Tamblyn RM (1980) Problem-based learning: An approach to medical education: Springer Publishing Company. 21. 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https://openalex.org/W4383053570	https://zenodo.org/record/8074286/files/Vanguard%20Node%20Status.pdf	English	null	Early adopter PoC nodes operational with synthetic data	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	134	Vanguard Node Status Jun 30, 2023 outlines the functionality provided by each Vanguard node on the above date. Green indicates the functionality is with links where possible. White means no information / not support, and orange indicates partially supported. This table outlines the functionality provided by each Vanguard node on the above date. Green indicates the functionality is provided, with links where possible. White means no information / not support, and orange indicates partially supported. Vanguard Node Data Discovery Data Reception Storage and Interfaces Access Management Tools Processing Belgium https://beacon-network-demo. ega-archive.org/members Using Galaxy (http://193.190.80.5:59980/) Using Galaxy (http://193.190.80.5:5 9980/) http://193.191.128.167: 3000/ Using Galaxy (http://193.190.80. 5:59980/) Finland https://beacon-network-demo. ega-archive.org/members https://gdi-demo.sd.csc.fi/hts get/reads/s3/.... https://gdi-demo.sd.csc. fi/ Luxembourg https://beacon-network-demo. ega-archive.org/members https://htsget.dev.gdi.lu/ https://login.dev.gdi.lu/ https://rems.dev.gdi.lu/ Netherlands Norway https://tryggve.tsd.usit.uio.no/ beacon https://tryggve.tsd.usit.uio.no/ retrieval.html https://rems.tsd.usit.uio. no Spain Sweden https://beacon-network-demo. ega-archive.org/members https://htsget.gdi.nbis.se/read s/s3/... https://login.gdi.nbis.s e/ https://rems.gdi.nbis.se/
https://openalex.org/W2948982792	https://jurnal.unimed.ac.id/2012/index.php/GDG/article/download/12257/11381	Indonesian	null	Minat Siswa Sekolah Menengah Atas dalam Mengikuti Jong Batak Art Festival IV di Taman Budaya Sumatera Utara	Gondang	2,019	cc-by	2,988	Minat Siswa Sekolah Menengah Atas dalam Mengikuti Jong Batak Art Festival IV di Taman Budaya Sumatera Utara The Interest of Students High School in the Jong Batak Art Festival IV in the North Sumatra Cultural Park Erfinaika Siringoringo, Uyuni Widiastuti & Lamhot Basani Sihombing Program Studi Pendidikan Musik Jurusan Sendratasik Fakultas Bahasa dan Seni Universitas Negeri Medan, Indonesia Diterima: Januari 2019; Disetujui: Mei 2019; Diterbitkan: Juni 2019. Diterima: Januari 2019; Disetujui: Mei 2019; Diterbitkan: Juni 2019. Abstrak Penelitian ini bertujuan untuk mengetahui minat siswa SMA YP. HKBP Sidorame dalam mengikuti Jong batak Art Festival IV di Taman Budaya Sumatera Utara serta faktor yang memengaruhi minat siswa SMA YP. HKBP Sidorame dalam mengikuti Jong batak Art Festival IV di Taman Budaya Sumatera Utara. Teori yang digunakan dalam penelitian ini adalah teori minat, macam-macam minat, faktor yang memengaruhi, jong batak yang menjelaskan rasa lebih suka dan rasa ketertarikan pada suatu hal atau aktivitas, tanpa ada yang menyuruh. Pada dasarnya minat adalah penerimaan akan suatu hubungan antara diri sendiri dengan sesuatu diluar diri, semakin kuat atau dekat hubungan tersebut maka semakin besar minat. Metode dalam penelitian ini adalah mixed methods. Pengumpulan data dilakukan berdasarkan studi kepustakaan, observasi, dan dukumentasi. Sampel dalam penelitian ini adalah siswa kelas XII IPS 1 yang berjumlah 32 orang. Berdasarkan hasil penelitian yang dilakukan, dapat diketahui bahwa penggunaan angket sebagai instrumen penelitian menunjukkan bahwa siswa berminat mengikuti Jong Batak Art Festival IV namun belum mencapai tingkat tertinggi dalam kategori penilaian, yaitu Sangat Berminat. Kata Kunci: Minat, Siswa, Jong Batak, Art Festival Kata Kunci: Minat, Siswa, Jong Batak, Art Festival Kata Kunci: Minat, Siswa, Jong Batak, Art Festival Gondang: Jurnal Seni dan Budaya, 3(1) (2019): 1-8. Gondang: Jurnal Seni dan Budaya, 3(1) (2019): 1-8. Abstract This study aims to determine the interest of YP high school students. HKBP Sidorame in following Jong Batak Art Festival IV in the North Sumatra Cultural Park and the factors that influenced the interest of YP High School students. HKBP Sidorame in following Jong Batak Art Festival IV in North Sumatra Cultural Park. The theory used in this study is the theory of interest, kinds of interests, influencing factors, jong batak which explains the feeling of being more like and feeling interested in something or activity, without being told. Basically, interest is the acceptance of a relationship between yourself and something outside of yourself, the stronger or nearer the relationship, the greater the interest. The method in this study is mixed methods. Data collection is based on library studies, observation, and documentation. The sample in this study were students of class XII IPS 1 totaling 32 people. Based on the results of the research conducted, it can be seen that the use of questionnaires as research instruments shows that students are interested in participating in the Jong Batak Art Festival IV but have not reached the highest level in the assessment category, which is Very Interested. Keywords: Interest, Students, Jong Batak, Art Festival Corresponding author: Corresponding author: E-mail: erfi12@gmail.com E-mail: erfi12@gmail.com PENDAHULUAN situasi yang mempunyai kaitan dengan dirinya. situasi yang mempunyai kaitan dengan dirinya. Jong Batak Art Festival IV dilaksanakan pada tanggal 25-28 Oktober 2017. kegiatan ini mengajak sejumlah seniman yang akan berkontribusi dalam acara ini, dan melibatkan sejumlah pelajar untuk mengikuti Acara tersebut. Selain dapat menikmati dan mengapresiasikan bakat, pelajar dan masyarakat semakin apresiatif terhadap budaya dan hendaknya ikut berperan dalam melestarikan budaya melalui acara tersebut. Dalam hal ini identitas serta kecintaan terhadap budaya dapat ditunjukan melalui sikap apresiasi, menampilkan sebuah kreativitas dan dapat berperan serta dalam perkembangan budaya di Sumatera Utara. SMA HKBP Sidorame merupakan salah satu sekolah swasta yang berada di Jl. Dorowati, Kota Medan. SMA HKBP Sidorame merupakan salah satu dari beberapa sekolah yang selalu aktif hadir dalam mengikuti Jong Batak Art Festival. Salah satu faktor yang mendorong pelajar mengikuti Jong Batak Art Festival IV adalah untuk menambah wawasan, pengetahuan, mengembangkan minat dan bakat. Apalagi kalau minat itu selaras dengan bakatnya, maka kekuatannya lebih luar biasa lagi. Berdasarkan observasi yang telah dilakukan, Jong Batak Art Festival ini sangat baik diadakan karena kegiatan ini mampu memotivasi pemuda/i khususnya Pelajar Kota Medan di Sumatera Utara untuk melestarikan budaya. Minat pelajar terhadap suatu pertunjukan yang berbau budaya seperti pertunjukan Jong Batak Art Festival kian lama kian ditinggalkan oleh pelajar yang disebabkan masuknya budaya Barat yang lebih modern. Minat bisa timbul karena adanya dorongan dari diri sendiri. Oleh karena itu, disimpulkan bahwa minat itu adalah sikap atau perasaan senang terhadap sesuatu yang diinginkannya. Jika seorang pelajar senang terhadap sesuatu, pelajar tersebut akan berusaha secara terus menerus untuk mendapatkannya. Minat merupakan kesadaran seseorang terhadap suatu objek, orang, masalah, atau Keywords: Interest, Students, Jong Batak, Art Festival Keywords: Interest, Students, Jong Batak, Art Festival How to Cite: Siringoringo, E. Widiastuti, U. & Basani, L. (2019). Minat Siswa Sekolah Menengah Atas dalam Mengikuti Jong Batak Art Festival IV di Taman Budaya Sumatera Utara. Gondang: Jurnal Seni dan Budaya, 3 (1): 1-9. ISSN 2549-1660 (Print) ISSN 2550-1305 (Online) *Corresponding author: E-mail: erfi12@gmail.com E-mail: erfi12@gmail.com 1 Erfinaika Siringoringo, Uyuni Widiastuti & Lamhot Basani, Minat Siswa Sekolah Menengah Atas rfinaika Siringoringo, Uyuni Widiastuti & Lamhot Basani, Minat Siswa Sekolah Menengah Atas METODE PENELITIAN Sesuai dengan judul penelitian ini, Minat SMA YP. HKBP Sidorame Dalam Mengikuti Jong Batak Art Festival IV di Taman Budaya Sumatera Utara. Maka lokasi penelitian dilakukan di Sekolah YP.SMA HKBP Sidorame di Kota Medan dan Waktu penelitian dilaksanakan pada bulan Juli-Agustus 2018. Minat merupakan suatu keadaan di mana seseorang mempunyai perhatian terhadap sesuatu dan disertai keinginan 2 2 Gondang: Jurnal Seni dan Budaya 3 (1) (2019): 1-8 untuk mengetahui dan mempelajari maupun membuktikannya lebih lanjut. Minat timbul karena adanya perhatian yang mendalam terhadap suatu obyek, di mana perhatian tersebut menimbulkan keinginan untuk mengetahui, mempelajari, serta membuktikan lebih lanjut. Untuk mengetahui minat tersebut, faktor-faktor yang mempengaruhi minat pada hakikatnya merupakan sebab akibat dari pengalaman. Minat tidak muncul dengan sendirinya akan tetapi ada beberapa faktor yang dapat mempengaruhi minat tersebut yaitu faktor internal dan eksternal.Seberapa besar minat dapat dilihat dari berbagai indikator/aspek mengenai minat yang kemudian diukur dengan pengukuran data melalui pernyataan berupa angket. Dilihat dari segi cara atau teknik pengumpulan data, maka teknik pengumpulan data dapat dilakukan dengan interview, kuesioner (angket), observasi (Sugiyono, 2006). Pengumpulan data dilakukan dengan cara sebagai berikut. 1) Observasi; 2) Angket; 3) Dokumentasi; 4) Instrument Minat Siswa mengikuti Jong Batak Art Festival IV Menurut Slameto (2010), “Minat adalah suatu rasa lebih suka dan rasa keterikatan pada suatu hal atau aktivitas, tanpa ada yang menyuruh.” Pada dasarnya minat adalah penerimaan akan suatu hubungan antara diri sendiri dengan sesuatu diluar diri.semakin kuat atau dekat hubungan tersebut, semakin besar minat. Sesuai pernyataan di atas maka kerangka konseptual ini bertujuan untuk menyusun topik penelitian yang berjudul ”Minat Siswa SMA YP. HKBP SidorameDalam Mengikuti Jong Batak Art Festival IV di Taman Budaya Sumatera Utara”. Menurut Hurlock (1999) dalam Khairani Makmun (2013) minat merupakan sumber motivasi yang mendorong orang untuk melakukan apa yang mereka inginkan bila mereka bebas memilih ketika seseorang menilai bahwa sesuatu akan bermanfaat, maka akan jadi berminat, kemudian hal tersebut akan mendatangkan kepuasan. Ketika kepuasaan menurun, maka minat juga akan menurun. Sehingga minat tidak bersifat permanen, tetapi minat bersifat sementara atau dapat berubah-ubah. Adapun populasi yang terjangkau dalam penelitian ini adalah kelas XII IPS SMA Sidorame Medan. Sampel diambil langsung dari kelas XII IPS SMA Sidorame yang berjumlah 32 siswa yang diambil secara sampling insidental. 3 Erfinaika Siringoringo, Uyuni Widiastuti & Lamhot Basani, Minat Siswa Sekolah Menengah Atas Erfinaika Siringoringo, Uyuni Widiastuti & Lamhot Basani, Minat Siswa Sekolah Menengah Atas rfinaika Siringoringo, Uyuni Widiastuti & Lamhot Basani, Minat Siswa Sekolah Menengah Atas Sedangkan menurut Crow and Crow (1984) dalam Khairani Makmun (2013) minat dapat menunjukkan kemampuan untuk memberi stimuli yang mendorong individu untuk memperhatikan seseorang, sesuatu barang atau kegiatan, atau sesuatu yang dapat memberi pengaruh terhadap pengalaman yang telah distimuli oleh kegiatan itu sendiri. kegiatan. Misalnya, kegiatan olahraga, pramuka, dan sebagainya yang menarik perhatian. Minat merupakan kecenderungan dalam diri individu untuk tertarik pada suatu objek, kesukaan atau kesenangan akan sesuatu. Minat sangat dibutuhkan oleh siapapun untukmeningkatkan motivasi dan kegairahan dalam melakukan sesuatu atau terhadapobjek tertentu.Dapat dikatakan bahwa minat merupakan indikator keberhasilan anak dalam melakukan suatu hal. Di samping itu menurut Jhon Holland, (2013) dalam Khairul Makmun minat merupakan aktivitas atau tugas- tugas yang membangkitkan perasaan ingin tahu, perhatian, dan memberi kesenangan atau kenikmatan. Bagi siswa, minat ini sangat penting.Hal tersebutdikarenakan dapatmenggerakkan perilaku siswa ke arah positif sehingga mampu menghadapi segalatuntunan, kesulitan serta menanggung resiko dalam studinya.Minat dapatmenentukan baik tidaknya dalam mencapai tujuan sehingga semakin besar minat, semakin besar kesuksesan yang dapat diraih oleh seseorang.Minat sebagai faktor batin berfungsimenimbulkan, mendasari dan mengarahkan perbuatan belajar. Minat Siswa mengikuti Jong Batak Art Festival IV Seorang yang besarminatnya akan giat berusaha, tampak gigih, tidak mau menyerah serta giatmengikuti setiap kegiatan.Minat dapat meningkatkan motivasi siswa mengikuti kegiatan Jong Batak Art Festival IV di Taman Budaya sehingga siswa dapat memeroleh prestasi yang maksimal. Berdasarkan pendapat tersebut, maka peneliti menyimpulkan bahwa minat merupakan rasa ketertarikan seseorang dan ingin tahu yang lebih dari sesuatu Objek yang diperhatikan sehingga minat dapat menyebabkan seseorang giat melakukan suatu kegiatan menuju ke sesuatu yang telah menarik minatnya. Seseorang dapat mengungkapkan minat atau pilihanya dengan kata-kata tertentu. Misalnya, seseorang mungkin mengatakan bahwa dirinya tertarik dalam mengumpulkan mata uang logam, perangko dan lain-lain. Seseorang dapat mengungkapkan minat bukan melalui kata-kata melainkan dengan tindakan atau perbuatan, yaitu ikut serta dan berperan aktif dalam suatu 4 Gondang: Jurnal Seni dan Budaya 3 (1) (2019): 1-8 Dari hasil penelitian diketahui minat siswa dalam mengikutikegiatan Jong Batak Art Festival IV di Taman Budaya Sumatera Utaraberminat dengan persentase sebesar62,5%. Hasil tersebut diartikan siswa sudah memiliki minatdan mempunyaiantusias dalam mengikuti kegiatan Jong Batak Art Festival IV di Taman Budaya Sumatera Utara.Hal tersebut dilihat dari hasil analisis data penulis berupa angket, dengan indikator minat siswa dalam mengikuti Jong Batak Art Festival, ketertarikan, perasaan senang, dan bakat siswa dalam hal seni. Minat tidak muncul dengan sendirinya, terdapat banyak faktor yang menimbulkan minat seseorang terhadap sesuatu yang diminatinya. Faktor-Faktor tersebut adalah sebagai berikut. Faktor internal adalah: 1) Bakat dan Bawaan; 2) Tingkat Perkembangan; 3) Kondisi dan psikis. Faktor internal atau faktor dari luar merupakan faktor yang terjadi ketika adanya rangsangan-rangsangan yang berpengaruh dalam memberikan sentuhan-sentuhan kejiwaan secara langsung yang dibuat atau sengaja dilakukan untuk menumbuhkan kesadaran, yang nantinya akan membangkitkan perhatian dan minat seseorang. Faktor luar tersebut adalah: 1) Motivasi; 2) Keluarga; 3) Fasilitas; 4) Kebutuhan. Minat seorang anak juga tidak lepas dari berbagai faktor yang memengaruhinya, yaitu faktor internal yang merupakan faktor yang berasal dari diri siswadan faktor eksternal yang merupakan faktor yang berasal dari luar diri siswa, yaitu lingkungansekolah dan lingkungan keluarga. Jika dua faktor ini bersinergi maka minat yang dimiliki siswa akan baik. Ada beberapa indikator mengenai minat. Indikator ini disusun berdasarkan aspek minat siswa. Menurut Djamarah (2011), aspek yang dimaksud adalah adanya kesukaan, ketertarikan, perhatian, dan keterlibatan. Faktor yang Memengaruhi Minat Siswa Seseorang menilai minatnya agar dapat diukur dengan menjawab sejumlah pertanyaan tertentu atau urutan pilihannya untuk kelompok aktivitas tertentu. Pertanyaan-pertanyaan disusun dengan menggunakan angket. Untuk mengetahui berapa besar minat siswa dapat diukur melalui: 1) Kesukaan; 2) Ketertarikan; 3) Perhatian; 4) Keterlibatan Faktor-faktor yang memengaruhi minat siswa dalam mengikuti Jong Batak Art Festival IV di Taman Budaya Sumatera 5 5 Erfinaika Siringoringo, Uyuni Widiastuti & Lamhot Basani, Minat Siswa Sekolah Menengah Atas Utara yaitu bakat, tingkat perkembangan, motivasi, kebutuhan, ketertarikan, perasaan senang, dan keluarga. daripada stay menghadiri acara tersebut. Kemudian ada juga yang menyatakan bahwa ia bosan selama acara berlangsung. Dengan begitu dapat disimpulkan bahwa motivasi siswa tersebut tidak memiliki motivasi dan minat yang tinggi dalam mengikuti kegiatan Jong Batak Art Festival IV di Taman Budaya, terlihat dari tidak adanya motivasi dari dalam dirinya untuk benar-benar serius mengikuti acara tersebut. Seorang anak yang memiliki bakat dalam hal seni serta didukung dengan tingkat perkembangan nya yang baik dalam seni, tentu akan sangat berminat mengikuti kegiatan seperti Jong Batak Art festival ini. Hal tersebut dibuktikan dengan adanya siswa yang berpartisipasi untuk menyukseskan Acara Jong Batak Art Festival IV di Taman Budaya Sumatera Utara. Adapun salah satu kegiatan yang mereka ikuti yaitu tari tortor. Terlihat jelas siswa yang memiliki bakat menari tradisi, berminat untuk menyukseskan acara tersebut dengan bakat yang mereka miliki sedangkan siswa lain yang kurang berbakat dalam hal tari memilih untuk menjadi penonton saja. Faktor kebutuhan juga salah satu cara melihat bagaimana minat siswa. Siswa yang merasa membutuhkan seni dalam hidupnya, yang juga memiliki bakat dalam hal seni, tentu akan senantiasa mengikuti kegiatan bermanfaat Jong Batak Art Festival IV di Taman Budaya. Dengan adanya kegiatan tersebut, maka pengetahuan akan hal seni terutama seni tradisi akan bertambah. Dengan bertambahnya pengetahuan siswa, maka akan semakin mendukung adanya bakat dalam dirinya.Terdapat beberapa siswa yang memiliki bakat seni dalam dirinya, sudah yakin memiliki minat untuk mengikuti kegiatan tersebut.Hal ini ditinjau dengan melihat hasil tes angket yang diisi oleh siswa. Kemudian faktor motivasi.Motivasi merupakan dorongan yang timbul pada diri seseorang secara sadar atau tidak sadar untuk melakukan suatu tindakan dengan tujuan tertentu.Motivasi dapat dipicu oleh beberapa hal, salah satunya adalah teman. Dalam hal ini, peneliti meninjau hasil tes angket siswa yang menunjukkan bahwa masih ada siswa yang terpengaruh oleh ajakan teman. Beberapa siswa menjawab bahwa ajakan teman meninggalkan acara Jong Batak Art Festival IV di Taman Budaya, lebih penting Ketertarikan juga merupakan hal penting lainnya. Faktor yang Memengaruhi Minat Siswa Anak yang memiliki ketertarikan akan suatu hal atau kegiatan, akan merespon kegiatan tersebut dengan 6 6 Gondang: Jurnal Seni dan Budaya 3 (1) (2019): 1-8 Dorongan seorang pendidik dapat menimbulkan minat siswanya dengan memberi arahan, membantu mengembangkan bakat, dan memberi keyakinan bahwa kesenian tradisi bukanlah hal yang membosankan, melainkan suatu kebudayaan yang memiiki nilai dan harus dilestarikan. Begitupula dengan dorongan dari orangtua yang sangat diperlukan oleh siswa untuk menimbulkan minat siswa. Orangtua mempunyai peran penting untuk selalu mengingatkan anak-anaknya bahwa budaya merupakan warisan yang harus dilestarikan dan diapresiasi.Dan dorongan teman, merupakan faktor eksternal lainnya yang sangat memengaruhi minat seorang anak. Hal tersebut terjadi karena pada umumnya anak-anak menghabiskan waktu bermain dengan temannya, sehingga faktor lingkungan ataupun teman menjadi salah satu pembentuk kepribadian dan minat seorang anak. positif serta mencari tau bagaimana ia dapat mengembangkan dirinya melalui ketertarikannya tersebut. Jika seorang anak tidak memiliki ketertarikan akan seni, maka akan sulit menumbuhkan minatnya untuk mengikuti kegiatan- kegiatan yang bersangkutan dengan seni, begitupula dengan siswa-siswi kelas XII IPS-1. Melalui hasil tes angket, peneliti dapat melihat seberapa tertarik dan antusias siswa-siswi tersebut mengikuti kegiatan Jong Batak Art Festival IV di Taman Budaya.Masih ada siswa yang hanya memiliki sedikit saja ketertarikan untuk mengikuti kegiatan tersebut.Hal itu dilihat dari hasil tes angket beberapa siswa yang masih terpengaruh oleh ajak teman untuk meninggalkan acara tersebut. Selain itu, masih ada siswa yang mengikuti kegiatan Jong Batak Art Festival IV di Taman Budaya hanya karena disuruh oleh guru. positif serta mencari tau bagaimana ia dapat mengembangkan dirinya melalui ketertarikannya tersebut. Jika seorang anak tidak memiliki ketertarikan akan seni, maka akan sulit menumbuhkan minatnya untuk mengikuti kegiatan- kegiatan yang bersangkutan dengan seni, begitupula dengan siswa-siswi kelas XII IPS-1. Melalui hasil tes angket, peneliti dapat melihat seberapa tertarik dan antusias siswa-siswi tersebut mengikuti kegiatan Jong Batak Art Festival IV di Taman Budaya.Masih ada siswa yang hanya memiliki sedikit saja ketertarikan untuk mengikuti kegiatan tersebut.Hal itu dilihat dari hasil tes angket beberapa siswa yang masih terpengaruh oleh ajak teman untuk meninggalkan acara tersebut. Selain itu, masih ada siswa yang mengikuti kegiatan Jong Batak Art Festival IV di Taman Budaya hanya karena disuruh oleh guru. Dorongan seorang pendidik dapat menimbulkan minat siswanya dengan memberi arahan, membantu mengembangkan bakat, dan memberi keyakinan bahwa kesenian tradisi bukanlah hal yang membosankan, melainkan suatu kebudayaan yang memiiki nilai dan harus dilestarikan. Begitupula dengan dorongan dari orangtua yang sangat diperlukan oleh siswa untuk menimbulkan minat siswa. Faktor yang Memengaruhi Minat Siswa Orangtua mempunyai peran penting untuk selalu mengingatkan anak-anaknya bahwa budaya merupakan warisan yang harus dilestarikan dan diapresiasi.Dan dorongan teman, merupakan faktor eksternal lainnya yang sangat memengaruhi minat seorang anak. Hal tersebut terjadi karena pada umumnya anak-anak menghabiskan waktu bermain dengan temannya, sehingga faktor lingkungan ataupun teman menjadi salah satu pembentuk kepribadian dan minat seorang anak. Selain ketertarikan, ada faktor perasaan senang. Jika seorang siswa memiliki perasaan senang, maka siswa tersebut merasa nyaman dan tenu memiliki minat yang tinggi untuk mengikuti kegiatan Jong Batak Art Festival IV di taman budaya sumatera utara. SIMPULAN Siswa berminat mengikuti Jong Batak Art Festival IV namun belum mencapai tingkat tertinggi dalam kategori penilaian, yaitu Sangat Berminat. Faktor-Faktor yang memengaruhi minat siswa dalam mengikuti Jong Batak Art Festival IV di Taman Budaya Sumatera Utara yaitu kurangnya rasa senang terhadap acara seni, tidak memiliki bakat, terpengaruh Selain itu, dorongan guru, orang tua, teman, cukup memengruhi minat siswa mengikuti Jong Batak Art Festival IV. 7 7 Erfinaika Siringoringo, Uyuni Widiastuti & Lamhot Basani, Minat Siswa Sekolah Menengah Atas Pamungkas, M.Z. (2015). Pengaruh Event Festival Budaya Terhadap Citra Kabupaten Purwakarta Sebagai Kota Budaya (Survey terhadap pengunjung Event festival budaya di purwakarta) ajakan teman, dan memilih hadir karena disuruh guru. Faktor tersebut berpengaruh pada tingkatan minat siswa SMA kelas XII IPS YP. HKBP Sidorame. Agus, N. (2013). Jong Batak Bond, Museum Sumpah Pemuda Yudana, N. (2014). Minat Siswa Kelas VIII SMP Negeri 1 Pangadegan terhadap jenis musik. Skripsi DAFTAR PUSTAKA Slameto. (2010). Belajar dan Faktor-Faktor Yang Mempengaruhi. Ardhana, A.R. (2016). Minat Remaja Terhadap Genre Musik Punk di Kota Kisaran Kecamatan Kisaran Timur. Skripsi Sugiyono. (2012). Metode Penelitian Pendidikan, Pendekatan Kuantitatif, Kualitatif, dan R & D. Bandung: Alfabeta. p Crow and Crow. (1984). Psikologi Belajar, Terbitan (KDT) Sugiyono. (2016). Metode Penelitian Pendidikan, Pendekatan Kuantitatif, Kualitatif, dan R & D. Bandung: Alfabeta. Djamarah. (2011). Psikologi Belajar.Rineka Cipta Harlock (1999) Psikologi Belajar, Terbitan (KDT Cwettherrington, H. (1983) Psikologi Belajar, Terbitan (KDT) Syahrum. (2014). Metode Penelitian Kuantitatif. Cita Pustaka Media ( ) Holland, J. (2013). Psikologi Belajar, Terbitan (KDT) Wahyudi. (2016). Minat Remaja Terhadap Orkes Melayu Di Kecamatan Marbau Kabupaten Labuhanbatu Utara. Khairani Makmun .2013. Psikologi Belajar, Terbitan (KDT) Khairani Makmun .2013. Psikologi Belajar, Terbitan (KDT) ( ) Manurung. (2012). Metodologi Penelitian. Halamanmoeka Budiarti, Y. (2011). Minat belajar siswa terhadap mata pelajaran bahasa Indonesia (studikasus di SMA PGRI 56 Ciputat). 8 8
https://openalex.org/W2921202339	https://www.matec-conferences.org/articles/matecconf/pdf/2019/21/matecconf_icfmce2019_01039.pdf	English	null	Analysis of the feasibility and necessity of special road construction for small passenger cars	MATEC web of conferences	2,019	cc-by	4,019	* Corresponding author: 747720426@qq.com Analysis of the feasibility and necessity of special road construction for small passenger cars Jin He, Pengfei Chang, and Yanjie Sun* Shanxi Luheng Communications Survey and Design Co., Ltd., Taiyuan 030006, China Abstract. The rapid development of social economy has caused many problems in highway transportation. The traffic on most roads is getting bigger and bigger. This trend not only increases traffic pressure but also poses a threat to the lives of travellers. In order to solve the current road traffic problem, this paper proposes a special route for the construction of small passenger vehicles. The basic idea of dedicated line construction is based on the secondary road and highway operation forms. Each line passes through 1 to 3 townships. The internal and external environment and advantages and disadvantages of the dedicated line construction were analyzed by SWOT method, and the SWOT matrix was constructed. The analysis results show that the small passenger dedicated line is feasible and necessary, and has a good development prospect. In this way, it provides a new idea for the development of China's highways. © 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/). , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 https://doi.org/10.1051/matecconf /201927201039 3.1 Advantage Analysis (S) According to the idea of the small passenger car line in this paper, it has the characteristics of rapidity and flexibility in transportation, has economical in the construction process, and has safety in the driving process. 2.2 Research methods The SWOT is a qualitative analysis method. It has the advantages of comprehensiveness, system, accuracy and conciseness. It can be used in various development strategies, plans and decision-making, etc. The factors are divided according to the internal and external influence relationship, then enumerate the various factors in the form of matrix, and use the system idea to cross-combine the above various factors to obtain decision-making conclusions. This paper establishes SWOT analysis matrix for the internal and external factors affecting the construction of small passenger car lines, and uses SWOT analysis method to analyze the rationality, necessity and construction ideas of the construction of small passenger car line. 2.1 Basic assumptions According to the yearbook of the length of China's sub-regional transportation routes, the total length of highways in China is 130,973km at the end of 2018. The secondary road is a type of highway grade in China. It is used after the first-class highway and before the third- grade highway. It is widely used in the actual road construction. Therefore, the small passenger car road is based on the secondary road. Special roads use the highway mode throughout the journey, but do not charge for passing vehicles. In order to radiate more areas, special roads require 1 to 3 townships. The main route modes are “city-county” and “county-county”. Dedicated roads are designed for small cars, with dual lanes and no other vehicles. The dedicated lanes are funded by the state and local governments. The traffic network of the special line in different periods is shown in figure 1. (b) (a) (b) Fig. 1. Specialized transportation network conditions in different development periods: a. early development; b. late development. (a) (a) (b) Fig. 1. Specialized transportation network conditions in different development periods: a. early development; b. late development. 1 Background With the rapid development of the market economy and the acceleration of the urban-rural integration process, China's transportation industry has made great progress. According to domestic and international research[1-3], mixed traffic has become an accident-prone area. Yan Shoujing[4] learned through gray correlation analysis that vehicle types are the most important factor in traffic accidents, and mixed traffic is an important source of fatal accidents[5]; Liang Guohua[6] Large vehicle mix rate and traffic stability The relationship is analyzed. The analysis shows that the higher the mixing rate of large vehicles, the worse the traffic stability. At the same time, Yang Jian[7] shows that on the straight road section, the speed of small cars decreases linearly with the increase of truck mixing rate. And with the increase of traffic volume, the “mobile bottleneck” effect is gradually becoming serious; Yang Zijie[8] applied evolutionary game theory to conclude that the game process of large- scale driver and small-car driver group belongs to asymmetric game, so In large-scale vehicles, the phenomenon of car pressing on small cars is more serious. . This paper proposes a construction idea for a special route for small passenger vehicles, aiming to improve the operating speed and safety of highway traffic. It is used to provide new ideas for the development direction and construction of highway traffic engineering in China. © 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/). , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 https://doi.org/10.1051/matecconf /201927201039 https://doi.org/10.1051/matecconf /201927201039 3.1.3 Flexibility The special line setting is mainly for the “city-county” and “county-county” cases, and passes through 1~3 townships. In the central and eastern regions of China, the population density is relatively large, and the distance between city-county-town is short. Many special lines can form a large transportation network, making transportation flexible and convenient. 3.1.4 Security In the process of transportation, the speed of various vehicles is not the same due to the difference between the models of large and small vehicles. When the traffic volume is large, it is difficult to overtake, which often causes the phenomenon of rolling and hinders the driving speed of the driving. According to the research results of Yang Yao[9], it can be known that the minimum safe car spacing of small cars is smaller than the minimum safe car spacing of large and small cars, which proves that the traffic safety situation of small passenger car lines is better than mixed traffic safety under the same driving conditions. The situation is better, at the same time alleviating the operational pressure of local traffic. 3.1.1 Rapidity The design speed of the small passenger car line is 80~120km/h, and the total length of the special line is not more than 80km. The expressway mode (no charge) is used and only small passenger cars are allowed to drive. Objectively speaking, it avoids the phenomenon 2 2 , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 https://doi.org/10.1051/matecconf /201927201039 of car pressing caused by the asymmetric game of large and small vehicles. Subjectively, it slows down the driving pressure of small passenger car drivers under mixed traffic and speeds up the driving speed of small passenger cars. of car pressing caused by the asymmetric game of large and small vehicles. Subjectively, it slows down the driving pressure of small passenger car drivers under mixed traffic and speeds up the driving speed of small passenger cars. 3.1.2 Economics The special line is built on the secondary road construction standard and adopts the expressway operation mode (free of charge). It has reached the operating standard of the expressway in terms of speed, but the construction cost is the construction standard of the secondary road. At the same time, because the special line prohibits the operation of large vehicles, the damage to the road caused by traffic operations is alleviated, and the service life of the road is prolonged. 4.1 Disadvantage Analysis (W) The idea of the small passenger car line is based on the secondary road construction standards and the highway operation mode (free of charge), and the radiation range is large in areas with large population density. Therefore, its existence has certain problems such as difficulty in funding sources, difficulty in operation and management, and limitation of radiation range. 4.1.2 Operation management is difficult. 4.1.1 Difficulties in funding The dedicated lanes are funded by the state and local governments. The national capital contribution ratio is 30%, and 70% of the capital is invested by the county. Due to the uneven development of various regions in China and the different regional geological and topographical conditions and people's education level, the attitudes and construction costs of the small passenger car line are different, and the source of funds in the more difficult areas will be more difficult. 4.1.2 Operation management is difficult. 4.2.2 Construction of the “New Silk Road Economic Belt” In September 2013, Chinese President Xi Jinping proposed the strategic concept of building the “New Silk Road Economic Belt”. On March 28, 2015, the National Development and Reform Commission, the Ministry of Foreign Affairs, and the Ministry of Commerce jointly issued the “Vision and Action for Promoting the Construction of the Silk Road Economic Belt” and “the 21st Century Maritime Silk Road”. The “New Silk Road Economic Belt” takes Xi'an as the starting point, and passes through Gansu and Xinjiang to countries along the route, radiating the western region of China, and bringing opportunities for the development of the western region of China. At the same time, it will definitely increase the development of transportation lines in the western region. 4.2.1 Development of artificial intelligence At present, China's expressways have two types of charging methods: manual bayonet and ETC. ETC is currently the most advanced road and bridge charging method in the world. Through the microwave-specific short-range communication between the on-board electronic tag, the computer network technology is used to perform background settlement processing with the bank, so that the vehicle does not need to pass through the road and bridge toll station. Parking can pay the road and bridge fees. 3 3 , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 https://doi.org/10.1051/matecconf /201927201039 As the entire line prohibits the entry of vehicles other than small passenger vehicles, the entire line is free of charge in order to save the economy. There is no special guard at both ends of the line, so there will be other vehicles mixed into the special line, which will bring inconvenience to traffic operation. 4.3.1 Competition in other modes of transportation The route proposed in this paper is exclusive to small passenger transport. The main purpose is to carry out rapid transportation between “city-county” and “county-county” in a short period of time, and alleviate local traffic pressure. At present, when the road is short, the modes of travel are mainly ordinary road transport, highway transport, ordinary rail transport, high-speed rail transport and waterway transport. These modes of travel are mature and accepted by the system. Compared with small passenger lines, high-speed rail transport has a faster running speed, and the expressway has formed a developed transportation network. In the early stage of the dedicated line operation, high-speed rail transportation and highways and transportation will pose serious threats to small passenger lines. 4.3.2 Imperfections in the legal system For the small car passenger dedicated line, it is only the envisaged stage. Due to the construction of the expressway and the ordinary secondary road, the problems that exist in the private line cannot be fully considered. The imperfection of the private line legal system makes it appear that there are more traffic problems during the operation. 4 https://doi.org/10.1051/matecconf /201927201039 , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 5 Strategic choice of dedicated line solutions The SWOT matrix is used to combine the analysis results into SO, WO, ST and WT strategies. The results are shown in table 1. The SWOT matrix is used to combine the analysis results into SO, WO, ST and WT strategies. The results are shown in table 1. Table 1. SWOT matrix of small passenger dedicated line. S W O SO WO SO1: Taking the national policy as an opportunity to accelerate the construction of small passenger dedicated lines and establish a perfect transportation network; SO2: Use artificial intelligence to improve the research and development of new technologies and new technologies for the construction of dedicated lines, and seize the market; SO3: With the national strategy as the background and the economic and safety of the small passenger dedicated line, the publicity of the special line construction will be strengthened. WO1: expanding the radiation range of the dedicated transportation network; WO2: Increase the source of funds for the construction of special lines in the context of national policies; WO3: Utilize the rapid development of artificial intelligence to improve the operation mechanism of the dedicated line. T ST WT ST1: Development of multimodal transport; ST2: Develop an operation mechanism with unique characteristics of small passenger lines; ST3: Learn from other road transportation experiences to improve the laws and regulations for the operation of the special line. WT1: Improve the construction of special line laws and strengthen the operation control of special lines; WT2: Increase the funding source of the dedicated line construction and increase the radiation range of the special line transportation network; WT3: Specially designed for small passenger vehicles to improve the competitiveness of the dedicated line. Table 1. SWOT matrix of small passenger dedicated line. WT1: Improve the construction of special line laws and strengthen the operation control of special lines; WT2: Increase the funding source of the dedicated line construction and increase the radiation range of the special line transportation network; WT3: Specially designed for small passenger vehicles to improve the competitiveness of the dedicated line. 5.1 Advantage Opportunity Strategy (SO Strategy) For the small passenger dedicated line, combined with its own internal advantages and the use of external opportunities, it can be divided into 3 advantages opportunity strategy. (1) Taking the national policy as an opportunity, such as the construction of the “New Silk Road Economic Belt”, the “Western Development” and the “Rise of the Midwest”, etc., using the national preferential policies to vigorously develop the special line construction, and it is easy to use the special line construction engineering experience. Quickly establish a relatively complete dedicated line operation network with reference to the characteristics of short construction period and short construction period, which makes the special line more flexible and flexible in the operation process; (2) Using artificial intelligence to develop new technologies and new technologies for the construction of dedicated lines on the basis of the original road construction, making it easier to serve traffic and help to seize the transportation market; (2) Using artificial intelligence to develop new technologies and new technologies for the construction of dedicated lines on the basis of the original road construction, making it easier to serve traffic and help to seize the transportation market; (3) Taking the national strategy as the background and combining the economics and safety of the small passenger dedicated line, increase the publicity of the special line construction and increase the competitiveness of the special line. 5.3 Advantage Threat Strategy (ST Strategy) While exerting internal advantages and avoiding external threats, there are mainly 3 dominant threat strategies for China's small passenger dedicated lines. (1) Develop multimodal transport. Small passenger transport lines are not as transportable as rail and water transport, and are not as fast as air transport. However, on a shorter distance, the special line can guarantee fast transportation and free of charge to make up for the shortage of other modes of transportation. Therefore, the small passenger dedicated line can be combined with other modes of transportation to give full play to the internal advantages of the small passenger line; (1) Develop multimodal transport. Small passenger transport lines are not as transportable as rail and water transport, and are not as fast as air transport. However, on a shorter distance, the special line can guarantee fast transportation and free of charge to make up for the shortage of other modes of transportation. Therefore, the small passenger dedicated line can be combined with other modes of transportation to give full play to the internal advantages of the small passenger line; (2) Develop an operation mechanism with unique characteristics of small passenger lines. Because of its numerous internal advantages, especially because of its low cost, good security, and the implementation of full-line free system and rapidity, it is a strong advantage. New technologies and new methods must be utilized to enhance these internal strengths and develop operational mechanisms that are unique to small passenger lines; g p p q p g ; (3) Learn from other road transportation experiences to improve the laws and regulations for the operation of special lines. Since the small passenger line is a kind of road transportation, in the operation and management of the special line, other laws and regulations on the form of road transportation can be used to restrict the behavior of the vehicle on the special line, and strengthen the control of the special line. 5.2 Inferior Opportunity Strategy (WO Strategy) 5 5 , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 https://doi.org/10.1051/matecconf /201927201039 Taking advantage of external opportunities and overcoming internal disadvantages, there are mainly 3 inferior opportunity strategies for the construction of China's small passenger dedicated lines. Taking advantage of external opportunities and overcoming internal disadvantages, there are mainly 3 inferior opportunity strategies for the construction of China's small passenger dedicated lines. (1) Expand the radiation range of the dedicated transportation network. Taking the construction of the “New Silk Road Economic Belt” as an opportunity to vigorously promote the construction of special lines in the western and underdeveloped areas, and relying on the development of urban and rural integration to accelerate the penetration of small passenger lines between urban and rural areas. Increase the radiation range of the small passenger dedicated line in the country, and give full play to the speed and flexibility of the dedicated line; ; (2) Increase the source of funds for the construction of special lines in the context of national policies. In the context of national policy, such as the construction of the “New Silk Road Economic Belt”, the “Western Development” and the “Rise of the Midwest”, the region’s economic development will be driven, including tourism, agriculture and industry. Economic development will certainly have a large number of transportation line construction needs. For the local economic development, local enterprises and tourism operators will increase their willingness to build transportation routes and enhance the local special line construction financing; p g (3) Utilize the rapid development of artificial intelligence to improve the operation mechanism of the dedicated line. With the rapid development of artificial intelligence, new technologies such as face recognition, ETC toll channel and two-dimensional code recognition have emerged. The dedicated line can use ETC channel technology (but free of charge) for vehicle control. Or use techniques such as face recognition and two-dimensional code recognition to control the passing vehicles. 6 Conclusion The SWOT analysis method was used to analyze the construction of special routes for small passenger cars in China. Advantages and disadvantages of the dedicated line were discussed. The SWOT matrix was used to analyze its solution. The main conclusions are as follows: (1) The internal advantages of dedicated line construction is far greater than the internal disadvantages. It can be solved by analyzing external defects, so the construction of small passenger lines is feasible. (2) The external opportunity for the construction of the dedicated line is greater than the external threat it receives. External opportunities allow the line to take full advantage of its internal strength while avoiding external threats. so the construction of small passenger lines is necessary. y (3) With the development of national policy background and artificial intelligence, the small passenger transport line has great support in economy and technology. The construction of small passenger transport line will have better development prospects. y (3) With the development of national policy background and artificial intelligence, the small passenger transport line has great support in economy and technology. The construction of small passenger transport line will have better development prospects. 5.4 Inferior Threat Strategy (WT Strategy) In overcoming the internal disadvantages of the small passenger-only route and at the same time avoiding external threats, there are mainly 3 disadvantage threat strategies for small dedicated routes. 6 , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 https://doi.org/10.1051/matecconf /201927201039 (1) Improve the construction of special line laws and strengthen the operation control of special lines. We will improve the laws and regulations of small passenger lines and restrict driving behaviors to avoid traffic problems caused by inadequate laws and regulations. Sound laws and regulations also constrain the incorporation of other types of mobile and non-motorized vehicles into small passenger-only routes; (1) Improve the construction of special line laws and strengthen the operation control of special lines. We will improve the laws and regulations of small passenger lines and restrict driving behaviors to avoid traffic problems caused by inadequate laws and regulations. Sound laws and regulations also constrain the incorporation of other types of mobile and non-motorized vehicles into small passenger-only routes; (2) Increasing the source of funds for the construction of special lines and increasing the radiation range of the special line transportation network; (2) Increasing the source of funds for the construction of special lines and increasing the radiation range of the special line transportation network; (3) Specially serve small passenger vehicles to improve the competitiveness of the special line. Since the dedicated line specializes in small passenger vehicles, it is necessary to establish a corresponding emergency response mechanism for small passenger vehicles to improve the service quality and then increase the competitiveness of the small passenger dedicated line and expand its development scope. 7. Yang Jian.2014 The Influence of Mixed Speed of Low Speed Freight Cars on Small Cars Traffic Standardization,vol 42,pp 73-75. 8. YANG Zijie, JIA Zhizhen. 2014 Evolutionary analysis of the behavior of large and small cars in the traffic bottleneck Journal of Taiyuan University of Science and Technology, vol 35,pp49-53. 9. Yang Yao. 2016 Research on the minimum safe driving distance of expressway Chang'an University. 7. Yang Jian.2014 The Influence of Mixed Speed of Low Speed Freight Cars on Small Cars Traffic Standardization,vol 42,pp 73-75. 8. YANG Zijie, JIA Zhizhen. 2014 Evolutionary analysis of the behavior of large and small cars in the traffic bottleneck Journal of Taiyuan University of Science and Technology, vol 35,pp49-53. 9 Yang Yao 2016 Research on the minimum safe driving distance of expressway References 1. Fred L. Mannering, Venky Shankar, Chandra R. 2016 Unobserved heterogeneity and the statistical analysis of highway accident data Analytic Methods in Accident Research ,vol 11. 1. Fred L. Mannering, Venky Shankar, Chandra R. 2016 Unobserved heterogeneity and the statistical analysis of highway accident data Analytic Methods in Accident Research ,vol 11. 2. Young Ai Kim, Boong Yeol Ryoo, Yong-Su Kim, et al. 2013 Major Accident Factors for Effective Safety Management of Highway Construction Projects Journal of Construction Engineering and Management, vol 139. 3. Jonathan C. Comer, Nicholas J. Rose, Leonard S. 2014 Bombom. Poisson Regression Analysis of Highway Fatality Accident Data in Oklahoma International Journal of Applied Geospatial Research (IJAGR), vol 5. 4. Yan Shoujing, Liu Jinghui, Yang Zezhong, et al. 2017 Analysis of urban road traffic accident rate Traffic and Transportation,pp215-218. 5. WANG Jianjun, BI Mingtao. 2004 Research and Countermeasures of Highway Mixed Traffic Problem Highway pp 95-99. 6. LIANG Guo-hua, CHENG Guo-zhu, WANG Chun-yan, et al. 2014 Relationship between the mixed rate of large-scale expressway and traffic flow stability Journal of Chang'an University (Natural Science Edition), vol 34 ,pp 120-126. 7 , 0 (2019) MATEC Web of Conferences 272 ICFMCE 2018 1039 https://doi.org/10.1051/matecconf /201927201039 7. Yang Jian.2014 The Influence of Mixed Speed of Low Speed Freight Cars on Small Cars Traffic Standardization,vol 42,pp 73-75. 8. YANG Zijie, JIA Zhizhen. 2014 Evolutionary analysis of the behavior of large and small cars in the traffic bottleneck Journal of Taiyuan University of Science and Technology, vol 35,pp49-53. 9. Yang Yao. 2016 Research on the minimum safe driving distance of expressway Chang'an University. 8. YANG Zijie, JIA Zhizhen. 2014 Evolutionary analysis of the behavior of large and small cars in the traffic bottleneck Journal of Taiyuan University of Science and Technology, vol 35,pp49-53. 9. Yang Yao. 2016 Research on the minimum safe driving distance of expressway Chang'an University. 8 8
https://openalex.org/W2982576455	https://europepmc.org/articles/pmc6872825?pdf=render	English	null	Longer small bowel segments are resected in emergency surgery for ileocaecal Crohn’s disease with a higher ileostomy and complication rate	Techniques in coloproctology	2,019	cc-by	5,566	Abstract Background Repeated intestinal resections may have disabling consequences in patients with Crohn’s disease even in the absence of short bowel syndrome. Our aim was to evaluate the length of resected small bowel in patients undergoing elective and emergency surgery for ileocolic Crohn’s disease. Methods A prospective observational study was conducted on patients undergoing surgery for ileocolonic Crohn’s disease in a single colorectal centre from May 2010 to April 2018. The following patients were included: (1) patients with first pres- entation of ileocaecal Crohn’s disease undergoing elective surgery; (2) patients with ileocaecal Crohn’s disease undergoing emergency surgery; (3) patients with recurrent Crohn’s disease of the distal ileum undergoing elective surgery. The primary outcomes were length of resected small bowel and the ileostomy rate. Operating time, complications and readmissions within 30 days were the secondary outcomes. Results One hundred and sixty-eight patients were included: 87 patients in the elective primary surgery group, 50 patients in the emergency surgery group and 31 in the elective redo surgery group. Eleven patients (22%) in the emergency surgery group had an ileostomy compared to 10 (11.5%) in the elective surgery group (p < 0.0001). In the emergency surgery group the median length of the resected small bowel was 10 cm longer than into the group having elective surgery for primary Crohn’s disease. Conclusions Patients undergoing emergency surgery for Crohn’s disease have a higher rate of stoma formation and 30-day complications. Laparoscopic surgery in the emergency setting has a higher conversion rate and involves resection of longer segments of small bowel. Keywords Crohn’s disease · Laparoscopic colorectal surgery · Ileocaecal resection · Inflammatory bowel disease · Emergency surgery Longer small bowel segments are resected in emergency surgery for ileocaecal Crohn’s disease with a higher ileostomy and complication rate V. Celentano1,2 · D. P. O’Leary1 · A. Caiazzo3 · K. G. Flashman1 · F. Sagias1 · J. Conti1 · A. Senap Received: 1 July 2019 / Accepted: 17 October 2019 / Published online: 29 October 2019 © The Author(s) 2019 Techniques in Coloproctology (2019) 23:1085–1091 https://doi.org/10.1007/s10151-019-02104-9 Techniques in Coloproctology (2019) 23:1085–1091 https://doi.org/10.1007/s10151-019-02104-9 ORIGINAL ARTICLE ORIGINAL ARTICLE * V. Celentano valeriocelentano@yahoo.it 1 Colorectal Unit, Queen Alexandra Hospital, Portsmouth Hospitals NHS Trust, Portsmouth, UK * V. Celentano valeriocelentano@yahoo.it 1 Colorectal Unit, Queen Alexandra Hospital, Portsmouth Hospitals NHS Trust, Portsmouth, UK 2 University of Portsmouth, Portsmouth, UK 3 University of Campania “Luigi Vanvitelli”, Naples, Italy 3 University of Campania “Luigi Vanvitelli”, Naples, Italy Primary and secondary outcomes Length of resected small bowel and ileostomy rate were the primary outcomes. The secondary outcomes were operat- ing time, length of hospital stay (LOS), complications, reoperations and rehospitalisation within 30 days, and were recorded prospectively. Introduction [2]. Almost 40–50% of patients undergoing surgery for CD are likely to need further operations within 10–15 years [3], with smoking, a penetrating phenotype and previous small bowel surgery increasing the likelihood of postoperative recurrence. Over 80% of patients diagnosed with primary ileocolic Crohn’s disease (CD), who are typically young adults, have a surgical resection within 10 years of their diagnosis [1]. Of these, 30–50% will have symptomatic recurrence of disease during the first 5 years and 50–80% by 10 years after surgery Short bowel syndrome is a rare sequela of repeated sur- gical resection for CD, with a cumulative risk of home parenteral nutrition of 1.5% at 20 years after diagnosis [4]. Nevertheless, repeated intestinal resections leading to impaired gastrointestinal functioning with nutritional and vitamin deficiencies may have disabling consequences for the patients even in the absence of short bowel syndrome. Bile salt diarrhoea may occur in up to half of patients with CD following bowel resection [5] and loss of the ileocecal (0123 1 3456789) 3 3456789) 3 Techniques in Coloproctology (2019) 23:1085–1091 1086 valve may increase the risk of small bowel bacterial over- growth [6]. hospital admission for an acute presentation with small bowel obstruction, peritonitis or intra-abdominal sepsis due to complications of CD. Surgery does not cure CD, and should be used restric- tively, although it should not be regarded as the last resort [7]. Specific indications for surgery include abscesses, complex perianal or internal fistulae that are unresponsive or insufficiently responsive to medical therapy, fibrosten- otic strictures with symptoms of partial or complete bowel obstruction, high-grade dysplasia, and cancer [8]. The majority of patients are treated with elective surgery; how- ever, patients with intestinal perforation, peritonitis, exces- sive bleeding or toxic megacolon require urgent surgery. The risk of major abdominal surgery within the first 5 years of diagnosis has declined due to the advances in medical man- agement of CD with the use of disease-modifying agents [9], unfortunately without this reflecting in shorter segments of bowel being resected when surgery occurs [10]. More importantly, inappropriate delay in surgery may increase surgical morbidity [11] and affect short- and long-term outcomes. The indication for surgical resection was discussed at a dedicated inflammatory bowel disease (IBD) multidis- ciplinary team meeting (MDT) involving gastroenterolo- gists, colorectal surgeons, radiologists, pathologists, IBD and stoma nurses. Preoperative assessment included colo- noscopy, magnetic resonance enterography and intestinal ultrasound. Study design All patients undergoing surgery for ileocolic CD from May 1st 2010 to April 30th 2018 were included in this single-centre observational study designed according to the strengthening the reporting of observational studies in epidemiology (STROBE) checklist [12]. Patients were divided into three groups to compare the length of resected small bowel, the stoma rate and the short-term outcomes of ileocolic resection in: (1) patients with first presentation of ileocaecal CD undergoing elective surgery; (2) patients with ileocaecal CD undergoing emergency surgery; (3) patients with recurrent CD of the distal ileum undergoing elective surgery. Data on patients undergoing laparoscopic surgery were collected prospectively on a dedicated database with an intention to treat analysis. Patients undergoing surgery for recurrent disease were also included, while patients undergoing surgery for colonic or rectal disease only were excluded. Data for patients who underwent open surgery during the study period were retrospectively retrieved from review of medical notes and via the National Emergency Laparotomy Audit (NELA) database. Emergency surgery was defined as surgical resection during the same unplanned Data collection Aim of this prospective study is to evaluate the length of resected small bowel in patients undergoing elective and emergency surgery for ileocaecal CD, and to assess the ile- ostomy rate and postoperative complications in these two groups. Preoperative, operative and postoperative data were recorded for each patient. Preoperative parameters included age, sex, body mass index (BMI), comorbidities, American Society of Anesthesiologists (ASA) status, albumin and haemoglo- bin concentration, smoking status, weight loss, indication for surgery and preoperative medical therapy, Montreal classification. Materials and methods i Operative data included operating time, intraoperative complications, estimated operative blood loss, conversion rate, reason for conversion and use of temporary ileostomy. Length of resected small bowel was detailed in the histopa- thology report. Postoperative data included LOS, time to tol- erate oral fluids and oral diet, time to resolution of ileus and postoperative complications according to the Dindo–Clavien classification [13]. Ileostomy rate and length of resected small bowel The study was conducted in accordance with the principles of the Declaration of Helsinki and ‘good clinical practice’ guidelines. The database was approved by the local ethics committee and informed consent was obtained from all patients. Patients undergoing emergency surgery were more likely to have a stoma fashioned at the time of the surgery. Eleven patients (22%) had an ileostomy in the emergency surgery group compared to 10 (11.5%) in the elective surgery group (p < 0.0001). At 24 months after surgery one patient in the emergency surgery group (2%) and two patients (2.3%) in the elective surgery group still had a stoma. Statistical analysis Categorical variables are presented as frequency or percent- age and were compared with the use of the Chi-square test or Fisher’s exact test, as appropriate. Continuous variables are presented as mean (± standard deviation) or median (first and third quartile) and were compared with the use of Student’s t test. The Mann–Whitney U test was used for continuous, not normally distributed outcomes. Because of possible confounders (i.e. anti-tumour necrosis factor (TNF) use, use of steroids, preoperative weight loss) the primary outcomes were also evaluated in a multivariate analysis. Statistical analysis was performed by using the Statistical Package for Social Sciences (SPSS version 16.0; SPSS, Chicago, IL, USA) and GraphPad Prism version 8.0.2 for Windows (GraphPad Software, La Jolla, CA, USA, www. 1 3 3 Techniques in Coloproctology (2019) 23:1085–1091 1087 graphpad.com). All reported p values were two-tailed, and p values of less than 0.05 were considered to indicate statisti- cal significance. more than 10 years older than patients undergoing primary resection, as expected, and more male patients underwent redo surgery compared to primary and emergency surgery. The rate of penetrating disease was similar in the emergency surgery and primary elective groups. Patient characteristics Emergency surgery for ileocolic CD carries a risk of a longer length of small bowel being resected (median length of resected small bowel 30.4 cm), with additional 10 cm of small bowel being resected compared to elective surgery for primary CD (median length of resected small bowel 19 cm, p < 0.0001). Significantly shorter segments of small bowel were resected in elective surgery performed for recurrent ileocolic CD (Table 2). One hundred and sixty-eight patients were included: 87 patients in the elective primary surgery group, 50 patients in the emergency surgery group and 31 in the elective redo sur- gery group. In the elective primary surgery group 39% of the patients were male and the median age was 32 years (range 26–48 years), while in the emergency group 42% were male and the median age was 50 years (range 33–65 years) and in the elective redo surgery group 64% were male and median age was 46 years (range 35–59 years). None of the patients in the emergency surgery group had any previous CD resection. The length of resected small bowel was longer than 40 cm in 14 patients (28%) in the emergency surgery group, com- pared to 7 (8%) and none in the elective surgery groups for primary and recurrent CD, respectively (p < 0.0001). Baseline patients’ characteristics are detailed in Table 1. Patient characteristics Patients undergoing redo surgery for recurrent CD were Data are expressed as number (percentage) and median (lower–upper quartile) BMI body mass index, ASA American Society of Anesthesiologists, TNF tumour necrosis factor Statistically significant with p value < 0.0001 † Preoperative weight loss of > 5% during last 6 months †† Previous abdominal surgery ††† 20 mg or more Elective primary (n = 87) Emergency (n = 50) Elective redo (n = 31) Age (years) 32.5 (26–48) 50.5 (33.7–65.2) 46.5 (35–59)* Male-to-female ratio 34:53 21:29 20:11 * ASA class I 5 0 4 II 55 23 16 III 9 10 7 Missing data 18 17 4 BMI 23.75 (20–29.1) 26 (20.5–32.5) 25 (21.7–28) Weight loss† 21 (24.1%) 10 (20%) 5 (16.1%) Previous surgery†† 16 (18.3%) 13 (26%) 31 (100%)* Penetrating disease 15 (17.2%) 11 (22%) 2 (6.4%)* Preoperative anti-TNF 41 (47.1%) 21 (42%) 7 (22.5%)* Preoperative steroids††† 15 (17.2%) 14 (28%) 1 (3.2%)* Table 1 Baseline patient characteristics Data are expressed as number (percentage) and median (lower–upper quartile) † Preoperative weight loss of > 5% during last 6 months 1 3 Techniques in Coloproctology (2019) 23:1085–1091 1088 Table 2 Short-term outcomes for emergency, recurrent and elective ileocolic resection for CD Table 2 Short-term outcomes for emergency, recurrent and elective ileocolic resection for CD Data are expressed as number (percentage) and median (lower–upper quartile) LOS length of hospital stay Statistically significant with p value < 0.0001 Elective primary (n = 87) Emergency (n = 50) Elective redo (n = 31) Open surgery 2 (2.3%) 15 (30%) 1 (3.2%) Laparoscopic surgery 85 (97.7%) 35 (70%)* 30 (96.8%) Conversion to open 3 (3.5%) 4 (11.4%)* 4 (12.9%)* Operating time (min) 140 (105–180) 170 (117.5–205)* 180 (143.8–198.8)* Ileostomy formation 10 (11.5%) 11 (22%)* 2 (6.4%) LOS (days) 6 (5–8) 8 (5–13)* 6 (4.7–10) Readmissions 11 (12.6%) 5 (10%) 6 (19.3%) Reoperations 1 (1.1%) 2 (4%)* 2 (6.4%)* 30-day complications 20 (22.9%) 17 (34%)* 9 (29%) Length of resected small bowel (cm) 19 (13–26) 30.4 (20–42)* 11 (8–17) Elective primary (n = 87) Emergency (n = 50) Elective redo (n = 31) CD Crohn’s disease 30‑day morbidity and mortality Conversion rate was 11.4% in the emergency surgery group, similar to 12.9% in the elective redo surgery group, but sig- nificantly different from 2.4% in the elective surgery group (p < 0.0001). Operating time was 30 min longer and LOS was 2 days longer in patients undergoing emergency sur- gery compared to elective primary ileocolic CD surgery (Table 2). Seventeen patients (34%) in the emergency surgery group, 9 in the elective redo surgery group (29%), and 20 (22.9%) in the elective primary surgery group experienced complica- tions (p < 0.0001). Complications are detailed in Table 3. There was no mortality. Table 3 Detailed 30-day morbidity and reoperations Elective surgery for primary ileocolic CD: 20 patients (22.9%) experienced a total of 26 complications CD Crohn’s disease Elective surgery for primary ileocolic CD: 20 patients (22.9%) experienced a total of 26 complications 8 Wound infection 7 Ileus requiring total parenteral nutrition 4 Intra-abdominal collection treated with radiological guided drainage 3 Bleeding: 2 requiring transfusions, 1 treated with laparotomy and washout 2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 requiring laparotomy and stoma formation 1 High-output stoma 1 Mechanical bowel obstruction due to internal hernia requiring laparotomy Emergency surgery: 17 patients (34%) experienced a total of 22 complications 6 Intra-abdominal collection requiring radiological guided drainage 3 Mechanical bowel obstruction: two treated conservatively, one requiring reoperation 3 Wound infection 3 High-output stoma 2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 treated with laparotomy and stoma formation 2 Ileus requiring total parenteral nutrition 1 Bleeding requiring transfusions 1 Enterocutaneous fistula 1 Parastomal hernia Elective redo surgery for recurrent ileocolic CD: 9 patients (29%) experienced a total of 12 complications 6 Wound infection 3 Ileus requiring total parenteral nutrition 2 Anastomotic leak requiring laparotomy and stoma formation 1 Bleeding requiring transfusions 8 Wound infection 7 Ileus requiring total parenteral nutrition 4 Intra-abdominal collection treated with radiological guided drainage 3 Bleeding: 2 requiring transfusions, 1 treated with laparotomy and washout 2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 requiring laparotomy and stoma formation 1 High-output stoma 1 Mechanical bowel obstruction due to internal hernia requiring laparotomy 1 Mechanical bowel obstruction due to internal hernia requiring laparotomy Emergency surgery: 17 patients (34%) experienced a total of 22 complications 1 Mechanical bowel obstruction due to internal hernia requiring laparotomy Emergency surgery: 17 patients (34%) experienced a total of 22 complications Emergency surgery: 17 patients (34%) experienced a total of 22 complications 6 Intra-abdominal collection requiring radiological guided drainage 3 Mechanical bowel obstruction: two treated conservatively, one requiring reoperation 3 d i f i g p 2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 treated with laparotomy and stoma formation 2 Ileus requiring total parenteral nutrition g p 2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 treated with laparotomy and stoma formation 2 Il i i t t l t l t iti 2 Ileus requiring total parenteral nutrition 1 Bleeding requiring transfusionsi 1 Enterocutaneous fistula i 1 Parastomal hernia Elective redo surgery for recurrent ileocolic CD: 9 patients (29%) experienced a total of 12 complications 6 Wound infection 3 Ileus requiring total parenteral nutrition 3 Ileus requiring total parenteral nutrition 2 A i l k i i l d f i 3 Ileus requiring total parenteral nutrition 2 Anastomotic leak requiring laparotomy and stoma formation Anastomotic leak requiring laparotomy and stoma formatio 1 Bleeding requiring transfusions 3 Techniques in Coloproctology (2019) 23:1085–1091 1089 Multivariate analysis Multivariate analysis was performed to test for potential con- founding factors in view of the small sample size. Length of resected small bowel and ileostomy rate in the emergency and elective groups were evaluated in a multivariable logis- tic regression model, with no statistically significant differ- ence demonstrated regarding age (p = 0.42), preoperative weight loss > 5% (p = 0.54), ASA grade (p = 0.88), BMI (p = 0.31), preoperative steroids > 20 mg (p = 0.8) and anti- TNF treatment (p = 0.68). Our study reported a rate of stoma formation of 11.5% when surgery is performed electively, compared to 22% in the emergency setting. The presence of a stoma can sig- nificantly affect patients’ quality of life [23, 24] and is also associated with a risk of complications and re-interventions [25]. A stoma rate up to 35% has been described for com- plicated CD [26] and a penetrating phenotype of CD may explain a more common use of stomas in selected patients, because of abscesses and intra-abdominal contamination, or due to complex fistulae requiring more than one intesti- nal resection. Nevertheless, stricturing disease is the most common indication for surgery in ileocolic CD [27] and no difference was found in our study in the rate of penetrat- ing phenotype between the emergency and elective surgery groups. A recent meta-analysis reported that a third of the ileostomies are reversed with a stapled technique, resulting in further small bowel resection [28], which needs to be taken into account when considering the cumulative bowel loss in patients undergoing emergency surgery and where the surgery is complicated by anastomotic leak requiring relaparotomy. Readmissions and reoperations this reason we considered recurrent CD as a separate group in this study. Five patients (10%) in the emergency surgery group were readmitted within 30 days from discharge, compared to 6 (19.3%) and 11 (12.6%) in the redo surgery and pri- mary elective surgery groups, respectively (p < 0.0001). The reoperation rate did not significantly differ amongst the three groups. Reasons for reoperations are detailed in Table 3. Prompt planning of elective surgery and high-volume IBD surgeons may impact on length of the resected speci- men, stoma rate and postoperative outcomes when surgery is performed in specialist referral centres, which may be more often the case in recurrent CD, with surgeons more aware of the risk of short bowel syndrome and more famil- iar with the use of bowel-preserving techniques (i.e. stric- tureplasties) [20]. These hypotheses, however, need to be addressed in larger prospective studies. During the 9-year study period 50 patients (29.7%) within the total population of patients requiring surgery for CD at our institution, had an emergency operation, which is in agreement with previously reported rates of 35% [21] and 42% [22] for non-scheduled surgery in CD, highlighting the difficulties in predicting the course of disease in a significant proportion of patients. It is important to consider how these rates may be affected by the adopted denotation of emergency surgery, which was broadly defined in our study as surgical resection during the same unplanned hospital admission for an acute presenta- tion of CD. Discussion Emergency surgery for primary ileocolic CD carries a risk of longer segments of small bowel being resected and a higher likelihood of an ileostomy being fashioned at the time of the surgery, compared to elective surgery for primary ileocolic CD. A median length of 23 cm of resected small bowel has been recently reported in patients undergoing surgery for CD, with a cumulative length of resected small bowel of 36 cm at 15 years [14]. Our study has similar findings, with a median of 30 and 19 cm of resected ileum in the emergency and primary elective surgery groups, respectively. It is important to note that the risk of short bowel syn- drome in CD is low [15] and is mainly due to repeated operations because of complications, rather than recur- rence, and that the length of remaining small bowel is more important than the extent of resection [16]. Nevertheless, multiple resections of the diseased bowel may result in func- tional diarrhoea, fat malabsorption, and ultimately short bowel syndrome, requiring parenteral nutrition treatment, and affecting patients’ quality of life with selective vitamin deficiencies and malnutrition. The Lémann index assesses globally the cumulative structural bowel damage that can occur in CD [17]. Surgical resection of the bowel, being irreversible, is considered the maximum level of bowel dam- age in this index. The significantly shorter length of resected small bowel in patients undergoing redo surgery for recur- rent ileocolic CD has been previously demonstrated [18] and may be explained by the fibrostenotic phenotype that typically involves anastomotic CD recurrence [19] and for Our study found a high complication rate following ileocolic CD surgery, varying from 22.9 to 34% in elec- tive and emergency surgery, similar to the published lit- erature [29]. The association between procedural volume and surgical outcomes is well-described throughout all types of surgery, including those for IBD [30] with up to a twofold in-hospital mortality increase in low-volume hospitals [31]. Surgery for CD is technically challenging, due to multifocal disease, a thickened mesentery and the potential for fistulae, abscesses, and large phlegmons [32] 1 3 1090 Techniques in Coloproctology (2019) 23:1085–1091 and the perioperative decision-making of when to oper- ate and whether to fashion an anastomosis or to create a stoma, requires highly trained surgeons [33]. Discussion Never- theless, the underlying mechanisms for the relationship between surgical volume and postoperative mortality are likely multifactorial and more complex than just surgeon experience as higher volume hospitals may have more institution-level-related resources and infrastructure such as operating room volume and intensive care unit beds that may facilitate surgery. Patients with CD require a multi- disciplinary approach [34] for an essential close and struc- tured integration of medical and surgical management to identify the right time for surgery with the aim of prevent- ing emergency surgery, postoperative complications and recurrence. It is a quality requirement that patients having surgery for IBD have it undertaken by a colorectal surgeon who is a core member of the IBD multidisciplinary team [35] auditing stoma rate, complications, re-interventions and mortality [36]. Author contributions VC: conception and design of the study, acqui- sition, analysis and interpretation of data, manuscript drafting. DPO: acquisition of data, manuscript drafting. AC: acquisition of data, manuscript drafting. KGF: acquisition of data, manuscript drafting. FS: analysis of data, critical review of the manuscript. JC: analysis of data, critical review of the manuscript. AS: interpretation of data, critical review of the manuscript. JK: interpretation of data, critical review of the manuscript. All authors have reviewed and approved the final version of the manuscript to be published. All authors agree to be accountable for all aspects of the study regarding the accuracy and integrity of the work presented. Author contributions VC: conception and design of the study, acqui- sition, analysis and interpretation of data, manuscript drafting. DPO: acquisition of data, manuscript drafting. AC: acquisition of data, manuscript drafting. KGF: acquisition of data, manuscript drafting. FS: analysis of data, critical review of the manuscript. JC: analysis of data, critical review of the manuscript. AS: interpretation of data, critical review of the manuscript. JK: interpretation of data, critical review of the manuscript. All authors have reviewed and approved the final version of the manuscript to be published. All authors agree to be accountable for all aspects of the study regarding the accuracy and integrity of the work presented. References 1. Bernell O, Lapidus A, Hellers G (2000) Risk factors for surgery and recurrence in 907 patients with primary ileocaecal Crohn’s disease. Br J Surg 87:1697–1701 2. Spinelli A, Sacchi M, Bazzi P, Leone N, Danese S, Montorsi M (2012) Laparoscopic surgery for recurrent Crohn’s disease. Gastroenterol Res Pract 2012:381017 3. Gordon PH, Nivatvongs S (2007) Informa Healthcare 2007: Crohn’s disease, New York, pp 820–907 4. Yadav S, Edakkanambeth Varayil J, Harmsen S (2014) Inci- dence and outcomes of home parenteral nutrition in patients with Crohn’s disease in Olmsted County, Minnesota. Am J Gas- troenterol 109(Suppl):S506i 5. Cosnes J, de Parades V, Carbonnel F et al (1994) Classification of the sequelae of bowel resection for Crohn’s disease. Br J Surg 81:1627–1631 6. Klaus J, Spaniol U, Adler G, Mason RA, Reinshagen M, von Tirpitz CC (2009) Small intestinal bacterial overgrowth mim- icking acute flare as a pitfall in patients with Crohn’s Disease. BMC Gastroenterol 9:61 Compliance with ethical standards Another limitation of our study is that no direct patient reported outcome measures were assessed and no data were collected on smoking status and rate and length of postoperative admission to intensive care amongst the three different groups of patients. Elective CD surgeries are all performed by dedicated colorectal surgeons in our department, while an emergency surgery subspecialty colorectal service provision was only introduced during the second half of the study period, introducing a bias on the contribution of training and expertise of the operating surgeons. Finally, the effect of emergency surgery on the risk of clinical and surgical recurrence cannot be evaluated because of the lack of long-term follow-up, and a larger prospective study, with patients stratified according to the risk of recurrence is desirable. 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. Compliance with ethical standards Conflict of interest The authors declare no conflict of interest. No funding has been received for this study. The abstract of this manu- script has been presented as an e-poster to the American Society of Colon and Rectal Surgeon Annual Meeting 2019 in Cleveland. Ethical approval The study was conducted in accordance with the principles of the Declaration of Helsinki and ‘good clinical practice’ guidelines. The database was approved by the local ethics committee. This is a single-centre study with patients being recruited within a study period of 9 years, and concerns about cases being performed at different stages of the learning curve might be raised, particularly with relation to conversion to open surgery. The cumulative conversion rate was 7.3% (11 cases out of 150), however 7 of the 11 conversions occurred in the last 3 years of the study period, suggesting more complex cases being approached laparoscopically rather than being due to the learning curve. Another limitation of our study is that no direct patient reported outcome measures were assessed and no data were collected on smoking status and rate and length of postoperative admission to intensive care amongst the three different groups of patients. Elective CD surgeries are all performed by dedicated colorectal surgeons in our department, while an emergency surgery subspecialty colorectal service provision was only introduced during the second half of the study period, introducing a bias on the contribution of training and expertise of the operating surgeons. Finally, the effect of emergency surgery on the risk of clinical and surgical recurrence cannot be evaluated because of the lack of long-term follow-up, and a larger prospective study, with patients stratified according to the risk of recurrence is desirable. Informed consent Informed consent was obtained from all patients. This is a single-centre study with patients being recruited within a study period of 9 years, and concerns about cases being performed at different stages of the learning curve might be raised, particularly with relation to conversion to open surgery. The cumulative conversion rate was 7.3% (11 cases out of 150), however 7 of the 11 conversions occurred in the last 3 years of the study period, suggesting more complex cases being approached laparoscopically rather than being due to the learning curve. Conclusions Madani R, Day N, Kumar L, Tilney HS, Gudgeon AM (2018) Hand-Sewn versus stapled closure of loop ileostomy: a meta-anal- ysis. Dig Surg. https://doi.org/10.1159/000487310 (Epub ahead of print) 14. Peyrin-Biroulet L, Harmsen WS, Tremaine WJ, Zinsmeister AR, Sandborn WJ, Loftus EV Jr (2016) Cumulative length of bowel resection in a population-based cohort of patients with Crohn’s Disease. Clin Gastroenterol Hepatol 14(10):1439–1444 29. Rosenfeld G, Qian H, Bressler B (2013) The risks of post-opera- tive complications following pre-operative infliximab therapy for Crohn’s disease in patients undergoing abdominal surgery: a sys- tematic review and meta-analysis. J Crohns Colitis 7(11):868–877 15. Uchino M, Ikeuchi H, Bando T et al (2012) Risk factors for short bowel syndrome in patients with Crohn’s disease. Surg Today 42(5):447–452 30. Nguyen GC, Steinhart AH (2014) The impact of surgeon vol- ume on postoperative outcomes after surgery for Crohn’s disease. Inflamm Bowel Dis 20(2):301–306 16. Nightingale JM, Lennard-Jones JE (1995) Adult patients with a short bowel due to Crohn’s disease often start with a short normal bowel. Eur J Gastroenterol Hepatol 7(10):989–991 l 31. Kaplan GG, McCarthy EP, Ayanian JZ, Korzenik J, Hodin R, Sands BE (2008) Impact of hospital volume on postoperative mor- bidity and mortality following a colectomy for ulcerative colitis. Gastroenterology 134:680–687fi 17. Pariente B, Mary JY, Danese S et al (2015) Development of the Lémann Index to assess digestive tract damage in patients with Crohn’s disease. Gastroenterology 148:52–63 32. Celentano V, Finch D, Forster L, Robinson JM, Griffith JP (2015) Safety of supervised trainee-performed laparoscopic surgery for inflammatory bowel disease. Int J Colorectal Dis 30:639–644 18. Pelletier AL, Stefanescu C, Vincent C, Etienney I, Mentré F, Soulé JC (2011) Is the length of postoperative recurrence on the neo ileum terminal ileum predictable in Crohn’s disease? J Crohns Colitis 5(1):24–27 l 33. Morar PS, Hollingshead J, Bemelman W et al (2017) Establish- ing key performance indicators [KPIs] and their importance for the surgical management of inflammatory bowel disease-results From a Pan-European. Delphi Consensus Study. J Crohns Colitis 11(11):1362–1368 19. Celentano V, Sagias F, Flashman KG, Conti J, Khan J (2019) Lap- aroscopic redo ileocolic resection for Crohn’s disease in patients with previous multiple laparotomies. Scand J Surg 108(1):42–48 34. IBD Standards Group (2013) Standards for the healthcare of peo- ple who have inflammatory bowel disease [IBD]. 2013 update. Oyster Healthcare Communications, St Albans, UKl 20. Conclusions 7. Baumgart DC, Sandborn WJ (2012) Crohn’s disease. Lancet 380(9853):1590–1605 Patients who have emergency surgery for CD have a higher rate of stoma formation and 30-day complications. Laparo- scopic surgery for CD in the emergency setting has a higher conversion rate and involves resection of longer segments of small bowel. 8. Larson DW, Pemberton JH (2004) Current concepts and contro- versies in surgery for IBD. Gastroenterology 126:1611–1619 9. Rungoe C, Langholz E, Andersson M et al (2014) Changes in medical treatment and surgery rates in inflammatory bowel dis- ease: a nationwide cohort study 1979–2011. Gut 63:1607–1616 9. Rungoe C, Langholz E, Andersson M et al (2014) Changes in medical treatment and surgery rates in inflammatory bowel dis- ease: a nationwide cohort study 1979–2011. Gut 63:1607–1616 1 3 Techniques in Coloproctology (2019) 23:1085–1091 1091 24. Knowles SR, Wilson J, Wilkinson A et al (2013) Psychological well-being and quality of life in Crohn’s disease patients with an ostomy: a preliminary investigation. J Wound Ostomy Continence Nurs 40(6):623–629f 10. de Groof EJ, Gardenbroek TJ, Buskens CJ et al (2017) The asso- ciation between intensified medical treatment, time to surgery and ileocolic specimen length in Crohn’s disease. Colorectal Dis 19(6):551–558 11. Iesalnieks I, Kilger A, Glass H, Obermeier F, Agha A, Schlitt HJ (2010) Perforating Crohn’s ileitis: delay of surgery is associ- ated with inferior postoperative outcome. Inflamm Bowel Dis 16(12):2125–2130 25. Scarpa M, Ruffolo C, Bassi D et al (2009) Intestinal surgery for Crohn’s disease: predictors of recovery, quality of life, and costs. J Gastrointest Surg. 13(12):2128–2135 26. Goyer P, Alves A, Bretagnol F, Bouhnik Y, Valleur P, Panis Y (2009) Impact of complex Crohn’s disease on the outcome of laparoscopic ileocecal resection: a comparative clinical study in 124 patients. Dis Colon Rectum 52:205–210 12. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP, STROBE Initiative (2008) The strength- ening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol. 61(4):344–349 27. Myrelid P, Marti-Gallostra M, Ashraf S et al (2014) Complica- tions in surgery for Crohn’s disease after preoperative antitumour necrosis factor therapy. Br J Surg 101(5):539–545 13. Dindo D, Demartines N, Clavien PA (2004) Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg 240:205–213 28. Conclusions Pellino G, Selvaggi F, Ghezzi G et al (2015) A think tank of the Italian society of colorectal surgery (SICCR) on the surgical treat- ment of inflammatory bowel disease using the Delphi method: Crohn’s disease. Tech Coloproctol 19(10):639–651 35. NICE Quality Standard [QS81]—Inflammatory Bowel Disease. http://www.nice.org.uk/guidance/qs81/chapter/quality-statement- 2-multidisciplinary-team-support. Accessed 30 Sep 2018 21. Ma C, Moran GW, Benchimol E et al (2017) Surgical Rates for Crohn’s Disease are decreasing: a population-based time trend analysis and validation study. Am J Gastroenterol 112(12):1840–1848 36. Calvet X, Panes J, Alfaro N et al (2014) Delphi consensus state- ment: quality Indicators for inflammatory bowel disease compre- hensive care units. J Crohns Colitis 8:240–251 22. Aquina CT, Probst CP, Becerra AZ (2016) Emergency surgery for inflammatory bowel disease in the 21st century remains life- threatening—a continual failure of decision-making? J Am Coll Surg 223(4):e84–e85f Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 23. Schiergens TS, Hoffmann V, Schobel TN et al (2017) Long- term quality of life of patients with permanent end ileostomy: results of a nationwide cross-sectional survey. Dis Colon Rectum 60(1):51–60 1 3 1 3 1 3 3
https://openalex.org/W4377989892	https://www.researchsquare.com/article/rs-2962885/latest.pdf	English	null	Elemental Profiling and mapping analysis of the Excreta of Trogopterus xanthipes (Wulingzhi) Collected from Different Regions using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Multivariate Statistical Analysis	Research Square (Research Square)	2,023	cc-by	8,972	Elemental Profiling and mapping analysis of the Excreta of Trogopterus xanthipes (Wulingzhi) Collected from Different Regions using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Multivariate Statistical Analysis Anqi Wang (  angelking521@163.c Peking University Zheng Li Beijing Institute for Drug Control Yiyi Zhao Beijing Institute for Drug Control Jinghui Wang Beijing Institute for Drug Control Xintong Fu Beijing Institute for Drug Control Yougen Chen Beijing Institute for Drug Control Min Ye Peking University Hongzhu Guo Beijing Institute for Drug Control Anqi Wang (  angelking521@163. Peking University Zheng Li Beijing Institute for Drug Control Yiyi Zhao Beijing Institute for Drug Control Jinghui Wang Beijing Institute for Drug Control Xintong Fu Beijing Institute for Drug Control Yougen Chen Beijing Institute for Drug Control Min Ye Peking University Hongzhu Guo Beijing Institute for Drug Control Research Article Keywords: Trogopterus xanthipes excreta, elemental profiling, Wulingzhi, inductively coupled plasma mass spectrometry, major and trace elements, multivariate statistical analysis Posted Date: May 24th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2962885/v1 Abstract Inorganic element content is an important quality and origin indicator for herbs. The excreta of Trogopterus xanthipes (“Wulingzhi” in Chinese; WLZ) is a well-known traditional Chinese medicine that has been widely used to treat amenorrhea, menstrual pain, and postpartum abdominal pain for thousands of years. However, there has been less emphasis on the analysis of trace element content in WLZ. This study aimed to investigate the elemental characteristics of WLZ obtained from different regions (Shānxi, Shănxi, Hubei, Hebei) and perform a risk and benefit analysis to assess its quality and safety. Thirty elements in sixty batches of WLZ and their products processed with vinegar (V-WLZ) were compared using inductively coupled plasma mass spectrometry (ICP-MS). Multivariate statistical techniques, including principal component analysis (PCA), correlation analysis, elemental mapping analysis, and metal pollution index (MPI) analysis, were applied to interpret the data. The box plots showed a heterogeneous distribution of almost all analyzed elements, indicating significant differences between the samples. This is the first study to provide the reference values for major, trace, and toxic elements in WLZ. Furthermore, the results revealed that the average value of heavy-MPI in V-WLZ (2.030) was slightly lower than that in WLZ (2.096). Combining the PCA data and the inorganic element spectra, we identified Fe, Al, Cu, Se, Pb, Rb, V, K, P, (Na), Cr, As, and Ni as the characteristic elements of WLZ and V- WLZ. DOI: https://doi.org/10.21203/rs.3.rs-2962885/v1 DOI: https://doi.org/10.21203/rs.3.rs-2962885/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/27 Page 1/27 1. Introduction Wulingzhi (WLZ), the dry excreta of Trogopterus xanthipes, is a widely used traditional Chinese medicine that plays a vital role in the clinical treatment of amenorrhea, menstrual pain, and dysmenorrhea by promoting blood circulation and preventing stasis [1, 2]. In clinical practice, the most commonly used form of WLZ is its product processed with vinegar (V-WLZ), which promotes the entry of active ingredients into the liver and enhances hemostasis. WLZ and V-WLZ have been reported to possess various pharmacological properties, such as the inhibition of platelet aggregation, immunity enhancement, control of antithrombin levels, cytotoxic activity, and anti-inflammatory activities [3, 4]. Although many studies on WLZ have been reported, there were less emphasis has been given to the analysis of trace metal content in WLZ [5]. Trace elements are essential because animals cannot synthesize them, and these elements are derived from the diet, water, and other sources [6]. Moreover, trace elements play structural and chemical roles, and their mobilization provides one of the most extensive forms of signaling for biological regulation at the cellular level [7, 8]. Studies have shown that essential trace elements, including V, Cr, Mn, Fe, Co, Cu, Zn, Se, Ni, As, Rb, and Sr have special physiological functions [9] and are required by the human body in very small quantities [10]. Notably, Cu, Ni, Cr, Zn, and Se may be toxic at high concentrations; however, they are necessary to maintain some decisive metabolic pathways in the human body [11, 12]. Other studies have reported that the element Zn, as a catalytic, structural, and regulatory ion, can enhance the body’s immunity, accelerate wound healing, and regulate various enzymes and receptors, thereby influencing the biological function of the brain [13]. Additionally, toxic elements, such as As, Pb, and Cd, are known to have detrimental effects on human Page 2/27 Page 2/27 health [14, 15]. Human consumption and uptake of traditional Chinese medicine contaminated with toxic heavy elements [16] result in alterations in the functioning of organs and the central nervous system, with the risk increasing proportionally with the quantity consumed [17]. Consequently, research on the concentration of inorganic elements in WLZ is crucial to reflect the properties of WLZ, provide a guideline for further research on its active ingredients, and offer scientific evidence for quality control. 1. Introduction In a previous study, research on the elemental composition of WLZs was minimal, coupled with a small sample volume and few trace elements involved, resulting in a lack of a holistic and systematic metallomic profiling. Therefore, performing a risk and benefit elemental analysis is important to assess the quality and safety of WLZs. This study aimed to reveal the elemental characteristics of WLZ in terms of major (23Na, 24Mg, 27Al, 31P, 39K, 44Ca) and trace (9Be, 51V, 52Cr, 55Mn, 57Fe, 59Co, 60Ni, 63Cu, 64Zn, 71Ga, 75As, 77Se, 85Rb, 88Sr, 107Ag, 114Cd, 118Sn, 121Sb, 133Cs, 138Ba, 202Hg, 205Tl, 208Pb, 238U) elements by utilizing ICP-MS. The presence of these elements in WLZ ranged from nanogram to microgram levels. Similar to the Kopi Luwak civet coffee, which is one of the most expensive coffees in the world, WLZ is the excreta of Trogopterus xanthipes resulting from the digestion of Cacumen Platycladi [18]. Based on the above information, we speculated that there may be a close relationship between WLZ and Cacumen Platycladi; therefore, it is meaningful to investigate element correlation and internal connection between WLZ elemental composition and Cacumen Platycladi. In this study, ICP-MS was used to characterize the inorganic components in 67 batches of WLZs and Cacumen Platycladi. Owing to its remarkable robustness, high sensitivity, high sample throughput, and simultaneous multi-element measurement capability, ICP-MS is the preferred analytical tool for rapid determination of metals at trace and ultra-trace levels in traditional Chinese medicine, typically pretreated with microwave-assisted digestion [19–22]. In addition, the ICP-MS method has been optimized to reduce spectral (polyatomic and isobaric) and non-spectral interference that can significantly affect multi- analyte determination [23–25]. The ICP-MS method proposed in this study was fully validated. The results suggest that WLZ is rich in K, Ca, Mg, P, Al, Fe, Na, with K being the most abundant element, followed by Ca. Notably, Na levels were significantly elevated after vinegar processing. Furthermore, the toxic elements in WLZs were analyzed to determine the maximum tolerable concentration limits in plants (Chinese Pharmacopoeia 2020). ICP-MS data were interpreted using chemometrics and subjected to Pearson’s correlation analysis and principal component analysis (PCA) to determine the relationships and similarities among the elements in WLZs and Cacumen Platycladi. Metal pollution index (MPI) and heavy-MPI (H-MPI) were used to investigate the concentrations of metal elements in WLZs. 1. Introduction The inorganic element spectra of WLZs and Cacumen Platycladi were depicted, and the characteristic elements reflecting the overall quality were comprehensively analyzed to provide a basis for their quality control. 2.1. Instrumentation A QCap inductively coupled plasma mass spectrometer (Thermo Fisher Scientific, USA) was used for simultaneous analysis, and the operating conditions of the instrument are listed in Table 2. In addition, the operating conditions for microwave digestion instrument (Mars-5; CEM, NC, USA) are listed in Table 2. A Milli-Q Synthesis Purification System (Millipore, Billerica, MA, USA) was used to obtain ultrapure water. The samples were weighed using an AE-240 precision electronic balance (Mettler Toledo, USA). All polytetrafluoroethylene (PTEE) digestion vessels (CEM Mars X, USA) were soaked in 15% HNO3 (v/v) for 24 h, rinsed with ultrapure water, and dried before use. 2. Experimental Page 3/27 Page 3/27 2.3. Standard and reagents Nitric acid (BV Ш) was purchased from Beijing Chemical Works (Beijing, China) and used for sample digestion. Ultrapure water prepared using a Millipore Milli-Q water purification system was used in all experiments. A mixed internal standard (ISTD) solution containing Bi, Ge, In, Li, Lu, Rh, Sc, and Tb (100 ug/mL in 10% HNO3) and a multi-element calibration standard solution containing Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, Tl, U, V, Zn (10 ug/mL in 5% HNO3) were purchased from Agilent Technologies. A single-element standard solution of Hg (10 ug/mL in 5% HNO3) was provided by Agilent Technologies. Four remaining elements (P, Sn, Sb, and Au) were obtained from the National Institute of Metrology. TUNE iCAP Q/RQ solution (2% [v/v] HNO3 + 0.5% HCl) was obtained from Alfa Aesar. 2.2. Sample collection A total of 60 batches of WLZs were collected from four provinces in China and identified by Professor Yougen Chen of the Beijing Institute for drug control. These five provinces are the major production areas of WLZs. Seven batches of Cacumen Platycladi were obtained from these five provinces. Detailed sample information is presented in Table 1. WLZ refers to the raw product, and V-WLZ is the processed product stir-baked with vinegar. P represents the Cacumen Platycladi. Page 4/27 Table 1 Specific information of the 67 batches of WLZs and Cacumen Platycladi. No Species Collection area (province) No Species Collection area (province) W1 WLZ Shanxi-1 V1 V-WLZ Shanxi-1 W2 WLZ Shanxi-1 V2 V-WLZ Shanxi-2 W3 WLZ Shanxi-1 V3 V-WLZ Hebei W4 WLZ Shanxi-1 V4 V-WLZ Shanxi-1 W5 WLZ Shanxi-1 V5 V-WLZ Shanxi-1 W6 WLZ Shanxi-1 V6 V-WLZ Shanxi-1 W7 WLZ Shanxi-1 V7 V-WLZ Shanxi-1 W8 WLZ Shanxi-1 V8 V-WLZ Shanxi-1 W9 WLZ Shanxi-1 V9 V-WLZ Shanxi-2 W10 WLZ Shanxi-1 V10 V-WLZ Shanxi-2 W11 WLZ Shanxi-1 V11 V-WLZ Shanxi-2 W12 WLZ Shanxi-2 V12 V-WLZ Shanxi-2 W13 WLZ Shanxi-2 V13 V-WLZ Shanxi-1 W14 WLZ Shanxi-2 V14 V-WLZ Hebei W15 WLZ Shanxi-2 V15 V-WLZ Hebei W16 WLZ Shanxi-2 V16 V-WLZ Hebei W17 WLZ Shanxi-2 V17 V-WLZ Shanxi-1 W18 WLZ Shanxi-2 V18 V-WLZ Shanxi-1 W19 WLZ Shanxi-2 V19 V-WLZ Shanxi-1 W20 WLZ Shanxi-2 V20 V-WLZ Shanxi-2 W21 WLZ Shanxi-2 V21 V-WLZ Shanxi-2 W22 WLZ QinLing V22 V-WLZ Shanxi-1 W23 WLZ QinLing V23 V-WLZ Shanxi-1 W24 WLZ QinLing V24 V-WLZ Shanxi-1 W25 WLZ QinLing V25 V-WLZ Shanxi-2 W26 WLZ QinLing P1 Cacumen Platycladi Yunnan Page 5/27 Page 5/27 No Species Collection area (province) No Species Collection area (province) W27 WLZ QinLing P2 Cacumen Platycladi Anhui W28 WLZ QinLing P3 Cacumen Platycladi Shanxi-1 W29 WLZ Hubei P4 Cacumen Platycladi Shandong W30 WLZ Hubei P5 Cacumen Platycladi Shanxi-2 W31 WLZ Hubei P6 Cacumen Platycladi Shanxi-1 W32 WLZ Hubei P7 Cacumen Platycladi Shandong W33 WLZ Hubei W34 WLZ Specimen W35 WLZ Specimen 2.5. Preparation of standards Multi-element mixtures with a concentration of 1µg/mL were prepared as described above, using the multi-element calibration standard solution with twenty-seven elements. Thereafter, 1–1000 µg/L multi- element mixtures were prepared using serial dilution for external calibration. From a single-element stock solution of P, Sn, and Sb, 1–1000 ug/L of P, Sn, and Sb were prepared. Furthermore, 0.1–10 µg/L of Hg was prepared from a 10 µg/mL Hg stock solution. All the calibration curves were plotted so that the concentration of each element was within the linear range of the curves. Moreover, blanks were run along with the standard solutions (n = 6 at each level). To reduce non-spectral interference and effectively correct temporal variations in the signal intensity, an internal standard containing Li, Sc, Ge, Rh, In, Tb, Lu, and Bi was added to all calibration points and samples at a final concentration of 1000 µg/mL. Au (1 µg/mL) was added to the standard solutions to enhance Hg stability. 2.4. Sample preparation For ICP-MS analysis, approximately 0.5 g of each air-dried and powdered sample was weighed in a dried screw-cap polytetrafluoroethylene (PTEE) digestion vessel. Then, 8 mL of HNO3 was immediately added to each sample, and the vessels were kept for 24 h as a pre-digestion step for sufficient digestion. A two- step program using the microwave instrument was performed twice to digest the samples (Table 2). After digestion, the digestion vessels were taken out to cool down to ambient temperature, then acid-driving treatment was implemented at 80 ℃ for about 30 min for acid removal. Subsequently, the clear solutions were cooled down to room temperature, quantitatively transferred to plastic volumetric flasks, and made up to 50 mL with 1% HNO3 (v/v); 200 µL of Au single-element standard solution (1 µg/mL) was then Page 6/27 Page 6/27 added as the stabilizing agent. All samples were analyzed in triplicate and blanks were used to check for cross-contamination or loss. Blank experiments (n = 3) were performed following the same procedure. Table 2 Operation conditions of ICP-MS and microwave digestion system. Digestion program ICP-MS parameters Stage 1 2 RF power (W) 1300 Temperature (℃) 150 200 Cool gas (L/min) 14 Ramp time (time) 10 2 Auxiliary gas (L/min) 0.94 Hold time (time) 5 8 Sample depth (mm) 140 Power 1 (W) 200 600 Peristaltic pump volume (r/min) 40 Power 2 (W) 400 800 ORS mode He Cool down time Automatic Detection mode 2.6. Statistical analysis of results Raw ICP-MS data were calculated using Microsoft Excel, considering the weights of the dry samples (Appendix. A). Multivariate and univariate statistical analyses were performed to assess WLZ characteristics. Box plots were used to represent the distribution of all the measured values. The data for the 29 variables were statistically processed using PCA to describe the variability of species in the lower dimensions of the factors. Eigenvalues greater than one were used to determine the number of factors. Relationships between the mean content of the studied elements in the same WLZ or V-WLZ were described using the Pearson product-moment correlation coefficient (r). Moreover, H-MPI was applied to examine the heavy metal concentrations (except for P, As, Se, Na, Mg, Al, K, and Ca, which are non-metal Page 7/27 Page 7/27 Page 7/27 and light metal elements) in WLZs and Cacumen Platycladi samples. This index was obtained by calculating the geometrical mean of concentrations of all the metals in the samples with the equation: and light metal elements) in WLZs and Cacumen Platycladi samples. This index was obtained by calculating the geometrical mean of concentrations of all the metals in the samples with the equation: MPI= (Cm1⨯Cm2⨯…Cmn)1/n where Cmn (mg/Kg) = concentration of metal n in the sample [26]. where Cmn (mg/Kg) = concentration of metal n in the sample [26]. 3. Method validation The validation of the method further included limits of detection (LOD), limit of quantitation (LOQ), precision, accuracy, stability, and recovery. Linearity was assessed using the correlation coefficients (R2) originating from the calibration curves of each element constructed according to the least squares linear regression method. The LOD and LOQ were estimated by analyzing the signal response values of 11 replicate aliquots of blanks and were defined as three and ten times the standard deviation of 11 consecutive measurements divided by the slope of the calibration curve for all elements. As illustrated in Table 3, almost all calibration curves showed a good linear response with R2 values not less than 0.99 (R2 > 0.99). The LOD values were observed as 0.0003–0.6650 and LOQ values as 0.0010– 2.2168. According to these results, the LOD and LOQ can be efficiently applied for the quantification of major and trace elements in whole samples. Precision and accuracy were evaluated based on consecutively measured WLZ samples (n = 6) on the same day using the same instrument and operator. Both parameters were expressed as relative standard deviations (RSD). As a result of the analysis (Table 4), the precision values ranged from 0.29–3.85%, and the accuracy was 0.15–3.76% for all investigated elements, thereby meeting the requirements of routine monitoring for elements. Stability was estimated by repeated analyses of WLZ sample at 0, 2, 4, 8, 16, and 24 h using the same instrument. The RSD values were in the range 0.69–3.61% for various elements, indicating that the content of each element was stable within 24 h. Recovery studies were performed by analyzing a WLZ sample with known element content. Six parallel samples (approximately 0.5 g each) were accurately weighed and spiked with a known amount of elements, digested, and analyzed alongside the same non-spiked sample. The recoveries of the spiked samples were calculated by dividing the recovered portion of the spiked analyte by the known amount of the spiked analyte. All samples were analyzed in triplicate. The mean recovery for all elements was in the range 83.32–113.90%, with RSD values of 0.24–4.95% in different analytes. The results demonstrate that this method is valid for quantifying these elements in WLZs. Page 8/27 Table 3 Table 3 Summary of calibration equations, R2, linear calibration range, LODs, and LOQs data of optimal method used for the analysis of WLZs and Cacumen Platycladi analysis. 3. Method validation Summary of calibration equations, R2, linear calibration range, LODs, and LOQs data of optimal method used for the analysis of WLZs and Cacumen Platycladi analysis. Summary of calibration equations, R2, linear calibration range, LODs, and LOQs data of optimal method used for the analysis of WLZs and Cacumen Platycladi analysis. Elements Linear Calibration range (ug/L) Regression Equation R2 LOD (ug/L) LOQ (ug/L) 9Be 0–20 Y = 85.850X + 1.098 0.9868 0.6650 2.2168 23Na 0-100 Y = 2.436×103X+2.056×103 0.9988 0.1000 0.3333 24Mg 0-200 Y = 5.835×102X+1.667×103 0.9986 0.1000 0.3333 27Al 0-200 Y = 2.059×102X+1.812×103 0.9993 0.1000 0.3333 31P 10-1000 Y = 13.125X + 29.952 0.9999 0.5417 1.8057 39K 10-1000 Y = 7.832×102X+1.399×103 0.9995 0.1000 0.3333 44Ca 0-200 Y = 4.210×102X+1.256×103 0.9891 0.2592 0.8640 51V 0-200 Y = 1.693×104X+3.260×102 0.9993 0.0095 0.0317 52Cr 0-1000 Y = 2.699×104X+1.122×103 1.0000 0.0113 0.0377 55Mn 0-1000 Y = 1.326×104X+6.401×102 0.9999 0.0054 0.0180 57Fe 0-1000 Y = 5.965×102X+4.051×102 0.9999 0.1829 0.6097 59Co 0–50 Y = 5.052×104X+2.006×102 0.9999 0.0013 0.0043 60Ni 0-200 Y = 1.349×104X+3.373×102 0.9998 0.0053 0.0177 63Cu 0-200 Y = 3.572×104X+1.447×104 0.9999 0.0206 0.0687 64Zn 0-1000 Y = 1.006×104X+4.822×104 0.9999 0.6641 2.2137 71Ga 0–50 Y = 6.138×104X+25.652 0.9999 0.0049 0.0163 75 0 100 3 0 9998 0 0148 0 0493 Page 9/27 Elements Linear Calibration range (ug/L) Regression Equation R2 LOD (ug/L) LOQ (ug/L) 77Se 0-100 Y = 61.113X + 16.635 0.9994 0.2779 0.9263 85Rb 0-200 Y = 1.011×104X+2.055×102 0.9998 0.0010 0.0033 88Sr 0-1000 Y = 1.491×104X+5.307×103 0.9998 0.0119 0.0397 107Ag 0–50 Y = 7.045×103X+1.146×102 0.9966 0.0005 0.0017 114Cd 0–50 Y = 2.590×104X+72.114 0.9996 0.0011 0.0037 118Sn 0–50 Y = 1.797×104X+4.945×102 0.9998 0.0009 0.0030 121Sb 0–50 Y = 1.781×104X+6.659×103 0.9997 0.0297 0.0990 133Cs 0–50 Y = 4.028×104X+34.301 1.0000 0.0009 0.0030 138Ba 0-1000 Y = 6.223×104X+4.604×103 0.9999 0.0062 0.0207 205Tl 0–50 Y = 2.541×105X+9.107×102 0.9999 0.0003 0.0010 208Pb 0-200 Y = 1.817×105X+7.883×103 0.9999 0.0053 0.0177 238U 0–50 Y = 3.888×105X+86.048 0.9998 0.0002 0.0007 202Hg 0–50 Y = 7.295×103X+1.190×102 0.9947 0.0008 0.0027 Page 10/27 Table 4 Analytical performance of the ICP-MS method. 4. Results and Discussion 4. Results and Discussion 4.1. Sample characteristics Sixty batches of WLZs were collected from the Shanxi-1 (Shānxi), Shanxi-2 (Shănxi), Hubei, and Hebei provinces of China, which are the major production areas for WLZs. Samples W1-W11were collected from Shanxi-1 province; W12-W21 were collected from Shanxi-2 province; W22-W28 were collected from the same manufacturer in QL city, Shanxi-2 province; W29-W33 collected from Hubei province; W34-W35, in which W34 was collected from Hubei province in 2010 and W35 was collected from Shanxi-2 province in 2012. V1, V4-V8, V13, V18-V19, and V22-V23 were collected from Shanxi-2 province; V2, V9-V12, V20-V21, and V24-V25 were collected from Shanxi-1 province; and V3 and V14-V17 were collected from Hebei province. 3. Method validation Elements Precision (RSD%) Accuracy (RSD%) Stability (RSD%) Recovery rate (n = 6) Values (%) RSD (%) 9Be 3.85 3.73 3.61 111.50 3.52 23Na 1.41 1.82 2.10 107.89 4.01 24Mg 2.30 0.94 3.33 94.63 2.42 27Al 1.41 2.43 2.57 103.81 4.95 31P 0.52 1.30 0.93 104.02 4.28 39K 2.21 1.07 0.91 100.52 2.99 44Ca 0.64 0.50 1.31 83.32 2.11 51V 0.81 1.27 2.62 99.29 1.01 52Cr 0.48 0.36 0.81 101.88 1.16 55Mn 0.66 0.69 1.02 104.17 1.27 57Fe 0.82 1.83 1.53 91.98 1.70 59Co 0.55 2.22 2.89 85.63 2.43 60Ni 0.48 0.53 1.05 93.86 3.22 63Cu 0.51 0.49 1.19 86.30 1.92 64Zn 0.54 0.86 0.69 105.21 1.27 71Ga 0.40 0.67 1.33 99.89 0.74 75As 0.36 1.54 2.24 87.27 3.95 77Se 3.29 1.71 1.62 97.16 4.11 85Rb 0.29 0.82 0.84 95.64 3.20 88Sr 0.41 2.31 2.71 101.56 1.59 107Ag 2.70 0.77 3.45 105.1 1.26 114Cd 0.52 0.15 0.82 88.78 2.04 Page 11/27 Page 11/27 Elements Precision (RSD%) Accuracy (RSD%) Stability (RSD%) Recovery rate (n = 6) Values (%) RSD (%) 118Sn 1.47 2.40 2.84 111.53 0.24 121Sb 0.79 3.76 3.01 97.24 1.91 133Cs 0.68 1.42 1.27 105.05 1.14 138Ba 0.54 0.74 1.57 110.34 0.36 205Tl 0.59 1.14 0.97 104.63 1.46 208Pb 0.38 1.59 1.21 113.90 1.42 238U 0.79 2.95 1.88 108.43 1.45 202Hg 1.37 3.37 2.81 84.38 2.14 4.2. Intuitionistic analysis of elemental composition of WLZs A summary of the concentrations of the 30 elements in WLZs and Cacumen Platycladi is shown in the Appendix. A. Considerable attention has been paid to pre-analytical methods. Among them, when more than 60% of the samples showed concentrations below LOQs, the result was given as an estimate rather than a value (the reference values proposed were only indicative, as below the LOQ). Twenty-nine elements (excluding Be) showed 100% of measured values above the LOQs, whereas Be showed 100% below the LOQ. In our method, 77Se and 114Cd isotopes were chosen because they are more abundant than 82Se and 111Cd isotopes. The mean values, median line, outlier data, and concentration range (25– 75% and 1.5 folds of Inter-Quartile Range) of all investigated elements are shown in Fig. 2. Hg, a toxic Page 12/27 Page 12/27 element, was analyzed separately because of its extremely low content. The concentration ranges of the studied elements were observed. Notably, the order of elemental content in Cacumen Platycladi was consistent with that of WLZ. The detailed analysis is as follows. 4.2.1. Major elements The major essential elements investigated were Na, Mg, K, Ca, and P; the sum of these seven major elements accounted for nearly 99% of the total investigated elements. Unlike other trace elements, Al and Fe are at a “macro” level in both WLZ and V-WLZ, therefore we classified Fe and Al as major elements for further analysis. According to the results depicted in Fig. 2, the major elements were found in the decreasing order of K > Mg > Ca > P ≈ Al > Fe > Na in WLZ, whereas the order of element mean concentrations in V-WLZ was K > Mg > Ca > P ≈ Al ≈ Na > Fe. K was the most abundant macro-element of WLZs, accounting for approximately 46.66% and 44.41% of all analyzed elements in WLZ and V-WLZ, respectively. Mg was the second most abundant macro- element, representing 14.51% and 14.07% of the total investigated elements, respectively. Hence, we can speculate that the reason why the K content was the highest could be related to K being a product of the digestive tract. The mean concentration of Na was significantly increased after being processed with vinegar, varying from 2.26% (WLZ) to 7.71% (V-WLZ). Additionally, the average concentration of Ca and Al were reduced, ranging from 11.86% and 9.71% (WLZ) to 10.74% and 8.33% (V-WLZ), respectively. The concentration of Fe displayed similar levels for WLZ and V-WLZ with a value of 5.44% and 5.48%, respectively. Generally, Fe levels in traditional Chinese medicines used for invigorating blood are high. 4.2.2. Trace elements The current Chinese pharmacopeia method for monitoring inorganic contaminants in pharmaceutical samples is defined in the general chapter < 2321>, including the elements Hg, Pb, As, Cd, and Cu. In contrast to other trace elements, these five heavy metals are potentially toxic for human health and their overdose may cause diseases. Consequently, we classified the trace elements as essential and potentially toxic for further discussion. Toxic elements The presence of potentially toxic metals in WLZs typically reflects an exogenous influence that may be correlated with dietary sources and environmental pollution at the feeding sites. According to the Chinese pharmacopeia 2020, Hg, Pb, As, Cd, and Cu are toxic heavy metals that can be detrimental to human health if they exceed certain limits. However, there are no limit standards for toxic elements in animal fecal drugs in the pharmacopeia. The permissible limits of some elements in herbal medicines are given in the Chinese pharmacopeia 2020 as reference values. The concentrations of Pb, Cd, As, Hg, and Cu were 5, 1, 2, 0.2, and 20 mg/kg, respectively. These elements were found in decreasing order of Pb ≈ Cu > As > Cd > Hg in both WLZs. The content of Hg in WLZ and V-WLZ was below 0.06 mg/kg (0.010–0.058 mg/kg), which indicates the low intake of Hg from diet and atmosphere. According to the reference values proposed by the Chinese pharmacopeia (2020), the sample Cd concentrations were within the prescribed limits in our study, ranging from 0.098 to 0.534 mg/kg. Almost all sample concentrations for As were within the normal value; only five samples were more than 2 mg/kg (2.049–5.840 mg/kg). The Pb content in almost all samples was present at elevated concentrations, ranging from 3 to 20 mg/kg. The results showed that the Pb contents in some samples were outside the limits. Moreover, we found a significant difference in Pb content (0.798–15.366 mg/kg) in Cacumen Platycladi; therefore, we speculate that the relatively high concentration found in the samples could be related to the consumption of Cacumen Platycladi. Consequently, toxic elements accumulated in plants can also threaten human health through the food chain. Furthermore, the toxicity of metallic and non-metallic elements in a drug depends on the chemical form and ligand complex in which they are present. less than 0.2 mg/kg, which suggests a negligible role of WLZs as a medicinal source of Ag, Be, Tl, Sn, and U. less than 0.2 mg/kg, which suggests a negligible role of WLZs as a medicinal source of Ag, Be, Tl, Sn, and U. Essential elements Compared to major elements, essential trace metals reliably reflect the feed strategy and environment of Trogopterus xanthipes, although their total content is less than 1% of all the analyzed elements. The essential trace elements were divided into three gradients according to their concentrations. In the first gradient (40–140 mg/kg), the average concentrations decreased in the order Mn > Sr > Zn ≈ Ba in both WLZs. Fe, Zn, Mn, and Cu have critical functional roles in hematopoiesis, immunity, and bone metabolism and might have direct and indirect effects on bone cells or bone mineralization. The second gradient included the elements Rb, Cr, Ni, V, Se, Co, Ga, Cs, and Sb, with mean concentrations ranging from 0.2–10 mg/kg. The third gradient, including Ag, Be, Tl, Sn, and U, resulted in the less abundant trace essential elements with comparatively lower levels, and the mean concentrations were Page 13/27 less than 0.2 mg/kg, which suggests a negligible role of WLZs as a medicinal source of Ag, Be, Tl, Sn, and U. 4.3. Correlation analysis In addition, significant positive correlations, including Sr-(Tl, K), Mn-(P, Tl), and Ca-Al, only existed in WLZ. Negative and significant correlations between the metal pairs were observed only in WLZ, whereas K-Na, P- (Ca, Al, Mg) were observed in V-WLZ. Furthermore, there were positive correlations between Cs, Cr, Se, V, Fe, U, Al, Tl, As, Sb, Pb, Hg, and Cd in Cacumen Platycladi, which have substantial similarities with WLZs. However, further negative correlations were observed in Cacumen Platycladi, including K- (Cs, Cr, Fe, V, Sb) and Ca-As. Hence, we can conclude that, the high correlation coefficients between the above elements (U, Tl, Cr, Ni, Cs, Fe, Al, Co, Se, Ga, V, As, Cu, Rb, Mg, and Pb) indicate that the increase (decrease) of one of the elements is associated with the increase (decrease) of other elements, which helps in controlling toxic elements to a certain extent. Moreover, compared with WLZ, the positive/negative correlations between partial elements in V-WLZ are more or less enhanced. The significant similarities and differences between WLZs and Cacumen Platycladi could be related to the digestion and metabolism of Trogopterus xanthipes. Figure 3 Correlation analysis of element content in WLZ, V-WLZ, and Cacumen Platycladi (Pearson, p < 0.05, p < 0.01) l 4.3. Correlation analysis Pearson’s correlation analysis results of the total element contents in WLZ, V-WLZ, and Cacumen Platycladi are shown in Fig. 3. Inter-element relationships provide important information on their sources, which can indicate whether there is a synergistic or antagonistic relationship between different elements. If r > 0 (< 0), there is a probable positive (negative) correlation; if r = 0, there is no relationship. Moreover, if r > 0.5 (< -0.5), the positive (negative) correlation is said to be strong, and weak when r < 0.5 (> -0.5). Page 14/27 As depicted in Fig. 3, there were robust positive correlations (r > 0.7, p < 0.01) among the ten elements, including U, Tl, Cr, Ni, Cs, Fe, Al, Co, Se, Ga, V, and As, in both WLZ and V-WLZ. A strong association (r > 0.35, p < 0.05) between Cu, Rb, Mg, Pb, Sb, Ag, and the above ten elements (except for As) are also shown in Fig. 3 (a) and (b). Although there is a slight difference in the correlation between the above four elements in WLZ and V-WLZ, the metal pairs between Cu, Rb, Mg, and Pb have probable positive correlations (r > 0.2) except for Cu-Mg (0.07) in V-WLZ. Additionally, significant positive correlations at the 95% confidence level were observed between K-P-Mn-Cu-Sr-Rb-Mn, Mg-Ca, Hg-Zn-Cd-Pb-Ba-Ag in both Page 14/27 Page 14/27 WLZ and V-WLZ, whereas significant positive correlations between other elements were only present in V- WLZ, such as Ba-(Cd, Zn, U, Co, Tl, Ag, Se, Ga), P-(Hg, Zn, K), Mn-(Sb, Cd, Zn), Sr-(U, Ag, Hg, Cd, Zn, Sn), K- (Cd, Al), and Sn-(Sb, Pb, Zn). In addition, significant positive correlations, including Sr-(Tl, K), Mn-(P, Tl), and Ca-Al, only existed in WLZ. Negative and significant correlations between the metal pairs were observed only in WLZ, whereas K-Na, P- (Ca, Al, Mg) were observed in V-WLZ. Furthermore, there were positive correlations between Cs, Cr, Se, V, Fe, U, Al, Tl, As, Sb, Pb, Hg, and Cd in Cacumen Platycladi, which have substantial similarities with WLZs. However, further negative correlations were observed in Cacumen Platycladi, including K- (Cs, Cr, Fe, V, Sb) and Ca-As. WLZ and V-WLZ, whereas significant positive correlations between other elements were only present in V- WLZ, such as Ba-(Cd, Zn, U, Co, Tl, Ag, Se, Ga), P-(Hg, Zn, K), Mn-(Sb, Cd, Zn), Sr-(U, Ag, Hg, Cd, Zn, Sn), K- (Cd, Al), and Sn-(Sb, Pb, Zn). 4.4. PCA analysis The PCA bilinear model constructed for variables gave an interpretable overview of the information on the most meaningful parameters that describe the whole multi-dimensional data set and enabled data reduction with a minimum loss of original [27, 28]. It allowed a reduction of twenty-nine variables (the element below LOQ was excluded) to six/five principal components (PC1-PC6)/(PC1-PC5) in WLZ and V- WLZ respectively, which were distinguished as important factors according to the eigenvalue-one-criterion (their eigenvalues were above 1). From Tables 5 and 6, we can see that these six/five PCs explained approximately 85% of the total variance, which was sufficient to describe the whole WLZs. Figure 4a shows that the PC1 explained more than 43.10% of the total variance and revealed an apparent clustering between elements (U, Tl, Cs, Ga, Co, As, Se, V, Ni, Cr, Fe, and Al), which were characterized by the high positive loading values on PC1. The PC2 was associated with positively correlated Zn, Hg, P, K Ag, Ba, Pb, Cu, and Cd and provided information enclosed in the next 12.69% of the total variance. However, major elements (Na, Mg, and Ca) had low positive loading values on PC1-2, suggesting individualized behavior. Figure 5a shows that most elements were positively correlated with PC1 and only Hg, P, and K were negatively correlated with PC. A strong association between Tl, U, Cs, Se, Co, Ga, V, Ni, Al, Cr, Cu, Fe, and Pb is also observed because they presented high positive loading values for PC1. The contribution of the second factor was 18.9%, which showed high loading values for Zn, Cd, Hg, K, Sr, Pb, Ba, and Mn, with weak loading values for Ag, P, and Sn. These results indicate that the correlations between the elements changed after processing with vinegar. Page 15/27 Page 15/27 Figure 4b and 5b show a graphic distribution of WLZ and V-WLZ (sampling sites) analyzed according to their component scores (PC1 vs. PC2). The samples on the right-hand side, with positive PC1 values were characterized by higher concentrations of Tl, U, Cs, Se, Co, Ga, V, Ni, Al, Cr, Cu, Fe, and Pb. The samples in the top part of the plot, with positive PC2 values featured higher concentrations of Zn, Cd, Hg, K, Pb, Ba, and Mn. 4.4. PCA analysis Therefore, by overlapping the loading and score plots, it is evident that there is an apparent clustering among Group C in WLZ, with negative values of PC1 and PC2, which featured the lowest concentration levels of almost all elements. This could be because the samples in Group C were bathed and unprocessed. Moreover, it can be interpreted that some samples in Groups A, B, and D have strong commonalities and show lower concentration levels because their sites plot on the negative side of the PC score plot. However, samples further showed different individualities from the same site, mainly due to the different ways in which humans raise Trogopterus xanthipes. Notably, Group E, including W34 and W35, was significantly different from the other samples. Remarkably, the heavy metals Hg, Ag, Cd, Pb, Ba, and Zn showed the highest concentrations in W35, which can be interpreted as being closely related to the ages of WLZ. Hence, it is necessary to specify the shelf life of traditional Chinese medicine. Page 16/27 Table 5 Eigenvalue and contribution in WLZ Principal Component Number Eigenvalue Percentage of Variance Cumulative (%) PC1 12.49 43.101 43.101 PC2 3.68 12.69 55.79 PC3 2.83 9.75 65.54 PC4 2.77 9.57 75.10 PC5 1.82 6.29 81.40 PC6 1.26 4.34 85.73 Table 6 Eigenvalue and contribution in V-WLZ Principal Component Number Eigenvalue Percentage of Variance Cumulative (%) PC1 12.54 43.23 43.23 PC2 5.48 18.91 62.14 PC3 2.80 9.65 71.79 PC4 2.09 7.20 78.99 PC5 1.65 5.68 84.68 5. MPI and H-MPI Table 5 Eigenvalue and contribution in WLZ Eigenvalue and contribution in WLZ Eigenvalue and contribution in V-WLZ 4.5. MPI and H-MPI The MPI was calculated to examine the overall metal content in the studied WLZs, excluding the non- metal elements P, As, and Se. The H-MPI was calculated to examine the heavy metal (except for P, As, Se, K, Ca, Na, Mg, and Al) contents in the studied WLZs. This method can comprehensively reflect the levels of bioaccumulation of heavy metals in WLZ and V-WLZ and can be further used to evaluate the degree of heavy metal pollution of the organism. A significant fluctuation range (4.129 to 20.980) was observed in WLZ (Fig. 6). Meanwhile, H-MPI varied over a large range (0.997 to 6.291) in WLZ, demonstrating that there are relatively major differences in elemental contents between WLZ from different sampling spots. 4.6.1. Detection of outlier data Some samples contained elements with extremely high abundances, as shown in the boxplot in Fig. 2. Taking Ba as an example, the maximum concentration was 494.31 mg/kg in WLZ, which was almost eight times the average concentration (57.25 mg/kg). This sample is an outlier. To obtain a more objective assessment of the average concentrations and content ranges for different elements and to balance the representativeness of the samples, we deleted two outlier samples from the sample data. The average concentrations of the 66 remaining samples, including 33 WLZ, 25 V-WLZ, and 8 Cacumen Platycladi, were calculated and are shown in Fig. 7. 4.4. PCA analysis Moreover, the specimens W34 and W35 exhibited the highest values of H-MPI and MPI, which is related to the ages of WLZ and has an extent of elemental accumulation; however, the mechanism of this hypothesis needs further research to be proved. On the contrary, the MPI varied within a comparatively narrow range (5.963 to 13.307) in V-WLZ. Therefore, we can speculate that the levels of metals in V-WLZ changed after processing with vinegar. Compared with WLZ, the average value of MPI (8.643) in V-WLZ is higher than that of WLZ (8.403); the reason could be that the vinegar processing adds other metal elements, such as Na, K, Fe, and Ca. The average value of H-MPI (2.030) in V-WLZ showed a slight decrease compared to that in WLZ (2.096); the lower H-MPI indicated that the samples were more secure for human beings, given the heavy metal contents only. Thus, the decrease of some heavy metal elements after processing could serve as evidence for the “toxicity-reducing” mechanism. 4.6.2. Spectra of inorganic elements Further statistical analyses were conducted to better understand the elemental characteristics of WLZs samples. Consequently, to establish the fingerprint of the inorganic elements, we synchronously reduced some elements (K, Ca, Mg, P, Al, Fe, Na, Mn, Sr, Zn, and Ba) at tremendously high concentrations of 1000x and obtained the same order of magnitude. The spectra of various sources of WLZs and Cacumen Platycladi are plotted in Fig. 7a-c. Additionally, the average contents of the studied elements in WLZ, V- WLZ, and Cacumen Platycladi were calculated and are shown in Fig. 7d. Page 17/27 Page 17/27 Page 17/27 The results in Fig. 7a-b show that the elemental spectra of WLZ and V-WLZ from different sources have similar peak shapes but different relative contents. The concentrations of K, Ca, Mg, P, Cr, V, Ni, Al, As, Fe, Cu, Se, Rb, and Pb varied dramatically, with relatively high contents of K, Ca, Mg, P, Al, Fe, and Cu. g p similar peak shapes but different relative contents. The concentrations of K, Ca, Mg, P, Cr, V, Ni, Al, As, Fe, Cu, Se, Rb, and Pb varied dramatically, with relatively high contents of K, Ca, Mg, P, Al, Fe, and Cu. Moreover, Na contained in different V-WLZs changed significantly, except for the above elements; this could be closely related to processing with vinegar. This is because vinegar contains a large amount of Na, and the processing of different manufacturers is distinct. Additionally, as shown in Fig. 7, it is easy to find out that the inorganic element spectra of WLZs in the two varieties is consistent, and the concentration levels of most elements are similar. In contrast, the contents of K, Ca, (Na), Mg, P, Cr, V, Ni, Al, As, Fe, Cu, Se, Rb, and Pb showed significant variations in WLZ (V-WLZ). Thus, we can conclude that the composition distribution of inorganic elements in WLZs has a certain regularity, and that the inorganic element chromatograms of different sources and varieties of WLZs have outstanding features and consistency, which can be used as a reference index to distinguish other medicinal materials. Furthermore, the results indicated that the concentrations of the studied elements in WLZs and Cacumen Platycladi had very similar trends, as depicted in Fig. 7d. We speculate that a close relationship may exist between WLZs and Cacumen Platycladi. 4.6.2. Spectra of inorganic elements Significantly higher sample levels of K, Mg, Na, P, Cr, V, Ca, Ni, Al, As, Fe, Cu, Rb, and Pb in WLZs could be explained to a certain extent by the continuous accumulation of these elements due to daily Cacumen Platycladi consumption. Several studies have shown that K, Ca, Mg, Cu, Zn, Fe, and Mn enhance the pharmacological effects of traditional Chinese medicine. In conclusion, by overlapping the PCA data and the inorganic element spectra, we identified Fe, Al, Cu, Se, Pb, Rb, V, K, P, Na, Cr, As, and Ni as the characteristic elements in WLZ and V-WLZ. 5. Conclusion The elemental concentrations in WLZ were in the following order: K > Mg > Ca > P ≈ Al > Fe > Na > Mn > Sr > Zn ≈ Ba > Rb ≈ Pb ≈ Cu ≈ Cr > Ni > V > Se > As > Co ≈ Ga > Cs > Cd > Sb > U > Sn > Be > Tl > Ag. The order of element concentration in V-WLZ is K > Mg > Ca > P ≈ Al ≈ Na > Fe > Mn > Sr > Ba ≈ Zn > Rb ≈ Pb ≈ Cu ≈ Cr > Ni > V > Se > As > Co ≈ Ga > Cd > Cs > Sb > Sn > U > Be > Ag > Tl. The profiles of WLZs and their processed product elements are useful for obtaining information on the function of WLZs in promoting blood circulation. In this study, the profiles of major and trace elements in WLZs and Cacumen Platycladi from different areas were elucidated in detail. This finding implies that Cacumen Platycladi is a significant source of chemical elements and provides the first reference value for major, trace, and toxic elements in WLZs, enabling us to carry out further investigations on animal-derived drugs. This similarity is mainly due to the dietary habits of Trogopterus xanthipes, which feed on Cacumen Platycladi. The differences mainly depend on individual variations in Trogopterus xanthipes and environmental factors of the habitats. Pharmacopoeia 2020 has not been considered because it primarily presents complex organic arsenical compounds in Chinese medicine, which are much less toxic than their inorganic counterparts. However, it should be noted that no speciation studies have been conducted to confirm these assumptions regarding Page 18/27 Page 18/27 Page 18/27 heavy metals in the samples. Therefore, the obtained data are speculative and must be carefully interpreted considering the degree of underlying uncertainty. In addition, H-MPI and MPI were used to evaluate the safety and metal element characteristics of WLZs. heavy metals in the samples. Therefore, the obtained data are speculative and must be carefully interpreted considering the degree of underlying uncertainty. In addition, H-MPI and MPI were used to evaluate the safety and metal element characteristics of WLZs. Competing Interest The authors have no relevant financial or non-financial interests to disclose. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Anqi Wang] and [Zheng Li]. The first draft of the manuscript was written by [Anqi Wang] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 5. Conclusion Overlapping the PCA data and inorganic element spectra, we considered Fe, Al, Cu, Se, Pb, Rb, V, K, P, Na, Cr, As, and Ni as the characteristic elements of WLZ and V-WLZ. These distribution rules can be used to differentiate WLZs from other medicinal herbs. Funding This work was supported by [National Key R&D Projects: Integrated Quality Control Technology of Chinese Patent Medicine] (2018 YFC1707302). Declarations Statements & Declarations Data Availability The datasets generated during and/or analysed during the current study are available in the supplementary information. References 1. B. Soyoon, X. Xuikui, M. 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Page 21/27 Page 21/27 Figures Figures Box plot of concentration of different elements in the (a) WLZ (b) V-WLZ (c) Cacumen Platycladi Page 22/27 Page 22/27 2 tion analysis of element content in WLZ, V-WLZ, and Cacumen Platycladi (Pearson, p < 0.05, *p < Correlation analysis of element content in WLZ, V-WLZ, and Cacumen Platycladi (Pearson, p < 0.05, **p < 0 01) Figure 2 on analysis of element content in WLZ, V-WLZ, and Cacumen Platycladi (Pe 0.01) Page 23/27 Page 23/27 Figure 3 PCA loading and score plot of the element content in WLZ (Group A: W1-W11, which were collected from Shanxi-1 province; Group B: W12-W21, which were collected from Shanxi-2 province; Group C: W22-W28, which were collected from the same manufacturer in QL city, Shanxi-2 province; Group D: W29-W33, which were collected from Hubei province; Group E: Specimens W34-W35, in which W34 was collected from Hubei province in 2010 and W35 was collected from Shanxi-2 province in 2012.) Figure 3 Characteristic spectra of inorganic elements in WLZs and Cacumen Platycladi Characteristic spectra of inorganic elements in WLZs and Cacumen Platycladi Characteristic spectra of inorganic elements in WLZs and Cacumen Platycladi Figure 3 PCA loading and score plot of the element content in WLZ (Group A: W1-W11, which were collected from PCA loading and score plot of the element content in WLZ (Group A: W1-W11, which were collected from Shanxi-1 province; Group B: W12-W21, which were collected from Shanxi-2 province; Group C: W22-W28, which were collected from the same manufacturer in QL city, Shanxi-2 province; Group D: W29-W33, which were collected from Hubei province; Group E: Specimens W34-W35, in which W34 was collected from Hubei province in 2010 and W35 was collected from Shanxi-2 province in 2012.) PCA loading and score plot of the element content in WLZ (Group A: W1-W PCA loading and score plot of the element content in WLZ (Group A: W1-W11, which were collected from Shanxi-1 province; Group B: W12-W21, which were collected from Shanxi-2 province; Group C: W22-W28, which were collected from the same manufacturer in QL city, Shanxi-2 province; Group D: W29-W33, which were collected from Hubei province; Group E: Specimens W34-W35, in which W34 was collected from Hubei province in 2010 and W35 was collected from Shanxi-2 province in 2012.) Figure 4 PCA loading and score plot of the element content in V-WLZ (Group A: V1, V4-V8, V13, V18-V19, and V22- V23, which were collected from Shanxi-2 province; Group B: V2, V9-V12, V20-V21, and V24-V25, which were collected from Shanxi-1 province; Group C: V3 and V14-V17, which were collected from Hebei province.) Figure 4 PCA loading and score plot of the element content in V-WLZ (Group PCA loading and score plot of the element content in V-WLZ (Group A: V1, V4-V8, V13, V18-V19, and V22- V23, which were collected from Shanxi-2 province; Group B: V2, V9-V12, V20-V21, and V24-V25, which were collected from Shanxi-1 province; Group C: V3 and V14-V17, which were collected from Hebei province.) Page 24/27 Page 24/27 Figure 5 Th MPI d H MPI l i WLZ d V WLZ The MPI and H-MPI values in WLZ and V-WLZ Page 25/27 Page 25/27 Page 25/27 Figure 6 Characteristic spectra of inorganic elements in WLZs and Cacumen Platycladi Page 26/27 Figure 6 Characteristic spectra of inorganic elements in WLZs and Cacumen Platycladi Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Appendixdate.docx Page 26/27 This is a list of supplementary files associated with this preprint. Click to download. Page 26/27 This is a list of supplementary files associated with this preprint. Click to download. Appendixdate.docx Page 27/27
https://openalex.org/W3003488558	http://www.scielo.br/pdf/hcsm/v26s1/en_0104-5970-hcsm-26-s1-0039.pdf	English	null	Limites da assistência oitocentista: o caso urbano de Juiz de Fora (MG)	História, ciências, saúde-Manguinhos	2,019	cc-by	9,315	The limits of healthcare assistance in the nineteenth century: the urban case of Juiz de Fora (Minas Gerais state) SIQUEIRA, Rogério; BARRETO, Maria Renilda. The limits of healthcare assistance in the nineteenth century: the urban case of Juiz de Fora (Minas Gerais state). História, Ciências, Saúde – Manguinhos, Rio de Janeiro, v.26, supl., dez. 2019. Available at: <http://www. scielo.br/hcsm>. Abstract The objective of this article is to discuss the mechanisms of healthcare organization in the city of Juiz de Fora (Minas Gerais state), in the second half of the nineteenth century. We investigated the arrangements organized to provide care for the sick and how this structure adapted to the demands of the socio-political and economic scenario. It is noticeable that, while the slave system lasted, the Charity Hospital in Juiz de Fora was irrelevant, since the coffee plantations had facilities capable of caring for the sick. The founding of the healthcare facility fulfilled the function of catapulting the plantation owner José Antonio da Silva Pinto into the Brazilian nobility, becoming the baron de Bertioga, and of accommodating the requirements of imperial legislation through personalist arrangements. Rogério Siqueirai i Instructor, Colégio Cave, Juiz de Fora. Juiz de Fora – MG – Brazil orcid.org/0000-0002-1356-4011 professorsiqueira@yahoo.com.br Rogério Siqueirai i Instructor, Colégio Cave, Juiz de Fora. Juiz de Fora – MG – Brazil orcid.org/0000-0002-1356-4011 professorsiqueira@yahoo.com.br Rogério Siqueirai i Instructor, Colégio Cave, Juiz de Fora. Juiz de Fora – MG – Brazil orcid.org/0000-0002-1356-4011 professorsiqueira@yahoo.com.br Keywords: history; assistance; healthcare; Juiz de Fora (MG); nineteenth century. 1 v.26, supl., dez. 2019 Rogério Siqueira, Maria Renilda Barreto A ssistance for dealing with social issues in nineteenth century Juiz de Fora, namely healthcare and the poor, was provided through three channels: the first combined elements of philanthropy from the elites and the actions of the municipality; the second occurred in the private domain, linked to slaveholding; and the last – charity − was long-term, part of what we could call “the salvation economy” (Castel, 1998; Geremek, 1986). Although the third form of assistance is not defined as the focus of this article, we will address it because we feel that it is an interesting component of the trio of forms of assistance in the state of Minas Gerais in the nineteenth century. A The principal sources upon which these reflections were based included documents from the Juiz de Fora City Council, probate inventories, local newspapers and the Senhor dos Passos Brotherhood Charter. Abstract This study focuses on the second half of the nineteenth century, a time of accommodation and crisis in in the Brazilian imperial political system, debates over the slave labor regime and a gradual change in the urban context, marked by the growth of cities, with displacement of the population due to the end of slavery and due to the arrival of immigrants. Capital from agriculture also financed the development of industry and the growth of services, significantly altering the urban landscape and the rearranging power, prestige and healthcare at the end of the nineteenth century and the early twentieth. In order to understand healthcare in the nineteenth century based on the establishment and maintenance of hospital facilities − small hospitals or infirmaries − we must ask: who received the medical, surgical and/or pharmaceutical care? Who paid for this care? What was the relationship between the hospital and the flow of people? Who organized care? How did the long period of slavery affect the arrangements for helping the sick in Brazil? Is the experience in the Zona da Mata in the state of Minas Gerais, Brazil, discussed in this article, a potential laboratory for uncovering the different facets of the Brazilian experience, since this agricultural region was involved in coffee production and relied extensively on slave labor? História, Ciências, Saúde – Manguinhos, Rio de Janeiro Both private and public Studying the history of healthcare obliges us to investigate both private, philanthropic efforts, and private institutions being built to benefit the public, given the strong connections between the public and private spheres (Lindemann, 1999). Based on this premise, the subject of healthcare in Minas Gerais in the nineteenth century leads us to José Antonio da Silva Pinto (1785-1870), the baron of Bertioga. Born in June 1785 in the Lage parish, now the Resende Costa municipality, in Minas Gerais state, he was one of 13 children. There are records of his presence in the main church in Simão Pereira, where he lived, as early as 1820. The owner of Soledade Plantation, he was one of the pioneer coffee farmers in the region, and his fortune appears to have arisen from this (Travassos, 1993). Around 1830, he moved to the village of Santo Antônio do Paraibuna, now the city of Juiz de Fora. José Antonio da Silva Pinto was one of the principal philanthropists in Paraibuna, involved in all of the movements related to city development, and recognized for using his economic História, Ciências, Saúde – Manguinhos, Rio de Janeiro 2 The limits of healthcare assistance in the nineteenth century and political capital to protect the poor (Travassos, 1993). Throughout his life, he accumulated titles and participated in various brotherhoods. He was commander of the Ordem da Rosa and of the Imperial Ordem de Cristo, and a member of the brotherhoods Ordem Terceira do Carmo of Rio de Janeiro, Ordem São Francisco de Paula of Rio de Janeiro, Santa Casa da Misericórdia of Rio de Janeiro, Santíssimo Sacramento of the Santa Rita Parish, Nossa Senhora dos Homens de Barbacena of Caraça, and Senhor Bom Jesus de Matosinhos of Congonhas, in addition to establishing and acting as lifetime superintendent of the Nosso Senhor dos Passos Brotherhood of Santo Antônio do Paraibuna (Azzi, 2000). Despite his involvement with these institutions, which involved regular monetary contributions, he also made significant donations to help the poor during epidemics, in addition to contributing to establishing a public cemetery and purchasing the building that would house the City Council, of which he was a member (Travassos, 1993; Oliveira, 2016). v.26, supl., dez. 2019 Both private and public Silva Pinto’s participation in various brotherhoods reveals the growing economic and social prestige of this coffee baron at the time, and corroborates the studies that indicate the existence of philanthropists in various social contexts, in addition to politics (Franco, 2015; Tomaschewski, 2015; Barreto, 2005; Boschi, 1986; Russel-Wood, 1981; Carvalho, 2018). Financial contributions, political support for the Emperor, and philanthropic work were part of the etiquette required of wealthy men who wished to obtain noble titles in Brazil (Holanda, 2010; Faoro, 2001; Barman, 1988). In 1854-1855, during a cholera epidemic that affected the Empire and arrived in the village of Santo Antônio do Paraibuna in the mid nineteenth century, José Antonio da Silva Pinto was responsible for founding a Charity House under the Senhor dos Passos Brotherhood charter and supported by it. Of all his initiatives, this work was described as his most important contribution to the city (Travassos, 1993). Recognition of his important work for the city of Juiz de Fora was demonstrated in the session of the City Council held on April 25, 1866, when city councilman doctor Avelino Milagres nominated the baron of Bertioga for special recognition for the Casa de Misericórdia [House of Mercy], and it was approved unanimously (Esteves, 1915). In addition to José Antonio da Silva Pinto and his wife, members of the Nosso Senhor dos Passos Brotherhood included the illustrious families of the municipality, based on the accounting records. They included the Halfeld family, specifically Commander Henrique and his wife Candida, the Tostes family, specifically Rita de Cássia, the Lage family, specifically Domingos Antonio Barbosa Lage, the Valle Amado family, specifically Domingos do Valle Amado, and the Horta family, namely Antonio Caetano Oliveira Horta. These families were an important part of the landholding elite that owned slaves and held government posts in the municipality. One particularly important individual was the influential politician João Nogueira Penido (father), deputy general of the Empire and federal representative of the Republic, from 1894 to 1899. The efforts of José Antônio da Silva Pinto, when proposing the foundation of the Charity Hospital, led to him being honored with the title baron of Bertioga, granted by Emperor Pedro II when the latter visited the city for the inauguration of the Union and Industry Highway on May 13, 1861 (Stehling, 1965). The noble title gave Silva Pinto social and political prominence, in addition to expressing the public recognition of the Emperor. Both private and public 3 3 Rogério Siqueira, Maria Renilda Barreto As mentioned by Eul-Soo Pang (1988), in order to receive a title, millions of réis was not enough. Good politics was also necessary. And, in the case study presented here, the good works consisted of providing the city with resources that should have been provided, a priori, by the government, such as healthcare for the sick. It is worth remembering that the imperial law of October 1828 established that City Councils were to provide villages and cities with establishments (charity houses) to cure “poor, sick people,” providing municipal physicians or surgeons.1 In addition to healthcare, the charity houses were to take care of abandoned children and provide vaccinations. When the baron founded a charity house to aid the sick, we see an overlap of the private and public since Silva Pinto takes upon himself the role of benefactor, whose “good” works will be recognized by the Emperor through a noble title, thus constituting a connection between the baron and the monarch, conferring distinction on Silva Pinto compared to the other landholders. We can conclude that the baron’s initiative to found a Charity House in Juiz de Fora through the Senhor dos Passos Brotherhood was an attempt to accommodate the recently established municipality to the provisions of imperial and provincial laws. As the lifelong superintendent of the Brotherhood and a member of the City Council, he took on the role of intermediary between private and public interests in rural, slaveholding Brazil. The other members of the City Council, belonging to the agricultural slave-holding elite, remained subordinate to the noble José Antonio da Silva Pinto, baron of Bertioga, and healthcare in the city was organized on a utilitarian foundation,2 balanced on the pillars of philanthropy, social virtue and political capital. Mary Lindemann (1999), when analyzing modern medicine and society, especially the establishment of hospitals, points out the need to take into account the complex social, economic and cultural forces that converged in the creation and administration of these healthcare facilities. História, Ciências, Saúde – Manguinhos, Rio de Janeiro The limits of healthcare assistance in the nineteenth century During the second half of the nineteenth century, this hospital was not a significant in terms of providing healthcare, nor did it aid the poor. After the death of the baron of Bertioga (1870), and with the onward march of the abolitionist movement, the equilibrium of the agreements entered into two decades earlier shattered. A few months after the death of the baron, the City Council established a commission to inspect the condition of the “jail, House of Mercy, and the slaughterhouse” (Câmara Municipal, 18--). The commission consisted of an engineer, a judge, an attorney and two physicians. In this report, the commission highlighted the inadequacy and precariousness of the building, which was in an advanced state of ruin. The report listed the assets of the Brotherhood, including buildings, slaves, credits, cash and imperial donation, totaling more than 115 million réis. According to the commission, the House of Charity was “built improperly,” “ruined,” but was the only option for treating the “numerous poor” in the municipality (Câmara Municipal, 18--). As a solution, the signers of the document proposed calling on the members of the Nosso Senhor dos Passos Brotherhood to carry out the necessary repairs and ask the Provincial Assembly to hold some lotteries, as was done with other “Misericórdias”3 in the Empire, or “any other provincial favor.” The council sent a letter to the secretary of the Brotherhood of Nosso Senhor dos Passos on June 2, 1870, calling for a meeting of the members of the brotherhood as soon as possible in order to elect a new superintendent to succeed the baron of Bertioga. Note that he died on May 6, 1870, and his testament was read and officially recognized in the month of his death. In his testament, the baron appointed his nephew, Elias Antonio Monteiro da Silva, superintendent of the brotherhood, a fact ignored by the city council members. This episode allows us to infer that the successor did not have sufficient political clout, nor leadership, nor social capital to take the place of his uncle. Or that the baron − lifetime superintendent of the Brotherhood − tried a last maneuver, believing that he could interfere in the future course of the brotherhood through his testament. v.26, supl., dez. 2019 Slavery and rural healthcare The historiography on healthcare in the eighteenth and nineteenth centuries has shown the importance of the establishment of hospitals to aid the poor, soldiers, local workers and the migrant population. For the Portuguese empire and its colonies, the establishment of the brotherhoods was the alternative way to promote healthcare, with the Misericórdia brotherhood the most prestigious (Abreu, 2001; Barreto, 2005; Franco, 2011, 2014; Sá, 1997; Lopes, 2012; Boschi, 1996; Gandelman, 2005; Araújo, 2009). Some brotherhoods served only their members, while others operated more broadly, including the poor, workers, orphans, widows, soldiers, prisoners, foreigners and the enslaved (Soares, 2002; Karasch, 2012). The Misericórdia Brotherhood, in particular, was an institution that united the interests of the State, the local elite, the Church and the city councils. In Juiz de Fora – then Vila de Santo Antonio do Paraibuna –, in the Senhor dos Passos Chapel, the Nosso Senhor Brotherhood was founded in 1854-1855, as an initiative of José Antonio da Silva Pinto, baron of Bertioga, with the intention of holding religious masses and aiding the poor. Spiritual support was provided by the Chapel, and the sick were aided by the House of Charity, also known as the House of Mercy, which had an infirmary. 4 História, Ciências, Saúde – Manguinhos, Rio de Janeiro The limits of healthcare assistance in the nineteenth century The limits of healthcare assistance in the nineteenth century The baron’s political power and his intention to perpetuate his influence in the brotherhood were explicit in the charter of the organization: Section 9 The founding member, Commander José Antonio da Silva Pinto, of his own volition, is the lifetime Superintendent of the Brotherhood and its Benefactor, and during his lifetime no election shall be held to fill that position (Compromisso..., 6 ago. 1854). Six years later, in 1876, establishment of the hospital with the resources donated by the baron had not yet occurred. When consulting the brotherhood’s register of revenues and expenses, we note an inconsistency in the accounting. In addition to not occurring annually, no review of accounts was carried out from 1865 to 1873, and in 1873 a single accounting was provided for the entire period. Note that, even after the death of the founder of the brotherhood, in 1870, no accounting was provided at the end of his term as superintendent. In later years, they were irregular again until 1886, when they became annual. In the accounting records, revenues are predominantly from annual fees, alms, rent, donations, credits and testaments. Expenses, however, are mainly payments to priests 5 5 Rogério Siqueira, Maria Renilda Barreto and churchwardens, communion wafers and procession ornaments, and in a few rare cases nominal payments, without a description of the nature of the expense. Only for the years 1887-1888 were there revenues and expenses specifically related to the House of Charity. It is clear that, during the reign of Emperor Dom Pedro II, the hospital of the Senhor dos Passos Brotherhood was not involved in a significant way in aiding the sick of the city of Juiz de Fora. Founded through a private initiative, linked to a brotherhood, associated with the government since it released the City Council from its obligation to provide assistance for the poor, its role was minimal. Why? In order to understand this dynamic, we need to understand the composition of the social segment that needed care. For the study in question, the main group to be aided was the enslaved population, and they did not reside in the city, but rather in the countryside. Historiography on modern hospitals has associated their existence with population mobility. The limits of healthcare assistance in the nineteenth century Cities with an intense flow of people, whether due to a port, agriculture or a concentration of workers, tend to establish hospitals with the capacity to aid those who are in transit and beyond their family and regional social networks (Lopes, 2017; Barreto, 2005, 2011; Correa, 2018). In Juiz de Fora, the most prominent flow of people was that of the enslaved. The increase of slave labor was related to the expansion of coffee farming. In the last decades of the eighteenth century and the first decades of the nineteenth, the first signs of an increase in coffee production in the South of the province, the future Minas Gerais Zona da Mata, became noticeable (Carneiro, 2008) During this period, coffee production grew in the provinces of Rio de Janeiro, São Paulo and Minas Gerais, along with the concentration of slaves in the region of the Paraíba valley, the midwest of São Paulo state, and the Minas Gerais Zona da Mata. From 1830 to 1850, there was considerable expansion in coffee production in the Paraibuna valley, reaching large-scale production levels. Starting as no more than rustic muleteer supply stops in the eighteenth century, the region underwent significant transformation, becoming the main producer and exporter of coffee in the province. Note that the Zona da Mata was the source of almost all coffee produced in Minas Gerais (Machado, 2002; Freire, 2011). The growth of the free and enslaved populations in the city of Juiz de Fora reflects the increase in importance of the production and outflow of coffee in the region. In 1833, the Paraibuna district had the third largest population in the province (15.2%), and the largest population of slaves (19.2%). Vittoretto (2012) estimated the quantity of the enslaved population in the Juiz de Fora region, where 30.49% of landholders had more than 20 slaves, by consulting 118 probate inventories from 1830 through 1854. This group had 1,648 slaves, of a total of 2,131, corresponding to 77.32% (Vittoretto, 2012). From 1855 to 1870, there was an expansion in the concentration of enslaved labor on the plantations in comparison to the prior period. There were 47 landholders with 20 to 50 slaves, corresponding to 16.04% of the probate inventories, and 19.01% of all slaves. Forty-five landholders had more than 50 slaves, therefore 15.37% of the total. This group had 4,475 slaves, corresponding to 61.15% of the total. História, Ciências, Saúde – Manguinhos, Rio de Janeiro The limits of healthcare assistance in the nineteenth century Grouping the mid-sized and large landholders (from 20 to over 150 slaves), there was a 77.32% increase in individuals enslaved 6 História, Ciências, Saúde – Manguinhos, Rio de Janeiro The limits of healthcare assistance in the nineteenth century by this group during the earlier period (1830-1854), and an increase of 80.16% during the later period (1855-1870) (Vittoretto, 2012). Table 1: Structure of slaveholding in Juiz de Fora, 1855-1870 SLAVES NO. OWNERS % TOTAL SLAVES % AVERAGE 1-5 92 31.39 252 3.44 2.73 6-10 62 21.16 475 6.49 7.66 11-19 47 16.04 716 9.8 15.23 20-50 47 16.04 1,397 19.1 29.72 51-100 29 9.9 1,934 26.44 66.68 101-150 10 3.41 1,168 15.96 11.68 + 150 6 2.06 1,372 18.75 228 TOTAL 293 100 7,314 100 24.96 Source: Vitoretto (2012). Table 1: Structure of slaveholding in Juiz de Fora, 1855-1870 Source: Vitoretto (2012). During the period studied, the ill were treated in the home, where the sick individual was cared for by family members, relatives and friends. Slaves were definitively outside this support network, given the specific characteristics of this diaspora population, especially in the rural environment. Thus, since the enslaved were the main group of workers distanced from family environments providing protection and solidarity, where were they treated? In rural hospitals and infirmaries. Recent historiographic research reveals that, in Brazil, the large and mid-sized plantations had hospitals and infirmaries. Keith Barbosa (2014b) also studied this issue, discussing the reality of the enslaved with respect to health and disease in the Cantagalo region, an important coffee producer in the second half of the nineteenth century in the Paraíba valley in the province of Rio de Janeiro. Using primary sources such as probate inventories, periodicals, medical reports, judicial collection of medical fees, in addition to medical manuals and theses published during the period, he portrayed the experiences of the ill and the actions of the plantation owners to prevent illness and treat illnesses. The relationship between healthcare and economic prosperity is clear in the editorial published in the Gazeta da Bahia newspaper on August 25, 1866, when the author urged landholders to protect all the lives needed for the prosperity of the country (Editais, 25 ago. 1866). v.26, supl., dez. 2019 The limits of healthcare assistance in the nineteenth century Rosilene Mariosa (2006) worked with the family archives of commander Manoel Antonio Esteves, owner of the Santo Antônio do Paiol plantation, located in the Rio de Janeiro Paraíba valley region, in the municipality of Valença, state of Rio de Janeiro. When studying the diseases and treatments of slaves on the Paiol plantation − which had 320 slaves − she shows that there was a hospital there, built on the highest point on the land, near the plantation house. The slaves were taken care of by the physician Ernesto Frederico da Cunha, and by pharmacists, pharmaceutical assistants and trained, enslaved assistants. Júlio César Pereira (2009, 2011) provides details of the Santa Cruz Imperial Plantation (Rio de Janeiro) during the second half of the nineteenth century. The plantation had had a hospital since 1700, built by the Jesuits to care for sick slaves. In 1820, the hospital 7 Rogério Siqueira, Maria Renilda Barreto consisted of one two-story building, where the ill were separated based on sex and age. The hospital also treated travelers and free men. There was an apothecary on the ground floor, and the clinical staff also included slaves skilled in the art of bleeding. It is important to note that the existence of hospitals to care for the enslaved was a recurring practice in other geographic regions with slave plantations, such as the Caribbean and the USA. Richard B. Sheridan (1985) analyzed slave medicine and the demographic experience of captives based on birth and mortality rates, longevity, slave diseases, and other aspects, and described the operation of Caribbean plantation hospitals. Plantation owners generally built hospitals that combined characteristics of infirmaries and prisons. The staff working in them varied according to the number of slaves on the plantation, the size of the hospital, and the owner’s willingness to train and employ slaves and freedmen in hospital service. On a large plantation, the hospital was staffed by a black man or woman skilled in the art of healing, an assistant, several nurses − usually sick women − cooks, a midwife, and nurses to work in the delivery room. There was also a specific slave to take care of the ward where the yaws victims were located. This ward was far away from the hospital (Sheridan, 1985). Todd L. História, Ciências, Saúde – Manguinhos, Rio de Janeiro The limits of healthcare assistance in the nineteenth century Savitt (1978) studied the relationship between landholders, slaves, and physicians as a component of the history of medicine in the South of the USA. According to the author, slaveholders dictated the living and working conditions of the captives, safeguarding or destroying the health of the enslaved population. Therefore, health, illness, and medical care helped shape the slave experience. More dated interpretations tended to describe the landholding elite as accustomed to easy “replacement” of slaves due to the continuing supply from human trafficking. Because of this, blacks suffered a routine of extreme violence, hard work and little or no concern for their health. Under these conditions, the sick slave tended to be seen as a hindrance, a burden to his owner, who was unwilling to invest in the resources that would aid his recovery. Recent studies, such as those by Almeida (2012), Loner et al. (2012), Pereira (2011) and Barbosa (2014a), in addition to those already mentioned, raise new questions by demonstrating that plantation owners established infrastructure for the care and preservation of their slaves’ health in various regions of Brazil and elsewhere. The agricultural manuals published beginning in the 1830s, in some cases with political support from the Brazilian government, sought to offer a solution to the dilemmas of large landholders − the target audience of these texts − regarding the management of their plantations (Marquese, 2001; Porto, 2008). They reflected on various issues such as healthcare, work, rest, housing, food, clothing, punishment, religiosity and family formation within the enslaved population (Oliveira, 2016). The agricultural handbooks reveal a scenario in which slaves’ precarious living conditions ultimately resulted in a high mortality rate. There were many factors triggering this scenario: food, inadequate housing, excessive work, insufficient rest, sexual excesses, excessive consumption of alcohol and the violence of captivity. They proposed detailed actions aimed at maintaining the slave system, but also promoting the expansion of its productive potential, by incorporating improvements related to healthcare, clothing, food, discipline, religion, housing, work, rest and family relationships among the enslaved into the routine of the plantations (Taunay, 1839; Imbert, 1834). The limits of healthcare assistance in the nineteenth century História, Ciências, Saúde – Manguinhos, Rio de Janeiro 8 The limits of healthcare assistance in the nineteenth century The change in the behavior of the large landholders and slaveholders regarding the treatment of their workers is seen as necessary and indispensable by the authors, as it was crucial for the survival of the system of slavery itself. To convince them of the need to incorporate new practices in the treatment of slaves, they argued that there was no other way to relate to slavery, that only through healthcare and maintenance could economic interests be ensured, combining them with the exaltation of benevolence toward other human beings. “If their own interests did not dictate this obligation, humanity would impose this duty on them” (Imbert, 1834). Taunay (1838, p.63) uses a very similar definition, attributing caring for sick slaves to interests and to humanity. Similarly, the physician David Jardim (1847, p.15) argues that “not only humanity, but also interests, guided the [slaveholders] to pay” greater attention to the treatment of ill slaves. In the discourse of these authors, we observed a common focus on the need for plantations to have facilities that were properly built and specifically designed for treating sick slaves. There are several references to the presence of infirmaries and/or hospitals as part of the fundamental infrastructure of large plantations. Keith Barbosa (2014b) described a landholding elite committed to expanding their infrastructure investments aimed at helping sick slaves. This infrastructure included infirmaries and hospitals, but also the hiring of apothecaries, municipal physicians, and surgeons, and the presence of slaves working as nurses and barbers, a strategy employed by the landholders to ensure the health of the breeding stock and avoid interrupting the profitability of the coffee plantation. The baron of Bertioga’s will records the granting of freedom to some of his slaves, provided that Manuel, a tailor, and his wife, Julia, worked for a period of six years in the Soledade plantation infirmary owned by the baron (Travassos, 1993). Six years was a long time considering the average life of a slave. In the case of Manuel and Julia, it is inferred that they could take care of the clothes used in the plantation infirmary, or perhaps clean, cook and serve the sick slaves. The fact is that, for this slave family, the road to freedom lay in services rendered for more than half a decade in an infirmary. v.26, supl., dez. 2019 The limits of healthcare assistance in the nineteenth century It is noteworthy that, by conditioning the granting of liberty on a period of work in the infirmary, the baron revealed the importance to him of this establishment and the deep need that the services provided there should not be interrupted. Thus, this need outweighed the benefit of freedom. Local newspapers also provided evidence of the existence of hospitals/infirmaries in the region by announcing property auction announcements, a doctor’s visit report, a slave escape, a medical services announcement, and an abolitionist article that, in different ways, mentioned the existence of infirmaries on plantations in the region. Given the importance of this information on the dynamics of organization of healthcare for the enslaved, please excuse our detailed description. In March 1876, the periodical Pharol published a letter from doctor Luiz de Mello Brandão addressing the influence of corn on the yellow fever epidemic, at the request of the Viscount of Prados. In his reports, he highlights his visit to take care of sick slaves in the plantation infirmary of Mr. Marcelino de Brito Pereira Andrade (Brandão, 12 mar. 1876). 9 9 Rogério Siqueira, Maria Renilda Barreto In 1884, the same newspaper contained the announcement of a pharmacist offering his services to landholders in the region, proclaiming himself skilled in bleeding and working in infirmaries (Aos Srs. Fazendeiros..., 15 abr. 1884). It is not explicit in the text that it is a slave infirmary, but since the announcement, in its title, is addressed to landholders in a slaveholding region, we believe this is not an unreasonable deduction. This same ad was published in some later editions. The emphasis on nursing-related skills indicates that this is a significant demand in the region, and hence highlighted in the text of the announcement. In 1888, Pharol reprinted an article originally published in Taubaté, upstate São Paulo, in favor of free labor. Among the various abolitionist arguments given in the article, it is worth noting the section in which the author points out: “Free labor, thus paid, does not require [the landholder] to keep guards, pay expenses related to escapes, an infirmary, a doctor, a pharmacist and other things. The difference is in favor of free labor” (Transformação..., 5 abr. 1888, p.1). Note that the writer sees an infirmary, a doctor and a pharmacy as essential, routine elements on plantations, highlighting their contribution to the costs of these plantations. The limits of healthcare assistance in the nineteenth century In 1883, Pharol published a City Council notice announcing the auctioning of the property used as a guarantee by José Bernardo da Silva Moreira, being foreclosed on by Commander José Pereira Darigue Faro and his wife. In the description and appraisal of the property, one notes the existence on his plantation of a “windowed, wooden-floored, pine- walled house with five rooms and two halls used as an infirmary” valued at 1:000$000 (one million réis) (Editais, 28 jun. 1883). In 1884, Pharol announced the auction of property pledged as a guarantee by José Rodrigues Goulart and his wife, foreclosed on by Forquim Jappers & Co. and Araújo Ferraz & Co., which lists a “13.2m infirmary with a tile roof and wooden floors” (Juízo Municipal, 12 jun. 1884, p.2). These announcements are important because they state the appraisal amount of the infirmary: as it was a first auction, the figure was five hundred thousand réis (500$000), falling to four hundred thousand réis (400$000) on the second call. Although they are the only ads indicating the specific value of an infirmary, they portray the average value attributed to these establishments in the Minas Gerais Zona da Mata region in the second half of the nineteenth century. The Pharol announced the auction of the pledged assets of Commander Antonio Lopes Coelho and his wife in a lawsuit brought by Banco do Brasil. Amongst the goods was a “series of houses 44m wide and 6.6m deep with a tile roof, with some wooden floors, serving as an infirmary and storeroom” valued at 1:400$000 (one million four hundred thousand réis), (Editais, 2 set. 1882). In the June 13, 1885 edition, Pharol announced the auction of Manoel Ribeiro Ferreira’s estate, in a lawsuit brought by doctor Antero José Lage Barbosa. Also in this case, among the listed goods was a “house with a width of 52.8m and depth of 5.5m serving as a slave cabin and infirmary” valued at 1:000$ (one million réis) (Editais, 13 jun. 1885). In March 1888, Pharol published a notice of the foreclosure by Banco do Brasil against the heirs of the baroness of São Mateus. Among the assets pledged and subject to auction was a “tile-roofed villa with wood floors and finished walls, divided into many rooms ... The limits of healthcare assistance in the nineteenth century with 23 10 História, Ciências, Saúde – Manguinhos, Rio de Janeiro The limits of healthcare assistance in the nineteenth century front windows and two doors, with attached infirmary and quarters for servants” valued at 7:000$000 (seven million réis). All assets belonged to the Boa Esperança plantation, in the parish of Vargem Grande, municipality of Juiz de Fora (Editais, 13 mar. 1888). Of all the examples described, only one of the ads did not include a slave auction. In cases where slaves were among the pledged assets, the numbers were considerable, ranging from 50 to 100 auctioned slaves (Editais, 5 jul. 1884; Editais, 24 jun. 1884). Following the model proposed by Rômulo Andrade (1991), relating land ownership to the number of captives, all reported cases probably involved large landholders. Thus, the existence of slave infirmaries was part of the infrastructure of the slave-based coffee plantations in the Minas Gerais Zona da Mata region in the second half of the nineteenth century. Another interesting analysis is the way in which these infirmaries were described. In four of the five examples, characteristics such as flooring, wall-covering and roof tiles are mentioned. In none of these cases were these buildings described as being in poor condition (as occurred for other types of buildings). Given their estimated price, they were considered important facilities and their presence contributed to raising the price of the properties in auctions. What these ads show is the undeniable existence of healthcare facilities for the enslaved population, organized in a manner very different from that of the Juiz de Fora Charity House. In rural areas there were apothecaries, physicians, nurses and infirmaries/hospitals. The large population flow of potential users of hospital care − due to harsh working conditions, the absence of a protective social fabric (even though new relationships and survival strategies were invented in captivity) and the commercial importance of an enslaved woman or man − was from the African diaspora to the countryside. We conclude that the urban groups in Juiz de Fora did not mobilize to maintain the urban hospital, since the wealthy families were rural landholders and the plantations had municipal doctors, hospitals and infirmaries. These healthcare facilities were part of the slave plantation infrastructure, as can also be seen in the Southern United States and Jamaica (Savitt, 1978; Sheridan, 1985). v.26, supl., dez. 2019 The limits of healthcare assistance in the nineteenth century Therefore, in Juiz de Fora, slaves and household members were cared for by municipal doctors in rural infirmaries/hospitals, and the urban poor were supported through other means: alms. Alms The urban charitable institutions in Juiz de Fora during the imperial period did not maintain infirmaries or hospitals that could assist patients excluded from the family and friend solidarity network. We defend the hypothesis that the plantations, with their hospitals and infirmaries, formed a sui generis model of healthcare for populations affected by the experience of enslavement. In the context of this investigation, we did not have access to quantitative data on those cared for, but we would not be surprised if household members − free, poor men and women − also received care in rural hospitals. However, it is in the urban context that the problem of poverty becomes most visible, and given the weaknesses of organized assistance, the poor availed themselves of informal aid and attitudes marked by Christian piety, such as almsgiving. 11 11 Rogério Siqueira, Maria Renilda Barreto Begging in Juiz de Fora became so intense that it was questioned on the pages of the Pharol newspaper in 1885, in the article entitled “Begging:” Begging in Juiz de Fora became so intense that it was questioned on the pages of the Pharol newspaper in 1885, in the article entitled “Begging:” We have already had occasion to refer, more than once in the columns of this newspaper, to the great number of individuals of both sexes who walk the streets of this city begging for public charity, with nothing to commend them to receive the compassion of their fellow men. These observations, which we have already made in the past, have been raised again by the huge number of beggars who appear on Saturdays, a day for almost everyone to hand out alms, and we wondered if there was a way to stop this speculation on the part of some, and the display of sores and illness on the part of others? (A mendicidade, 18 jan. 1885). The Correio de Juiz de Fora newspaper, in January 1886, also complained about the “inconvenience” of tolerating the “abuse” of the “deluge of beggars.” The writer promptly related begging “to the torrential source of criminal speculation based on the generosity and philanthropy of the people” (A mendicidade, 31 jan. 1886). Even with these questions, which reflected society’s criticism of this practice, it still persisted, fueled by the search for an objective logic to providing anonymous, silent charity, despite progressive attempts at control. História, Ciências, Saúde – Manguinhos, Rio de Janeiro Final considerations The organization of healthcare in the Minas Gerais Zona da Mata during the reign of Dom Pedro II was organized in a disparate way in the countryside and the city. On the plantations, there were medical care facilities for the enslaved population and, most likely, for the household members and relatives who, even if not being cared for in the same physical space in the infirmaries, could be seen by the doctor on his regular visits to the plantation and benefit from the drugs prepared in the apothecary. The costs of this assistance were borne by the landholder, but some of the nursing staff were from the enslaved population. Until slavery was abolished in Brazil, the healthcare facilities of small towns in agricultural regions − the agrarian elite had homes in the city but lived in the countryside − were of little significance. In Juiz de Fora, the creation of the House of Charity was more of a political bargaining chip than a modern hospital. From 1889 on, during the republican period, the Santa Casa de Misericórdia hospital − as it was called despite being linked to the Senhora dos Passos Brotherhood − underwent significant changes. The new providers were linked to medicine, law, engineering, commerce, pharmacy, industry, journalism, and investments. Although some still maintained their ties to agricultural production, this was not their main source of income and prestige. It is noteworthy that plantation owners still remained in the brotherhood, but ceased to be hegemonic as had been the case during the Empire. The hospital received material and human investments: it opened new infirmaries and operating rooms with modern equipment, furnished specialized care in obstetrics and gynecology, brought in the nuns of Santa Catarina to take care of hospital administration and expanded the public served − Brazilian workers and immigrants − exponentially (Fonseca, 2018). All of these changes are related to the reconfiguration of healthcare assistance after abolition. During this period, it was the urban worker who fell into poverty due to some physical, moral, or intellectual disability or old age who needed assistance. This is the definition of the deserving poor expressed in the Juiz de Fora Health Code (1911), written by physician Eduardo de Menezes (Fonseca, 2018). The limits of healthcare assistance in the nineteenth century The limits of healthcare assistance in the nineteenth century The will of Maria José de Oliveira Coelho, published in the Pharol on February 13, 1881, stated that the amount of 1:000$ (one million réis) in alms of 5$000 (five thousand réis) would be distributed after seventh-day memorial mass for her, at the main church. In these circumstances, alms attracted the needy of all types to funerals and masses. This was a strategy of the poor and a practice of the rich, where assistance was not mediated by a charity or philanthropy. Assistance was grounded and fed on the direct relationship between those assisted and the benevolent. v.26, supl., dez. 2019 Alms The expression “begging” represents diverse actions that ranged from alms collectors authorized to request charity for institutions that aided orphans and abandoned children, to the immediate support for the afflicted that circulated on the streets. Despite the express prohibition of begging imposed on loafers and beggars and the defense of the deserving poor, widespread begging took root in Juiz de Fora society. Even with various control measures, confusion between authorized and illicit begging was constant. It was common for alms to be given without any evidence of real need for the assisted. The intensity of the practice reached such a magnitude in the city that Pharol published, in the January 1885 edition (A mendicidade, 18 jan. 1885), a calculation of the amounts collected as alms in the municipality. Given, from reports of beggars, an average collection of 4$000 (four thousand réis) for an estimated total of at least one hundred people begging at least on Saturdays, the newspaper concluded that, in Juiz de Fora, the monthly amount spent was 1:600$ (one million six hundred thousand réis), that is, 19:200$ (nineteen million two hundred thousand réis) per year. According to the newspaper, with this amount distributed with discernment, “no one would need to beg in this city.” The wills of the baron and baroness of Bertioga support this claim. The baroness, in addition to the 25 masses with 2$000 (two thousand réis) given in alms, 50 masses for her soul, 25 masses for those of her parents and another 50 masses for those of her slaves, and 2:000$000 (two million réis) for ten orphans, she also gave 500$000 (five hundred thousand réis) for poor people at the discretion of the baron, her executor. In his will, the baron of Bertioga arranged for 50 masses for his soul, 25 for those of his parents and 25 for his siblings, in addition to 50 for those of the slaves. Two million réis for poor, honest orphans in the municipality. Five thousand reis for every poor person at his funeral up to 2:000$ (two million réis) (Travassos, 1993).4 12 História, Ciências, Saúde – Manguinhos, Rio de Janeiro notes 1 Given the definitions of the Dicionário da língua portuguesa [Dictionary of the Portuguese Language], edited by father Rafael Bluteau and revised by Antônio de Moraes Silva (1890), as well as the Manual do Agricultor Brasileiro [Brazilian Farmer’s Manual] (Taunay, 1839), we can establish a broad interpretation of the meaning of “municipal physicians” [médicos de partido in Portuguese], associating the concept with those who worked for rewards or for payment, in both public and private institutions. 2 It is understood that actions in the field of healthcare in this case study were intended to provide immediate answers to social questions other than that of treating the ill, namely as an element of political negotiation in relations between plantation owners and the Imperial Court. The establishment of hospitals and/or Houses of Charity was a rational form of philanthropy, much like the social, political, and religious thinking of the agrarian elite of nineteenth century Brazil. 3 We use the term Misericórdias as found in the document, but we believe that in this case it was used as a synonym for brotherhoods. 4 The wills of the baron and baroness of Bertioga were transcribed and published in Travassos (1993). Final considerations The history of the Minas Gerais Zona da Mata reveals the unique characteristics of a region where the slave-holding experience transformed and redefined the forms of healthcare organization in nineteenth century Brazil. 13 13 v.26, supl., dez. 2019 Rogério Siqueira, Maria Renilda Barreto REFERENCeS ABREU, Laurinda. BARBOSA, Keith. V.O. BARBOSA, Keith. V.O. Escravidão, saúde e doenças nas plantations cafeeiras do Vale do Paraíba fluminense, Cantagalo (1815-1888). 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S.l.: Cedeplar, Universidade Federal de Minas Gerais. p.1-25. Disponível em: <http://www.cedeplar.ufmg.br/ diamantina2002/textos/D10.PDF>. Acesso em: 4 dez. 2019. 2002. COMPROMISSO... Filantropia e assistência à saúde em Juiz de Fora: as Irmandades dos Passos e da Santa Casa de 15 v.26, supl., dez. 2019 Rogério Siqueira, Maria Renilda Barreto LINDEMANN, Mary. Medicine and society in early modern Europe. Cambridge: Cambridge University Press. 1999. Cruz, na segunda metade do século XIX. Tese (Doutorado em História das Ciências e da Saúde) – Fundação Oswaldo Cruz, Rio de Janeiro. 2011. LONER, Beatriz Ana et al. Enfermidade e morte: os escravos na cidade de Pelotas, 1870-1880. História, Ciências, Saúde – Manguinhos, v.19, supl.1, p.133-152. 2012. PEREIRA, Júlio César Medeiros da Silva. Práticas de saúde, doenças e sociabilidade escrava na Imperial Fazenda de Santa Cruz na segunda metade do século XIX. Histórica (São Paulo. 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In pursuit of honor and power: noblemen of the Southern Cross in nineteenth-century Brazil. Tuscaloosa: The University of Alabama Press. 1988. TOMASCHEWSKI, Cláudia. Composição social dos irmãos e dirigentes da Santa Casa de Misericórdia de Pelotas, Rio Grande do Sul, Brasil (1847-1922). In: Sanglard, Gisele et al. (Org.). Filantropos da nação: sociedade, saúde e assistência no Brasil e em Portugal. Rio de Janeiro: FGV. p.55-74. 2015. PEREIRA, Júlio César Medeiros da Silva. Trabalho, folga e cuidados terapêuticos: a sociabilidade escrava na Imperial Fazenda Santa PEREIRA, Júlio César Medeiros da Silva. Trabalho, folga e cuidados terapêuticos: a sociabilidade escrava na Imperial Fazenda Santa 16 História, Ciências, Saúde – Manguinhos, Rio de Janeiro The limits of healthcare assistance in the nineteenth century The limits of healthcare assistance in the nineteenth century The limits of healthcare assistance in the nineteenth century VITTORETTO, Bruno Novelino. 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https://openalex.org/W4200420997	https://riviste.unimi.it/index.php/irlh/article/download/16894/14858	Italian	null	Tanti piccoli Führer? Il “peso” della decretazione personale nel governatorato generale di Polonia	Italian Review of Legal History	2,021	cc-by	16,431	Italian Review of Legal History, 7 (2021), n. 12, pagg. 419-449 https://riviste.unimi.it/index.php/irlh/index ISSN 2464-8914 – DOI 10.54103/2464-8914/16894. Articolo pubblicato sotto Licenza CC-BY. TANTI PICCOLI FÜHRER? IL “PESO” DELLA DECRETAZIONE PERSONALE NEL GOVERNATORATO GENERALE DI POLONIA MANY “LITTLE FÜHRERS”? THE “WEIGHT” OF PERSONAL DECREE IN THE GENERAL GOVERNMENT OF POLAND Emiliano Vitti Dottore di Ricerca Università degli Studi di Pavia Emiliano Vitti Abstract English: The system based on National Socialist political doctrine was born neither accompanied by a legal cultural basis of its own, nor with the premise of “relying on” the State in order to reorganize German social and economic life. In fact, it was the numerous National Socialist jurists who elaborated a sort of “structure of legal sources” which constituted a reserve of legal instruments to be used to reach a point of “fusion” between institutional structure and political doctrine.ifi The distorting effects that the new “National Socialist Law” had in the management of territories, especially in those occupied during the war, manifested themselves with greater intensity on Polish territory or, more precisely, on that portion of territory renamed Generalgouvernement für die besetzten polnischen Gebiete, more simply the General Government of Poland; both in the high levels of the Zivilverwaltung and in the administration of the local districts (Kreise), the personalisms of the officials manifested themselves strongly, also thanks to the use of forms of personal decree with different effectiveness. The objective of the “codification” of the regime thus became the object of exploitation, both by the upper echelons of the party (including Hitler), and by those who worked in the administration of the GG, especially some local officials. They, in different forms and with different intensity, fulfilled their functions as petty dictators, using juridically different and territorially limited versions of the personal decree.ii f Frank found himself in the position of having to justify to his Führer the leadership of the «Gau of the Vandals» and his being at the same time a man of law. Hitler expressed his contempt for the category of jurists whom he considered “traitors to the people”, calling them «mentally handicapped from birth or destined to become so with the passage of time» and declared in a speech to the Reichstag on April 26, 1942 that he would have no «peace until every German was convinced that being a jurist was a shameful disgrace». TANTI PICCOLI FÜHRER? IL “PESO” DELLA DECRETAZIONE PERSONALE NEL GOVERNATORATO GENERALE DI POLONIA MANY “LITTLE FÜHRERS”? THE “WEIGHT” OF PERSONAL DECREE IN THE GENERAL GOVERNMENT OF POLAND Curiously, the actual exercise of power by the dictator, based on his undisputed personal and political authority, was justified on the juridical level by the doctrinal interpretation of some singular normative instruments, the Führererlasse, the maximum expression of “compromise”, between justification of existing power and an attempt to codify its strength and contents in a peculiar Nazi “constitutional” structure. Frank found himself in the position of having to justify to his Führer the leadership of the «Gau of the Vandals» and his being at the same time a man of law. Hitler expressed his contempt for the category of jurists whom he considered “traitors to the people”, calling them «mentally handicapped from birth or destined to become so with the passage of time» and declared in a speech to the Reichstag on April 26, 1942 that he would have no «peace until every German was convinced that being a jurist was a shameful disgrace». Curiously, the actual exercise of power by the dictator, based on his undisputed personal and political authority, was justified on the juridical level by the doctrinal interpretation of some singular normative instruments, the Führererlasse, the maximum expression of “compromise”, between justification of existing power and an attempt to codify its strength and contents in a peculiar Nazi “constitutional” structure. They were never formally part of the German legal order, but were de facto an element approved and amply justified by the National Socialist juridical doctrine, which influenced the life of the Reich and the territories it controlled, feeding and amplifying that system 420 Emiliano Vitti of double institutional levels. which had its most chaotic realization in the GG.ti of double institutional levels. TANTI PICCOLI FÜHRER? IL “PESO” DELLA DECRETAZIONE PERSONALE NEL GOVERNATORATO GENERALE DI POLONIA MANY “LITTLE FÜHRERS”? THE “WEIGHT” OF PERSONAL DECREE IN THE GENERAL GOVERNMENT OF POLAND which had its most chaotic realization in the GG.ti The attitude of absolute sovereign on the part of the Kreishauptmänner was typical in the local districts; even from these childish behaviors and from the ostentation of a pomp which was often the result of robbery and looting, it was possible to recognize both the amateurism of a large slice of the administrative staff, and the almost unlimited possibilities caused by the use of personal instruments in the hands of district chiefs.i The command practice was based on recommended and unpunished violence: the existence of some very rare “oases of normal administration” depended exclusively on the will of the individual local leaders and on the resonance that their actions could have at the top of the state nomenclature. The way the population was treated was aimed at leaving a mark on German occupation policy and showing the efficiency of the administrators, through construction work (Aufbauarbeit) of a testimony of the historische Mission in Ostgebiet.i The objective of this paper is to relate the instrument of personal decree with the powers of which they were at the same time cause and consequence, and the behavior of the individual officials who operated in the various institutional levels taken into account, mentioning some cases of an atypical administrative entity like GG. Keywords: Führererlasse; Zivilverwaltung; Kreise; Hans Frank; Kreishauptmann; Stadthauptmann. Abstract Italiano: Il sistema basato sulla dottrina politica nazionalsocialista non nacque accompagnato né da una base culturale giuridica propria, né con il presupposto di “ap poggiarsi” allo Stato per poter riorganizzare la vita sociale ed economica tedesca. In effet ti, furono i numerosi giuristi nazionalsocialisti ad elaborare una sorta di “struttura delle fonti giuridiche” che costituisse una riserva di strumenti giuridici da utilizzare per rag giungere un punto di “fusione” tra impianto istituzionale e dottrina politica di indirizzo.ftiti Abstract Italiano: Il sistema basato sulla dottrina politica nazionalsocialista non nacque accompagnato né da una base culturale giuridica propria, né con il presupposto di “ap poggiarsi” allo Stato per poter riorganizzare la vita sociale ed economica tedesca. In effet ti, furono i numerosi giuristi nazionalsocialisti ad elaborare una sorta di “struttura delle fonti giuridiche” che costituisse una riserva di strumenti giuridici da utilizzare per rag giungere un punto di “fusione” tra impianto istituzionale e dottrina politica di indirizzo. 1 In seguito GG. 2 Secondo Hitler, il Governatorato non sarebbe dovuto essere né una parte del Reich te desco, né un distretto amministrativo del Reich. 3 Ex avvocato personale di Hitler, membro fondatore del Nationalsozialistische Rechtswahrerbund, o NSRB (Associazione dei giuristi nazionalsocialisti), e a capo della Akademie für Deutsches Recht (Accademia del diritto tedesco). 4 Ritter (ed.), 1952, p. 225. 5 Frank, 1953, p. 116. TANTI PICCOLI FÜHRER? IL “PESO” DELLA DECRETAZIONE PERSONALE NEL GOVERNATORATO GENERALE DI POLONIA MANY “LITTLE FÜHRERS”? THE “WEIGHT” OF PERSONAL DECREE IN THE GENERAL GOVERNMENT OF POLAND Gli effetti distorsivi che il nuovo “Diritto Nazionalsocialista” ebbe nella gestione dei terri tori, specie in quelli occupati durante la guerra, si manifestarono con maggiore intensità sul territorio polacco, o meglio su quella porzione di territorio rinominato Generalgou vernement für die besetzten polnischen Gebiete; sia negli alti livelli della Zivilverwaltung, sia nell’amministrazione dei distretti locali (Kreise), i personalismi dei funzionari si ma nifestarono con forza, anche grazie all’uso di forme di decretazione personale dotate di differente efficacia. titi Gli effetti distorsivi che il nuovo “Diritto Nazionalsocialista” ebbe nella gestione dei terri tori, specie in quelli occupati durante la guerra, si manifestarono con maggiore intensità sul territorio polacco, o meglio su quella porzione di territorio rinominato Generalgou vernement für die besetzten polnischen Gebiete; sia negli alti livelli della Zivilverwaltung, sia nell’amministrazione dei distretti locali (Kreise), i personalismi dei funzionari si ma nifestarono con forza, anche grazie all’uso di forme di decretazione personale dotate di differente efficacia. titi L’obiettivo di questo scritto è porre in relazione gli strumenti giuridici di decretazione personale con l’operato dei molti “piccoli Führer” che operarono in una entità ammini strativa atipica come il Governatorato generale di Polonia mostrando, talvolta prima con l’inadeguatezza che con la brutalità, il volto peggiore della Zivilverwaltung. Parole chiave: Führererlasse; Zivilverwaltung; Kreise; Hans Frank; Kreishauptmann; Stadthauptmann. Sommario: 1. Introduzione. – 2. Lo strumento di decretazione personale: i Führererlasse. – 3. Eccessi e incongruenze nell’amministrazione locale: l’impatto col GG. – 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità. – 5. Quando il “diritto” è allo sbando. – 6. Conclusioni. Tanti piccoli Führer? 421 1. Introduzione Il sistema basato sulla dottrina politica nazionalsocialista non nacque accompa gnato né da una base culturale giuridica propria, né sul presupposto di “appog giarsi” allo Stato per poter riorganizzare la vita sociale ed economica tedesca. In effetti, furono i numerosi giuristi nazionalsocialisti ad elaborare una sorta di “struttura delle fonti giuridiche” che costituisse una riserva di strumenti giuridici da utilizzare per raggiungere un punto di “fusione” tra impianto istituzionale e dottrina politica di indirizzo.ftiti Gli effetti distorsivi che il nuovo “Diritto Nazionalsocialista” ebbe nella gestio ne dei territori, specie in quelli occupati durante la guerra, si manifestarono con maggiore intensità sul territorio polacco, o meglio su quella porzione di territo rio rinominato Generalgouvernement für die besetzten polnischen Gebiete, o più semplicemente Governatorato generale di Polonia1; sia negli alti livelli della Zivil verwaltung (amministrazione civile), sia nell’amministrazione dei distretti locali (Kreise), i personalismi dei funzionari si manifestarono con forza, anche grazie all’uso di forme di decretazione personale dotate di differente efficacia. L’obietti vo della “codificazione” del regime divenne quindi oggetto di strumentalizzazio ne, sia da parte delle alte sfere del partito (Hitler compreso), sia da parte di colo ro che operarono nell’amministrazione del GG, specialmente di alcuni funzionari locali che, in forme differenti e con diversa intensità, esercitarono le loro funzioni come dei piccoli dittatori, utilizzando versioni della decretazione personale giuri dicamente differenti e limitate territorialmente.ti Dopo l’invasione della Polonia, gli individui assoggettati che rimasero nelle zone amministrate dai tedeschi furono sottomessi ad una forma di Stato (-apparato e -ordinamento) particolare, il GG appunto, istituito per decreto il 12 ottobre 1939 ed operativo dal 26 ottobre dello stesso anno2.tii Il Governatore Hans Frank3 si trovò di fatto nella posizione di dover giustificare di fronte al suo Führer la guida del «Gau dei Vandali» e il suo essere al contempo un uomo di legge. Hitler esternò il proprio disprezzo verso la categoria dei giuristi che considerava «traditori del popolo», definendoli «minorati mentali dalla na scita o destinati a diventarlo con il passare del tempo»4 e dichiarò in un discorso al Reichstag del 26 aprile 1942 che «non si sarebbe dato pace finché ogni tedesco non si fosse convinto che essere giuristi era una vergognosa ignominia»5. 1. Introduzione Curio samente, l’effettivo esercizio del potere da parte del dittatore, impostato sulla 422 Emiliano Vitti sua indiscussa autorità personale e politica, venne giustificato sul piano giuridico dall’interpretazione dottrinale di alcuni singolari strumenti normativi, i Führe rerlasse, la massima espressione di “compromesso”, tra giustificazione del potere esistente e tentativo di codificarne la forza e i contenuti in una peculiare struttura “costituzionale” nazista. Essi non fecero mai parte formalmente dell’ordinamento tedesco, ma furono de facto un elemento approvato e ampiamente giustificato dalla dottrina giuri dica nazionalsocialista, che andò ad influenzare la vita del Reich e dei territori da esso controllati, alimentando ed amplificando quel sistema di doppi livelli istitu zionali che ebbe nel GG la più caotica delle realizzazioni. 6 Queste cinque categorie rappresentarono circa il 90% di tutte le direttive di Hitler, men tre gli atti collegiali del governo del Reich furono il 10% circa; in Moll (ed.), 2011, p. 17. 7 Per un quadro generale, cfr.: Majer, 1987; Böckenförde (ed.), 1985; Echterhölter, 1970. 8 RGBl., 1934, I, s. 375. 9 Moll, 2011, p. 17. 10 Copia dell’ordine emesso su carta intestata del Reichskanzler, in BAB, R 43, II/141, p. 12. Lettera accompagnatoria di Lammers al Reichsminister del 27 luglio 1934, in Moll, 2011, p. 11, in cui si parla di un “Erlass”. 11 Legge sul capo di stato del Reich tedesco del 1° agosto 1934, RGBl., 1934, I, p. 747. Decreto del Cancelliere per l’attuazione della legge sul capo di stato del Reich tedesco del 1 ° agosto 1934, operativa dal 2 agosto 1934. Moll, 2011, p. 751. Questo decreto venne stranamente inteso come un messaggio al Ministro degli Interni e inizia con le parole «Signor Ministro degli Interni!» (Herr Reichsinnenminister!). 12 Moll, 2011, p. 18. 2. Lo strumento di decretazione personale: i Führererlasse La divisione della quotidianità tra tedeschi e slavi fu realizzata attraverso un pia no normativo basato su una “fonte del diritto” di tipo “superprimario”, ossia il decreto personale. Nel quadro normativo nazionalsocialista, per decreto per sonale si intendeva innanzitutto (o, per meglio dire, unicamente) il Führererlass ma, durante gli anni della guerra, l’ulteriore personalizzazione di questo strumen to assunse i caratteri della “settorialità” e della “territorialità”: il primo riguardò principalmente il settore della sicurezza e la polizia ed ebbe come soggetto pro mulgatore il Reichsführer delle SS Himmler; nel secondo caso invece, il GG offrì un fertile terreno istituzionale per l’affermazione dell’autorità politica e giuridica dei funzionari della Zivilverwaltung, da Frank in giù.itii Considerando la superiorità gerarchica dei provvedimenti e degli atti normati vi collegati alla carica di Hitler, ossia ordine-comando (Befehl), decreto (Erlass), regolamento (Verordnung), ordine-disposizione (Anordnung) e ordinanza (Verfü gung)6, mi sembra opportuno fare alcune osservazioni per determinarne la na tura e la funzione, considerando che questi atti legislativi costituirono una novità nella storia “costituzionale” tedesca. I pronunciamenti di Hitler, tralasciando il ruolo di comandante in capo della Wehrmacht, ricordano quelli di un monar ca assoluto. Possono essere dunque facilmente inquadrabili come strumenti, di vario livello ed efficacia, caratterizzanti l’aspetto giuridico della dittatura hitle riana, sia se utilizzati direttamente dal Führer, sia se “presi a prestito” da molti funzionari di primo piano della nomenclatura nazista e impiegati, in riferimento al livello e alla “forza giuridica” necessari, per conferire autorevolezza ed effica cia immediata ai singoli provvedimenti, tanto “settoriali” (ossia nei rapporti tra funzionari amministrativi, di pari o differente livello, civili e/o di polizia) quanto “territorali” (attuati negli Ehemalige Deutsche Ostgebiete)7. 6 Queste cinque categorie rappresentarono circa il 90% di tutte le direttive di Hitler, men tre gli atti collegiali del governo del Reich furono il 10% circa; in Moll (ed.), 2011, p. 17. 7 Per un quadro generale, cfr.: Majer, 1987; Böckenförde (ed.), 1985; Echterhölter, 1970. 423 Tanti piccoli Führer? Fino all’estate del 1934, non vi fu traccia delle ordinanze e dei decreti personali di Hitler tra le pagine della Reichsgesetzblatt8. Vi erano solamente regolamenti e circolari dei singoli ministri, oppure leggi della Reichsregierung nelle quali Hitler è citato come «Cancelliere del Reich». 2. Lo strumento di decretazione personale: i Führererlasse Nel primo anno e mezzo di governo, le sporadiche decretazioni “di tipo personale” prodotte da Hitler, come il decreto sulle funzioni e sui compiti del Reichsministeriums für Wissenschaft, Erziehung und Volksbindung (Ministero della Scienza, dell’Istruzione e dell’Educazione Na zionale) dell’11 maggio 1934, non configurano la persona del Führer come vero e proprio legislatore; egli agiva sempre in un quadro di raccordo con il Presidente del Reich, quasi come un organo esecutivo9.i i Manca inoltre la (successiva) tipica intestazione di «Decreto del Führer e Can celliere del Reich». Dopo un dibattito e una sorta di “conflitto di attribuzioni” con il governo, Hitler emanò un ordine il 27 luglio 1934 con il quale il dittatore, pochi giorni prima della morte di Hindenburg e senza alcun ricorso ad una leg ge di delega, dispose la partecipazione di Rudolf Hess, vice di Hitler e ministro del Reich senza portafoglio («Stellvertreter des Führers» und Reichsminister ohne Geschäftsbereich) alla preparazione di tutti i disegni di legge (Gesetzesentwürfe) nella posizione di «ministro del Reich interessato»10.ti Il 2 agosto 1934, alla morte di Hindenburg, venne infatti emanato il «Decreto del Presidente del Reich per l’attuazione della legge sul capo di stato del Reich tedesco del 1 ° agosto 1934»11. Si possono individuare alcuni elementi caratte rizzanti questa nuova forma di decretazione: l’intestazione «Erlass des Reichs kanzlers» (decreto – personale – del Cancelliere del Reich), l’assenza di un rife rimento a qualunque base normativa che autorizzasse o limitasse tale tipologia di decretazione, così come una terminologia che denotava un tono di sicurezza della propria posizione e un personalismo “insolito” per un testo di natura giuri dica (tra le espressioni adoperate , «Io voglio che…» e «Io esigo che…»)12.iii Lo status “costituzionale” del Führer non era giustificato da uno stato emergen ziale, ma dalla volontà di accentramento totale dell’autorità politica. Ne furono un esempio altri tre Führererlasse datati 2 agosto 1934 riguardanti: l’istituzione del Reichs-Justizprüfungsamt (l’ufficio del Reich per il controllo sulla giustizia), una disciplina in materia di insediamenti e alloggi, nonché il diritto alla grazia 424 Emiliano Vitti nelle cause penali13. Essi costituirono indirettamente il punto di svolta sostanziale del potere hitleriano, conferendo al dittatore la giustificazione necessaria per il ricorso costante alla decretazione personale14. 13 RGBl., 1934, I, pp. 845, 1069, 1225. 14 Per un quadro generale, cfr. Hubert, 1992. 15 Hubert, 1992, p. 16. 16 Hubert, 1992, p. 60. 17 RGBl., 1934, I, p. 75. Anche la cosiddetta legge delega (Ermächtigungsgesetz) del 24 marzo 1933 (RGBl., 1933, I, p. 141), prorogata poi nel 1937, 1939, 1943, standardizzò i poteri del governo come organo collegiale. Con riferimento all’ultima proroga della leg ge, del 10 maggio 1943 (RGBl., 1943, I, p. 295), cfr. le considerazioni di Hubert in Hubert, 1992, pp. 140-2; 205-6. 13 RGBl., 1934, I, pp. 845, 1069, 1225. 14 Per un quadro generale, cfr. Hubert, 1992. 15 Hubert, 1992, p. 16. 16 Hubert, 1992, p. 60. 17 RGBl., 1934, I, p. 75. Anche la cosiddetta legge delega (Ermächtigungsgesetz) del 24 marzo 1933 (RGBl., 1933, I, p. 141), prorogata poi nel 1937, 1939, 1943, standardizzò i poteri del governo come organo collegiale. Con riferimento all’ultima proroga della leg ge, del 10 maggio 1943 (RGBl., 1943, I, p. 295), cfr. le considerazioni di Hubert in Hubert, 1992, pp. 140-2; 205-6. 18 Circolare di Lammers ai membri del Consiglio di Difesa del Reich e ai Ministri del Reich del 20 Febbraio 1940, in BA R 43 II/1648, p. 146; viene riportato nel documento il pronun ciamento di Hitler sul tema. 19 Per un riferimento generale, cfr. Schaefer A., 1985, pp. 89-105. 19 Per un riferimento generale, cfr. Schaefer A., 1985, pp. 89-105. 18 Circolare di Lammers ai membri del Consiglio di Difesa del Reich e ai Ministri del Reich del 20 Febbraio 1940, in BA R 43 II/1648, p. 146; viene riportato nel documento il pronun ciamento di Hitler sul tema. 13 RGBl., 1934, I, pp. 845, 1069, 1225. 2. Lo strumento di decretazione personale: i Führererlasse Peter Hubert pose l’interessante quesito sul perché, fino al 1945, non fu concesso per legge il monopolio del pote re legislativo al dittatore, sebbene nei primi anni di regime la giurisprudenza na zionalsocialista si fosse impegnata nel configurare una pur discutibile tutela giu ridica al suo operato e allo strumento della decretazione personale15. Per ragioni che peraltro lo stesso Hubert non riesce a dettagliare sul piano tecnico, non vi fu una puntuale codificazione di tipo costituzionale basata sul Führerprinzip, poiché il Reichstag mantenne il potere legislativo per tutti i dodici anni di regime16.tb iti Anche la cosiddetta legge sulla ricostruzione (Neuaufbaugesetz) del 1° gennaio 1934, deliberata dal Reichstag, autorizzò la produzione normativa del governo del Reich «in quanto organo collegiale», senza indicare il Führer o un singolo mi nistro17. È interessante notare che, poco dopo lo scoppio della guerra, Hitler si espresse sull’opportunità di riservare alcuni compiti legislativi al governo del Rei ch: «In ogni caso, le norme giuridiche, che per la loro importanza devono recare la firma del Führer, dovrebbero essere varate dal governo del Reich sotto forma di legge del Reich»18. Tuttavia, nel corso dei mesi questa linea di indirizzo fu ap plicata sempre meno, a partire dalle prime settimane successive alle fine della Campagna di Polonia.i La situazione giuridico-costituzionale, all’inizio della guerra, era solo formal mente caratterizzata da una certa “debolezza” del Führer, che sul piano del diritto vigente non aveva alcuna potestà legislativa, né esclusiva né concorrente (con il Reichstag, il governo e il popolo, attraverso i referendum). Hitler non si lasciò confinare nel ruolo previsto, più o meno direttamente, dal diritto, e non rinunciò alla “libertà d’azione”, “guadagnata” dopo la morte di Hindenburg.t Durante la guerra vi fu il costante ricorso ai Führererlasse; si assistette conte stualmente al declino del decreto del governo come “strumento giuridico alter nativo”19. I decreti personali di Hitler rappresentarono dunque il perno giuridico e operativo su cui il regime nazionalsocialista costituì il funzionamento del sistema 18 Circolare di Lammers ai membri del Consiglio di Difesa del Reich e ai Ministri del Reich del 20 Febbraio 1940, in BA R 43 II/1648, p. 146; viene riportato nel documento il pronun ciamento di Hitler sul tema. Tanti piccoli Führer? Tanti piccoli Führer? 20 Hubert, 1992, p. 61. 21 Si cita come esempio il dibattito sulla inedita decretazione di Hitler in materia di raffor zamento della germanicità del 7 ottobre 1939, in Huber, 1941, pp. 530-79 (qui p. 560). 22 Capo della Cancelleria del Reich dal 30 gennaio 1933 al 24 aprile 1945. 23 Huber, 1941, p. 561. 24 Huber, 1941, p. 561. Osservazioni sul crescente numero e sull’importanza dei cosiddetti Führerakte si trovano anche in Weber, 1942, pp. 101-37 (qui p. 101). 25 Cfr. inoltre i progetti per un decreto di Hitler «sul suo temporaneo alleggerimento da questioni di governo e amministrative in» in BA R 43 II/958, pp. 32-3. 26 Huber, 1941, p. 553. 27 Cfr. Korte, 1942, pp. 473-6, 498-501 (qui pp. 499-501); Weber, 1942, pp. 123-7. Nel caso, la decretazione personale venne presentata come un dogma, in cui i singoli atti presentavano spesso una singolare formulazione, indicata come “buntes Bild” (immagine variopinta), alla quale non era sempre attribuibile un significato squisitamente giuridico. 28 Korte, 1942, p. 498.ff pp (q p ) 31 Cfr. Best, 1938, pp. 44-50; cit. in Herbert, 1996, p. 179. 2. Lo strumento di decretazione personale: i Führererlasse 425 statuale, sia all’interno del Reich sia nei territori occupati, in particolare il GG, mediante un sistema di legislazione eccezionale permanente20.tiii statuale, sia all’interno del Reich sia nei territori occupati, in particolare il GG, mediante un sistema di legislazione eccezionale permanente20.tiii Non mancarono tuttavia i tentativi di noti giuristi del regime per armonizzare questa discrepanza giuridica.t Le incongruenze formali del sistema giuridico nazionalsocialista furono oggetto di studio da alcuni giuristi di regime, riscontrabile in alcuni articoli di riviste te desche specializzate degli anni Trenta e Quaranta21. A partire dagli anni Novanta, lo studio del diritto durante il nazismo ha registrato una serie di fondamentali contributi, come nel caso di Michael Stolleis; per questo motivo, è necessario sottolineare la singolarità del quadro italiano, caratterizzato da una sostanziale indifferenza verso questi testi da parte della quasi totalità degli storici.iii f qii p q g I cambiamenti nella prassi costituzionale e amministrativa del Reich furono accuratamente registrati e analizzati dalla dottrina, sebbene non siano stati in terpretati come segnali involutivi di disintegrazione del sistema costituzionale, ma come una forma di consolidamento della leadership hitleriana. L’adozione di strumenti giuridici come il disegno di legge e la bozza di regolamento (nell’ambito di una sorta di “tecnica di rotazione degli atti normativi”), attraverso i quali Hans Lammers22, nei primi due anni circa di guerra, assurse al livello di “Ministro della Legge”, anziché delle consuete precedenti deliberazioni all’interno del Gabinetto del Reich (presieduto da un “distratto” Hitler), trovò alcuni attenti osservatori. A fronte di un’autentica frammentazione degli uffici amministrativi e di governo del Reich (Huber menziona un totale di 42 uffici governativi, di cui 38 all’inter no del territorio tedesco e 4 nei territori occupati, tra cui lo stesso GG23) e la conseguente moltiplicazione degli atti normativi emanati dalle varie autorità, il decreto personale del Führer rappresentò il fulcro giuridico attorno al quale gra vitarono tutti gli altri atti giuridici; «l’unità dell’amministrazione per la guida del Reich» e la tenuta politica del paese in guerra, furono strutturate sulla centrale figura di Hitler24. La conseguente parziale riattivazione degli organi collegiali go vernativi consentì di “alleggerire” il peso dei lavori del Consiglio dei ministri per la difesa del Reich (Ministerrat für die Reichsverteidigung), piuttosto attivo all’i nizio della guerra25. p 29 Best, 1941, pp. 15-6. L’autore afferma come l’azione statale sia l’affermazione della volontà di un leader «attraveso un singolo ordine o la determinazione e accettazione di regole che siano giuridicamente vincolanti indipendentemente dalla forma». 30 H b 1935 202 29 ( i 228) 26 Huber, 1941, p. 553. 27 Cfr. Korte, 1942, pp. 473-6, 498-501 (qui pp. 499-501); Weber, 1942, pp. 123-7. Nel caso, la decretazione personale venne presentata come un dogma, in cui i singoli atti presentavano spesso una singolare formulazione, indicata come “buntes Bild” (immagine variopinta), alla quale non era sempre attribuibile un significato squisitamente giuridico. 28 Korte, 1942, p. 498. 29 Best, 1941, pp. 15-6. L’autore afferma come l’azione statale sia l’affermazione della volontà di un leader «attraveso un singolo ordine o la determinazione e accettazione di regole che siano giuridicamente vincolanti indipendentemente dalla forma». 30 Huber, 1935, pp. 202-29 (qui p. 228). 31 Cfr. Best, 1938, pp. 44-50; cit. in Herbert, 1996, p. 179. 2. Lo strumento di decretazione personale: i Führererlasse Nonostante l’ampia attività del Consiglio, Hitler mantenne uno stretto controllo su tutte le funzioni di governo; perciò Ernst Rudolf Huber, luminare del diritto costituzionale nazista, sostenne l’inesistenza del pericolo di 426 Emiliano Vitti separazione tra governo e leadership dello stato, in ottemperanza ai dettami del Führerprinzip26.tit Data l’incoerenza formale degli atti di Hitler rispetto al sistema giuridico vigente e l’impossibilità di mascherarla da parte della dottrina nazionalsocialista attra verso semplici circonlocuzioni27, Huber e altri studiosi decisero di inserire aperta mente nel panorama della giurisprudenza “costituzionale” tedesca delle formule giuridiche che accentuassero il carattere preminente della concentrazione del potere nella figura del Führer e nella personalità espressa attraverso la decreta zione personale nel quadro del Führerstaat28. Dal momento che Hitler rappresentava l’unica carica suprema col potere di decidere sulle questioni di pace o guerra, il dibattito tra giuristi sulla natura e sull’origine degli atti normativi diretti del Führer e sulla possibilità di considerarli autonomi o derivativi risultò irrilevante. Tanto più che le varianti tecniche dei de creti personali mantenevano l’elemento essenziale, ossia l’esercizio diretto della volontà del Führer, indipendente e strutturalmente superiore a qualunque stru mento giuridico. Venne addirittura negata, da parte del Dott. Werner Best, avvo cato e vice di Reinhard Heydrich presso il quartier generale della Gestapo, ogni distinzione tra norme forti e deboli, tra legge costituzionale e legge ordinaria, tra leggi, ordinanze e decreti, tra diritto pubblico e privato, poiché: «La volontà da parte della guida politica, in qualunque forma essa trovi espressione, crea la legge e modifica la legge esistente»29.t La dottrina sostenne sempre l’assoluta onnipotenza di Hitler sul piano giuridico e la sua insindacabile facoltà di adottare decisioni attraverso leggi, decreti o qua lunque altro atto normativo. Huber riassunse così il principio del Führermacht: «Il potere del Führer è esclusivo, onnicomprensivo e illimitato; esso è sovrano»30; ciò ha portato alla parificazione delle tipologie di atti adottate dal Führer. Secondo questo principio, come riportato da Werner Best, «dal momento che la legge, così come l’ordinanza governativa, l’ordinanza amministrativa, l’ordine di servi zio, la disposizione unica [costituiscono] in definitiva un Führerbefehl (ordine del Führer), non è possibile attribuire alla legge alcuna priorità»31. Ogni parere critico dei giuristi a questo orientamento esistente negli anni Trenta scomparve durante Tanti piccoli Führer? 32 Il noto costituzionalista di regime Otto Koellreutter si espresse così su questo argomen to: «Questi modelli, come la legge e l’ordinanza, hanno ciascuno le proprie peculiarità. Pertanto nello stato nazionalsocialista non tutte le espressioni della Führung agiscono come una legge. Per essere efficace come una legge, il Führerwille dovrà vestire i panni giuridici della legge». Koellreutter, 1938, p. 15. 2. Lo strumento di decretazione personale: i Führererlasse 427 la guerra32.i la guerra32.i A partire dal 1943 il tema della maggiore efficacia della legge rispetto agli altri atti normativi emanati da Hitler cessò di suscitare interesse per i giuristi tedeschi e fu considerata una “ovvietà” sul piano giuridico33.tifti In merito al dibattito storico sulla effettiva centralità di Hitler nel regime nazi sta, l’interpretazione della natura dei Führererlasse e il loro inserimento nel qua dro “costituzionale” dello stato possono essere d’aiuto nel definirne i parametri34. La costruzione di una visione d’insieme sulla natura giuridica delle decisioni prese da Hitler è sicuramente molto difficile, così come la conseguente analisi del grado di esclusività che avrebbero dovuto avere i decreti personali; non è infat ti possibile affidarsi solamente alle pubblicazioni sul Reichsgesetzblatt (Gazzetta del Reich). Di conseguenza, la proliferazione della decretazione personale nei territori po lacchi occupati, con riferimento all’amministrazione civile e alle SS, può essere generalmente intesa sotto due profili: il primo, di natura personalistica, garantì a ciascun funzionario la possibilità di affermare la propria autorità politica, tanto verso i propri subordinati quanto verso i colleghi di pari livello e nei confronti delle popolazioni assoggettate; la “straordinarietà” delle condizioni li rese titolari di un potere quasi illimitato, esercitato in un contesto di tipo quasi “feudale”. Il secondo, di carattere “tecnico”, ebbe la indiretta conseguenza di rafforzare uno strumento giuridico che accrebbe l’efficacia dell’azione amministrativa sul terri torio. ii Al fine di determinare le ragioni di un esercizio così personalistico del potere, cui furono così funzionali questi strumenti giuridici, sembra dunque opportuno valutare il contesto territoriale, l’impatto col nuovo “ambiente di lavoro” dei fun zionari chiamati nel GG, il processo di “integrazione” che li accompagnò nell’ope ra di gestione, ma soprattutto il peso delle tematiche razziali nei distretti.i Per indicare le dinamiche interne alle amministrazioni locali, credo risulti op portuna una presentazione di alcuni casi significativi, che meglio qualificano il rapporto tra soggetti, sistema giuridico e territorio. g 34 Zitelmann, 1992, pp. 491-506. g ggt 33 Cfr. a riguardo Rüthers, 1988. 3. Eccessi e incongruenze nell’amministrazione locale: l’impatto col GG L’analisi del lavoro degli amministratori locali non può prescindere da un approc cio di tipo “istituzionale”, fondamentale per cercare di comprendere i rapporti sia 32 Il noto costituzionalista di regime Otto Koellreutter si espresse così su questo argomen to: «Questi modelli, come la legge e l’ordinanza, hanno ciascuno le proprie peculiarità. Pertanto nello stato nazionalsocialista non tutte le espressioni della Führung agiscono come una legge. Per essere efficace come una legge, il Führerwille dovrà vestire i panni giuridici della legge». Koellreutter, 1938, p. 15. 428 Emiliano Vitti tra i diversi livelli di amministrazione sia al loro interno. L’architettura istituzionale del GG garantiva, più per inadeguatezza che per formazione, una certa signifi cativa libertà d’azione ai singoli capi-area, le cui efficienza e credibilità dipende vano dalle personalità dei singoli Kreishauptmänner (capi dei distretti locali) e Stadthauptmänner (capi dei distretti cittadini).ttit Rispetto all’esercizio delle attività di occupazione, si tratta di comprendere la predisposizione degli amministratori tedeschi nel prestarsi automaticamente a processi di radicalizzazione nel trattamento delle popolazioni dominate, come polacchi ed ebrei, con le distinzioni derivanti dalla dottrina razziale. Inoltre va intesa la portata dei margini di manovra e della discrezionalità di cui essi gode vano, una libertà d’azione, tacitamente concessa e autogestita, le cui conseguen ze furono incompatibili con un esercizio equilibrato dell’attività amministrativa all’interno della peculiare forma istituzionale del GG. i Quanto contarono le motivazioni ideologiche? Quanto la predisposizione alla violenza dei singoli soggetti e il carrierismo privo di scrupoli diffuso nella buro crazia nazista? Fino a che punto la radicalizzazione degli occupanti trasse origine dall’interno della macchina amministrativa?iii Con la destituzione di molte delle autorità politiche e istituzionali locali, il paese ha perso parte di quelle figure che mantenevano il legame (peraltro flebile) tra le diverse identità che componevano la società polacca, separandone di fatto alcuni settori e isolando sia la classe degli intellettuali polacchi sia la comunità ebraica. Questa “atomizzazione delle relazioni sociali” venne accelerata dall’operato degli occupanti con una brutalità senza precedenti e con la gerarchizzazione, su base etnica, del modello amministrativo e sociale nazionalsocialista35. Il corpo eterogeneo degli amministratori locali tedeschi del GG rivelò spesso gli aspetti più duri del dominio tedesco nei confronti dei polacchi, disprezzati e umiliati durante la gestione della quotidianità36. 35 Młynarczyk, 2004, pp. 145-6. 36 Riferimenti in Harten, 1996, pp. 86-7. 37 Lettera di Heinz Doering alla madre del 28 aprile 1942, in Roth, 2009, p. 7. Riferimenti più ampi sulla figura di Doering, principalmente riguardo il ruolo di giurista e amministra tore, in Friedrich, Heim, 2013, pp. 274 segg. 38 La documentazione sull’afflusso di personale, civile e SS, dal Reich alla Polonia occupa ta, e sui successivi smistamenti all’interno del GG, è vasta e frastagliata. Riferimenti sul tema sono presenti in molti archivi, tra cui Bundesarchiv Berlin (BAB) e Instytut Pamięci 35 Młynarczyk, 2004, pp. 145-6.i y y , , pp 36 Riferimenti in Harten, 1996, pp. 86-7. 3. Eccessi e incongruenze nell’amministrazione locale: l’impatto col GG «Sterile, vuota, desolata, una stazione in macerie, sporcizia, puzza, gentaglia, una lingua straniera, a malapena una parola di tedesco, [questa] è stata la prima accoglienza»37; questo fu il primo impatto di Goebbels alla vista di Cracovia. Heinz Doering, giurista bavarese in servizio presso il distretto di Mühldorf, trascorse solo un paio di giorni nella capitale del Governatorato generale, come riportato nelle lettere alla madre dell’aprile del 1942. Con riluttanza seguì la moglie nel GG, mostrandosi disgustato da «tutta questa gentaglia orientale semiumana (Halb menschentums)»38. Tanti piccoli Führer? 429 Il disagio di molti funzionari tedeschi “colpiti” da trasferimento si spiegava con la pessima reputazione di cui godeva il GG: il condizionamento prodotto dalla propaganda di Goebbels era talmente forte che quasi ogni funzionario rimase inizialmente traumatizzato dall’impatto col nuovo ambiente di lavoro, con le abi tudini e con lo stato dei servizi, degli alloggi e delle infrastrutture. «Il buco era nero di sudiciume e puzzava. L’intonaco si staccava, il lavandino era rotto, i letti di ferro sembravano essere […] rottam[i] [recuperati in] alcune baracche, i mate rassi avevano macchie di merda [vecchie di] generazioni»39; e ancora: «inganna la servile […] gentilezza, con la quale i polacchi si mostrano di facciata. Alle spalle sono [invece] pericolosi»40.i Dopo i primi giorni di disperazione, i residui propagandistici e il trauma del trasferimento lasciarono spazio a più ottimistiche considerazioni sulle effettive condizioni riservate ai funzionari amministrativi tedeschi in terra polacca. Nello specifico caso di Doering, possiamo parlare di uno dei (non molti) casi di sposta mento fondato sulla effettiva esigenza della Zentralregierung (governo centrale) di inserire personale competente “ariano” nel quadro di comando delle ammi nistrazioni locali, che vedevano una componente polacca largamente maggio ritaria. Dopo alcuni giorni di soggiorno a Cracovia, Doering scrisse alla madre: «Non lasciarti sfuggire nulla! Siamo così abbondantemente forniti di tutto […] [da sentirci] come in paradiso e certamente non abbiamo bisogno di nulla». In effetti i benefits dei coniugi Doering furono molti e di varia natura: ricevettero «un ap partamento, mobili, biancheria e molto altro, godevano di un buon [trattamento economico]» dal quale trassero un alto tenore di vita e si trovarono in condizione di «accumulare ricchezza»41.i Questo risvolto vantaggioso accrebbe lo spirito di iniziativa e le intenzioni di dominio di molti funzionari: «prima di tutto», asserì Doering, «non siamo qui per divertimento, ma come coloni!»42. Narodowej (Istituto di Memoria Nazionale – IPN). In particolare si ricorda l’iter seguito da Karl Heinrich Wilhelm Koppe nella sua carriera dalla Germania a Posen (maggio 1940 – giugno 1941), poi tra Posen e Chelmno (giugno 1941 – 8 novembre 1943) e poi a Cracovia (9 novembre 1943 – 9 dicembre 1944). Circolare indirizzata al Procuratore Capo presso la corte distrettuale di Bonn, al secondo tribunale penale della corte distrettuale di Bonn, atto d’accusa, Bonn, 10 settembre 1961, in IPN, BU-2586-329, pp. 4-6. La circolare fa ri ferimento all’arresto di Koppe, avvenuto nel 1960 a Bonn, dove l’ex gerarca viveva con lo pseudonimo di Wilhelm Karl Heinrich Lohmann, avendo assunto il cognome della moglie.t p , g 9 Lettera di H. Doering alla madre del 28 aprile 1942, Roth, 2009, p. 7.t Lettera di H. Doering alla madre del 28 aprile 1942, Roth, 2009, p. 7. 0 Lettera di H. Doering alla madre del 5 maggio 1942, Roth, 2009, p. 8. p 43 Riferimenti in Schwaneberg, 2009, pp. 133-54, https://ipn.gov.pl/pl/szukaj t g gg p 1 Lettera di H. Doering alla madre del 5 maggio 1942, Roth, 2009, p. 8. 2 Roth, 2009, p. 8. Lettera di H. Doering alla madre del 5 maggio 1942, Roth, 2009, p. 8. 1 Lettera di H. Doering alla madre del 5 maggio 1942, Roth, 2009, p. 8. 40 Lettera di H. Doering alla madre del 5 maggio 1942, Roth, 2009, p. 8. 41 Lettera di H Doering alla madre del 5 maggio 1942 Roth 2009 p 8 3. Eccessi e incongruenze nell’amministrazione locale: l’impatto col GG Ritorna dunque la considerazione del ter ritorio del Governatorato come di una “terra delle opportunità” per i nazisti, da conquistare tramite lo sfruttamento intensivo delle risorse umane e materiali che il nuovo stato metteva a disposizione degli occupanti43. Narodowej (Istituto di Memoria Nazionale – IPN). In particolare si ricorda l’iter seguito da Karl Heinrich Wilhelm Koppe nella sua carriera dalla Germania a Posen (maggio 1940 – giugno 1941), poi tra Posen e Chelmno (giugno 1941 – 8 novembre 1943) e poi a Cracovia (9 novembre 1943 – 9 dicembre 1944). Circolare indirizzata al Procuratore Capo presso la corte distrettuale di Bonn, al secondo tribunale penale della corte distrettuale di Bonn, atto d’accusa, Bonn, 10 settembre 1961, in IPN, BU-2586-329, pp. 4-6. La circolare fa ri ferimento all’arresto di Koppe, avvenuto nel 1960 a Bonn, dove l’ex gerarca viveva con lo pseudonimo di Wilhelm Karl Heinrich Lohmann, avendo assunto il cognome della moglie.t 430 Emiliano Vitti Emiliano Vitti Il percorso professionale del giurista bavarese può essere considerato un esem pio di piccolo carrierismo tipico dei funzionari locali: promosso a capo reparto dopo sole due settimane di permanenza nel Distrikt Krakau, fu progressivamente spostato verso i centri periferici del distretto fino alla realizzazione del suo obiet tivo più importante, ossia la nomina a Kreishauptmann. Dall’agosto 1943 all’ago sto 1944 ricoprì infatti la carica di Stadthauptmann di Opatów44, nel distretto di Radom, in sostituzione del Landrat (presidente del distretto rurale) Heinz Ritter (in carica dal gennaio 1940)45.fi Le condizioni di lavoro favorevoli agli amministratori tedeschi non furono suffi cienti a nascondere la natura e le conseguenze dell’indottrinamento nazista nel rapporto col territorio occupato. L’ostilità e l’esercizio della violenza nei confronti (principalmente) di polacchi ed ebrei costituì una sorta di tacita conditio sine qua non per una “corretta” applicazione dei principi amministrativi del nazionalsocia lismo.i «D'altra parte [dobbiamo], almeno in via provvisoria, rinunciare alla politica dei trasferimenti e di estinzione, perché non abbiamo abbastanza person[ale] e quindi per il momento [c’è] ancora bisogno de[lla manodopera] polacc[a] […]. Chi […] altrimenti, per esempio, [potrebbe] lavor[are] nei campi?»46. L’insieme di pragmatismo e radicalismo nell’approccio degli amministratori tedeschi alla loro “nuova missione” rivelò tutto il condizionamento ideologico, che non lasciava spazio alla pietà o alla compassione. Al contrario: la dottrina li aiutò a respingere questi impulsi, giustificandosi attraverso un’autodifesa razionalmente costruita. , g , , , p , 6 Lettera di H. Doering alla madre del 5 maggio 1942, Roth, 2009, p. 9.iit ch=4061521& sort=2&order=1&ile=20 (20-05-2021), documento caricato sull’archivio online dell’IPN il 16 aprile 2016. 44 Jehke (ed.), Territoriale Veränderungen in Deutschland und deutsch verwalteten Geb ieten 1874-1945, in http://www.territorial.de/gg/opatow/kreish.htm (20-05-2021), cari cato il 20 agosto 2009. 45 du Prel M. F., 1940, Das deutsche Generalgouvernement Polen: ein Überblick über Geb iet, Gestaltung und Geschichte, Krakau, BVO, p. 101, 133-4. 46 Lettera di H. Doering alla madre del 5 maggio 1942, Roth, 2009, p. 9. 47 fi b fi ß ( ) ht // h k l bl t 7 Riferimenti biografici su Gramß (o Gramss) in http://jewishsokolow.blogsp 45 du Prel M. F., 1940, Das deutsche Generalgouvernement Polen: ein Überblick über Geb iet, Gestaltung und Geschichte, Krakau, BVO, p. 101, 133-4. 3. Eccessi e incongruenze nell’amministrazione locale: l’impatto col GG Oltre al disprezzo, vi fu lo scherno e la repulsione per le pessime condizioni del la popolazione, che venivano imputate agli stessi polacchi, visti come colpevoli della loro scarsa qualità di vita. Con gli ebrei però l’accanimento fu più marcato: inquadrati come un “non essere”, non vi era alcuna limitazione, neppure forma le, agli attacchi perpetrabili contro di essi. Le operazioni di sterminio vennero anzi ricondotte all’interno di “parametri legali”: gli amministratori tedeschi non furono, in linea generale, solamente gli esecutori materiali di circolari e direttive provenienti dall’alto, ma parteciparono ampiamente alla progettazione di alcuni provvedimenti, personalizzandone talvolta gli aspetti relativi alla situazione dei singoli Kreise. ersone come Ernst Gramß47, funzionario e membro del partito trasferitosi con Tanti piccoli Führer? 431 la moglie dalla Germania nel novembre 193948, vissero la ricollocazione nel GG come una sorta di shock culturale. Sua moglie descrisse disgustata il viaggio in treno verso Cracovia: «il primo impatto era di grande povertà, un quadro deso lante, ma [apparentemente] non per gli effetti della guerra […] nei singoli villaggi ebrei (Judendörfer) un’immagine semplicemente indescrivibile […] la sporcizia […] i tipi dii i ebrei da coprirsi il volto alla sola vista, in parte deformati da nanismo, [dal] l’obesità»49. Presto Gramß trasse conclusioni radicali. Da Varsavia scrisse a sua moglie: «il quartiere ebraico è una vergogna, 300.000 ebrei […] spettacoli criminali in ab bondanza […] estirpar[li] sarebbe un vantaggio per l’umanità»50. Poco tempo dopo si ebbe il primo ebreo impiccato: le accuse più comuni prese a pretesto dai nazisti erano di alterare i prezzi e fare affari sul mercato nero; peraltro si trattava dello stesso tipo di affari da cui i funzionari tedeschi locali e molti soldati della polizia di sicurezza traevano grandi guadagni personali51.i Gramß non era un caso isolato; numerosi tedeschi chiamati a lavorare in Po lonia rimasero “scioccati”. Un caporale descrisse le proprie impressioni in una lettera del 11 settembre 1940: «giunti a destinazione, ci trovammo in una città la cui popolazione era per l’80% ebrea. Anche solo uno di loro […] è uno spettacolo disgustoso, ma migliaia di questo ceppo sono eccessivi. Quale aiuto si può dare, questa gente non vuole e non può migliorare»52. 53 Verbale della riunione dei governatori distrettuali, dei sindaci e dei commissari cittadini del distretto di Radom, 25 novembre 1939, in BAL, B 162/Dok-Slg. Polen, Ordn. 344, p. it/2011/08/ernst-gramss.html (16-06-2021), archivio online dell’associazione culturale ebreo-polacca Jewish Sokol´ów Podlaski. 48 Ernst Gramß ricoprì, dal 30 novembre 1939 al 6 maggio 1940, la carica di capo del dipartimento per le forniture di cibo e l’agricoltura nel distretto di Varsavia. In http:// jewishsokolow.blogspot.it/2011/08/ernst-gramss.html (23-05-2021). 49 Stralcio di una lettera senza data di Gramß a sua moglie, risalente presumibilmente al novembre o al dicembre 1939, in BAL, B 162/Dok-Slg. Polen, Ordn. 344, p. 45. 50 Lettera senza data di Gramß alla moglie del dicembre 1939, sugli inizi del suo periodo di lavoro a Varsavia, Roth, 2009, p. 46. 51 Engelking, Leociak, 2009, pp. 384-9. 52 M h k 1995 17 50 Lettera senza data di Gramß alla moglie del dicembre 1939, sugli inizi del suo periodo di lavoro a Varsavia, Roth, 2009, p. 46. j g p g 49 Stralcio di una lettera senza data di Gramß a sua moglie, risalente presumibilmente al novembre o al dicembre 1939, in BAL, B 162/Dok-Slg. Polen, Ordn. 344, p. 45.t g g 52 Manoschek, 1995, p. 17. , , , p 51 Engelking, Leociak, 2009, pp. 384-9. 3. Eccessi e incongruenze nell’amministrazione locale: l’impatto col GG Come riportato prima da Robert Seidel e poi da Markus Roth, nel novembre 1939 Frank ricevette i Kreishauptleute (dirigenti dei distretti locali) e Stadthauptleute (dirigenti dei distretti cittadini) del Distretto di Radom per essere informato sul lavoro svolto nelle prime settimane di vita del GG.itii Il primo a esporre i risultati dell’attività amministrativa fu Karl Glehn, anch’egli arrivato dal Reich: con superbia espose un resoconto estremamente “gonfiato” sul tipo di “lavoro pioneristico” svolto: «In principio il chaos. Tutti fuori dalle fab briche a lavorare per strada, furti, contrabbando, truffe, terreno ideale per gli ebrei. Tutto [necessitava di] controll[o] […]»53. 53 Verbale della riunione dei governatori distrettuali, dei sindaci e dei commissari cittadini del distretto di Radom, 25 novembre 1939, in BAL, B 162/Dok-Slg. Polen, Ordn. 344, p. 432 Emiliano Vitti Emiliano Vitti Glehn discusse sulla necessità di attuare il processo di arianizzazione dei terri tori con un costante sfruttamento dei terreni agricoli e della popolazione locale a favore del Reich, per preparare il territorio polacco sul piano economico alla completa germanizzazione e normalizzazione del sistema produttivo. Una parte di questo programma prevedeva pene molto severe per i polacchi che facevano ricorso al mercato nero e a traffici illegali per occultare parte dei raccolti e trarne guadagni personali (esattamente come facevano da impuniti i funzionari tede schi). Glehn concluse il suo intervento dicendo che, nel rispetto di quei presup posti, «il lavoro regala gioia»54. Il raggio d’azione dei singoli funzionari locali era così ampio che Glehn diceva spesso con sarcasmo di potersi nominare egli stesso giudice ed eseguire con danne a morte senza conseguenza alcuna per la sua persona. Riferite a Frank, queste affermazioni non incontrarono opposizione da parte del Governatore, che invitò i suoi capi locali a governare col pugno di ferro per il bene del Reich, anziché esercitare con accuratezza la funzione di raccordo tra gli amministratori. Frank incoraggiava apertamente i funzionari locali a superare il concetto di stret ta competenza e a ricorrere a deviazioni e scorciatoie per assolvere al compito principale: «Per come stanno le cose ora, questo rappresenta il primo tentativo di una nuova modalità di gestione dell’insediamento tedesco, che attraverso le esperienze si sta costruendo una nuova via […] Sono convinto che l’utilizzo di altre teorizzazioni arrechi solo disturbo [all’amministratore]». 3. Eccessi e incongruenze nell’amministrazione locale: l’impatto col GG Frank incoraggiò ad agire in funzione anti-polacca e anti-ebraica nelle politiche di occupazione, impedendo che un solo polacco conservasse la libertà di circolazione: «nessun polacco potrà mai più osare [mischiarsi] con dei signori tedeschi. A questo scopo è compito dell’amministrazione essere disposta a sopprimere ogni polacco pro veniente dal Reich». Il GG secondo Frank avrebbe dovuto svolgere la già citata funzione di “pattumiera sociale del Reich” senza subire diktat da Berlino e dalle SS che minassero la “autonomia” del nuovo stato e l’aura di “autorevolezza” che il Governatore credeva di possedere. Il raggio d’azione dei singoli funzionari locali era così ampio che Glehn diceva spesso con sarcasmo di potersi nominare egli stesso giudice ed eseguire con danne a morte senza conseguenza alcuna per la sua persona. Riferite a Frank, queste affermazioni non incontrarono opposizione da parte del Governatore, che invitò i suoi capi locali a governare col pugno di ferro per il bene del Reich, anziché esercitare con accuratezza la funzione di raccordo tra gli amministratori. t 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità La condotta eccessivamente violenta all’interno del GG, tanto delle SS quanto dei membri della Zivilverwaltung, venne apertamente criticata da molti funzio nari amministrativi. Tale presa di posizione non aveva (salvo rarissime eccezioni) fondamenti di tipo “umanitario”: la tortura inflitta quasi per gioco venne ritenuta un elemento superfluo e dagli effetti controproducenti; era come se l’abuso della forza potesse sminuire la “funzione sociale” che i nazisti credevano di svolgere 447. 54 BAL, B 162/Dok-Slg. Polen, Ordn. 344, pp. 449, 458. Tanti piccoli Führer? 433 nell’opera di “purificazione dei territori dagli influssi del veleno ebraico”55. Un esempio è dato da una lettera scritta da Heinz Doering alla madre in relazione alla vicenda di un sorvegliante che mostrava, a suo dire, divertimento nel frustare alcuni ebrei: «Così non va bene. Con lo sterminio degli ebrei sono piuttosto d’ac cordo, ma questo sadismo è troppo. Contro la crudeltà da macello si sono fatte regole. Perciò si dovrebbe, a mio parere, eliminare gli ebrei, ma non torturarli per divertimento […] Io sarei per la eliminazione totale»56. I Kreishauptmänner godevano di un potere molto esteso, comprensivo di pos sibilità pressoché illimitate nel ricorrere a varie forme di illegalità. Quando Ernst Gramß fu messo a capo della contea di Sokołów nel giugno 1940 si sentì al setti mo cielo, non solo per l’avanzamento di carriera, ma perché prese coscienza di quali altre forme di abuso erano concesse ai funzionari: principalmente il furto, la corruzione e lo sfruttamento delle strutture appartenute alle istituzioni polacche o a ex-cittadini facoltosi. Univa dunque l’euforia da potere per la possibilità di ge stire settori come economia, agricoltura, gestione del territorio, foreste, lavoro, trasporti, libera circolazione, polizia (in concorrenza con le SS) alla sostanziale vita da nababbo grazie a privilegi come cavalli per l’equitazione, un parco per sonale con piscina per il nuoto, la sauna, una schiera di servitori al suo servizio (bambinaie, cuoche, camerieri). Poco dopo la sua nomina scrisse alla moglie che il lavoro era così piacevole da volere «rimanere per sempre»57.ti L’atteggiamento da sovrano assoluto di Gramß era tipico dei responsabili di molti distretti locali nel GG: anche da questi comportamenti infantili e dall’osten tazione di uno sfarzo pagato da altri si riconosceva il dilettantismo di una larga fetta del personale amministrativo58.tti I funzionari tedeschi vennero così progressivamente attratti dal lavoro nel GG e nel Warthegau col miraggio di privilegi materiali. 55 Ricorrente espressione utilizzata da Hitler per definire la funzione della Soluzione Fi nale. 56 Lettera di H. Doering alla madre del 29 settembre 1942, in Roth, 2009, p. 49. 57 Lettera di Gramß alla moglie, s.i.d., probabilmente risalente al 2 giugno 1940, in Roth, 2009, p. 50. 58 BAL, B 162/II 211 AR-Z 73/66, parte 2, pp. 16, 140. Per riferimenti ai comportamenti dei funzionari vedi Madajczyk, 1987, p. 179. 59 Lettera di H. Doering alla madre del 30 luglio 1942, in Roth, 2009, p. 52. j y p 9 Lettera di H. Doering alla madre del 30 luglio 1942, in Roth, 2009, p. 52. 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità Doering era «convinto di [lavo rare] nell’amministrazione» perché «stufo di fare l’avvocato»; si dichiarava sod disfatto e riteneva gratificante poter vivere con sua moglie nel territorio che lo vedeva Kreishauptmann, anche se l’incarico era meno remunerativo della sua attività di giurista. I “grandi progetti” che dichiarava di avere erano generati dalla consapevolezza di una missione, permeati dunque tanto dall’ideologia quanto dall’ambizione personale, la stessa che a suo dire gli impedì di godersi assieme alla moglie i soldi guadagnati da avvocato e di avere «una vita migliore [composta da] poco lavoro, molto tempo libero e tanti soldi»59. 434 Emiliano Vitti Emiliano Vitti Emiliano Vitti Come riportato da Markus Roth, «non solo l’autonomia e la vita confortevole dei capi-distretto locali rese questa carica cosi appetita; vi si aggiunsero un errato idealismo e una fanatica e personale visione pioneristica del loro ruolo in Orien te»60.tt I capi-distretto e quelli cittadini si trovarono a ricoprire una funzione di gover no del territorio orientale in «organismi [attivi] in una missione temporalmen te limitata»61. Nel nuovo Lebensraum im Osten, «serv[iva] comandare, non […] governare». Joachim Nehring, nel suo articolo sulla Krakauer Zeitung, riportava: «Qui nulla è scritto. Qui valgono solo i fatti. Qui nessuno può dire non è affar mio poiché non rientra nelle proprie responsabilità. Ovunque ti trovi, sei nel Reich! In tutto ciò che accade, un polacco vede in te il Reich! Perciò non vi è che un limite alle vostre competenze e responsabilità: gli interessi del Reich. Energici pionieri, liberi da regole burocratiche […], devono marcatamente ispirarsi [nel loro lavoro] in Oriente alle storiche origini tedesche»62. La prassi di comando si basava su una violenza consigliata e impunita: l’esistenza di alcune rarissime “oasi di normale amministrazione” dipendeva esclusivamente dalla volontà dei singoli capi locali e dalla risonanza che le loro azioni potessero avere ai piani alti della nomenclatura dello stato. La modalità di trattamento del la popolazione era finalizzata a lasciare un segno nella politica di occupazione tedesca e mostrare la propria efficienza come amministratori attraverso lavori di costruzione (Aufbauarbeit) di una testimonianza della historische Mission nell’O stgebiet. Hans-Adolf Asbach predispose il piano di ricostruzione e di organizzazio ne urbanistica della città di cui fu posto a capo, Brzeżany. 60 Discorso del 20 giugno 1943 di Viktor von Dewitz, Kreishauptmann di Stryj (Distrikt Galizien), in Diensttagebuch (DTB), IfZM, Fb 105/30, p. 7466. 61 Nehring, 1940, Dienst im Osten, in „Krakauer Zeitung“, 11 giugno, n. 136; http://bc.ra dom.pl/dlibra/plain-content?id=23554 (26-05-2021). Archivio online della biblioteca cit tadina di Radom. 62 Nehring, 1940. Frank durante un incontro dei presidenti dei distretti locali del distretto di Cracovia dell’8 novembre 1939, DTB, IfZ, Fb 105/1, Bl. 40. La valutazione sul rapporto tra le opportunità concesse ai singoli funzionari di distretto e il “sacrificio” di dover ri siedere in terra polacca accomunava molti capi distretto locali. Baedeker, 1945, passim. 63 Hans-Adolf Asbach, «Von Krakau bis Brzeżany (1 Januar 1940 – 13 Februar 1943)», in Roth, 2009, p. 54. 62 Nehring, 1940. Frank durante un incontro dei presidenti dei distretti locali del distretto di Cracovia dell’8 novembre 1939, DTB, IfZ, Fb 105/1, Bl. 40. La valutazione sul rapporto tra le opportunità concesse ai singoli funzionari di distretto e il “sacrificio” di dover ri siedere in terra polacca accomunava molti capi distretto locali. Baedeker, 1945, passim. 63 Hans-Adolf Asbach, «Von Krakau bis Brzeżany (1 Januar 1940 – 13 Februar 1943)», in Roth, 2009, p. 54. 60 Discorso del 20 giugno 1943 di Viktor von Dewitz, Kreishauptmann di Stryj (Distrikt Galizien), in Diensttagebuch (DTB), IfZM, Fb 105/30, p. 7466. 61 Nehring, 1940, Dienst im Osten, in „Krakauer Zeitung“, 11 giugno, n. 136; http://bc.ra dom.pl/dlibra/plain-content?id=23554 (26-05-2021). Archivio online della biblioteca cit tadina di Radom. 62 Nehring, 1940. Frank durante un incontro dei presidenti dei distretti locali del distretto di Cracovia dell’8 novembre 1939, DTB, IfZ, Fb 105/1, Bl. 40. La valutazione sul rapporto l i à i i li f i i di di tt il “ ifi i ” di d i 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità Incaricò due architetti di Dresda di elaborare la nuova pianificazione urbana, sia per gli edifici pubblici sia per molte abitazioni private; il risultato avrebbe dovuto essere inteso in prospet tiva storica: «Se oggi questi piani ci sembrano favolosi, posso solo dire che nulla è impossibile per i tedeschi»63. Per i suoi piani, Asbach scelse come banco di pro va un quartiere ebraico relativamente in buono stato, con case ben conservate; chiese al Judenrat un contributo per i lavori (Kontributionszahlung) in cambio del rilascio (temporaneo) di 600 ebrei. La truffa si realizzò crudelmente poco dopo il pagamento, quando i prigionieri rilasciati furono condotti qualche chilometro La prassi di comando si basava su una violenza consigliata e impunita: l’esistenza di alcune rarissime “oasi di normale amministrazione” dipendeva esclusivamente dalla volontà dei singoli capi locali e dalla risonanza che le loro azioni potessero avere ai piani alti della nomenclatura dello stato. La modalità di trattamento del la popolazione era finalizzata a lasciare un segno nella politica di occupazione tedesca e mostrare la propria efficienza come amministratori attraverso lavori di costruzione (Aufbauarbeit) di una testimonianza della historische Mission nell’O stgebiet. Hans-Adolf Asbach predispose il piano di ricostruzione e di organizzazioitti Tanti piccoli Führer? 435 fuori dalla città e li giustiziati sul posto64. fuori dalla città e li giustiziati sul posto64. Durante un discorso del 2 marzo 1940, Frank mostrò un certo “pragmatismo”, lontano dai radicalismi di certe frange del regime nazionalsocialista: «Noi non possiamo, dopo tutto, uccidere quattordici milioni di polacchi! […] [poiché] non disponiamo di person[ale sufficiente] per costituire un apparato [amministrati vo]». Sei giorni dopo parlò della responsabilità del GG nel garantire un regolare svolgimento della vita quotidiana per tutti i «non-tedeschi» residenti nel terri torio dello stato: «Come potrebbero [altrimenti] le altre nazioni desiderare di [finire] sotto la protezione tedesca?»65.tit it Al di là della condotta nei confronti delle popolazioni assoggettate, vi furono anche altre “abitudini” dei funzionari amministrativi tedeschi che finirono con l’essere giustificate dall’abuso della propria autorità, e della strumentazione giu ridica a loro disposizione. La corruzione, l’arricchimento personale, i vizi, le abi tudini sessuali, tutti elementi che smentivano la già peraltro discutibile difesa dei funzionari tedeschi da parte di Frank. 64 BAL, B 162/202 AR-Z 76/61, parte 2, pp. 469, 506. 65 Präg, Jacobmeyer, 1975, pp. 146-9. 66 Präg, Jacobmeyer, 1975, pp. 146-9. 67 In seguito SD. 68 Lettera di H. Doering alla madre del 29 giugno 1942, in Roth, 2009, p. 59. 69 Roth, 2009, p. 59. Il giudizio del SD fu sfruttato da Krüger per screditare l’immagine di 69 Roth, 2009, p. 59. Il giudizio del SD fu sfruttato da Krüger per screditare l’immag g 8 Lettera di H. Doering alla madre del 29 giugno 1942, in Roth, 2009, p. 59.t 68 Lettera di H. Doering alla madre del 29 giugno 1942, in Roth, 2009, p. 59. 69 Roth, 2009, p. 59. Il giudizio del SD fu sfruttato da Krüger per screditare l’immagine di 69 Roth, 2009, p. 59. Il giudizio del SD fu sfruttato da Krüger per screditare l’immagine di 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità Heinz Doering analizzò correttamente lo scenario, causato da una cattiva selezione e dalla diffusa incompetenza dei fun zionari; indignato, scrisse a casa: «se penso a ciò che era una volta un ministero bavarese, le sue attività, l’ordine, la sua pulizia interna e la disciplina! E soprattut to le persone! [Oggi] sembra che la Germania abbia mandato tutta la feccia e la sporcizia qui, a far niente, ciascuno peggiore di dieci negri nudi. Quasi tutti coloro che sono qui fanno schifo e sono stati cacciati [dai rispettivi luoghi di lavoro] e spediti qui. Invece di colonizzare con decoro, si trattano i territori orientali come una specie di mucchio di spazzatura, sul quale poter scaricare lo sporco»66. Tali lamentele si potevano riscontrare ad ogni livello dell’amministrazione, tanto che lo stesso Sicherheitsdienst67 (servizio di sicurezza) sottolineò, non senza una dose di sarcasmo: «[era] come se si cercassero soltanto corrotti nel Governatorato ge nerale»68.f Doering riconosceva che, oltre alla corruzione e all’incompetenza diffuse, una grossa responsabilità dell’andamento generale fosse imputabile alla debolezza della leadership di Frank, poiché il Governatore passava buona parte del suo tem po tra Berlino e la sua tenuta di Kressendorf (Krzeszowice), lasciando i suoi subor dinati liberi di fare praticamente ciò che volevano. Il capo del SD del distretto di Galizia criticò apertamente il comportamento di Frank, accusandolo di scarso im pegno lavorativo e di non riconoscere alcun tipo di legame morale o istituzionale con lo Stato, poiché ogni funzionario corrotto poteva appoggiarsi al Governatore e alla sua famiglia69. 64 BAL, B 162/202 AR-Z 76/61, parte 2, pp. 469, 506. 65 Präg, Jacobmeyer, 1975, pp. 146-9. 66 Präg, Jacobmeyer, 1975, pp. 146-9. 67 In seguito SD. 68 Lettera di H. Doering alla madre del 29 giugno 1942, in Roth, 2009, p. 59. 69 Roth, 2009, p. 59. Il giudizio del SD fu sfruttato da Krüger per screditare l’immagine di 436 Emiliano Vitti Uno dei casi più interessanti di contrasto fittizio alla corruzione, ma di fattiva condotta corruttiva da parte di funzionari locali fu quello di Karl Valentin, capo della contea di Ostrów, nel distretto di Varsavia. Giunto nella Polonia occupata da soldato dopo una militanza nelle SA, in poco tempo Valentin divenne collabo ratore personale del governatore del distretto di Varsavia Ludwig Fischer70; il 19 gennaio 1942 fu nominato Kreishauptmann di Ostrów71. 3 Protocollo della riunione del 28 aprile 1942, in APW, KHm Ostrów 75, p. 23. 72 Protocollo della riunione del 1° aprile 1942, Archiwum Państwowe w Warszawie (APW), KHm Ostrów 75, p. 8. Frank. Resoconto del HSSPF Krüger sulla situazione nel GG nel 1943, BAB, NS 19/2664, pp. 6-10. 70 Lettera di Karl Valentin al Ministero della Giustizia del Reich del 7 maggio 1938, BAB (ehem. BDC), SA, Valentin, Karl, 1° agosto 1895. 71 Il 17 settembre 1942, l’amministrazione interna di Ostrów Mazowiecka ordinò l’arresto, eseguito dal capo locale della Gendarmerie Pfüttner, e la deportazione di Moses See mann al campo di Treblinka, avvenuta il 18 settembre, Doc 141 del 17 settembre 1942, in Friedrich, 2014, p. 440, nota 2. 72 Protocollo della riunione del 1° aprile 1942, Archiwum Państwowe w Warszawie (APW), KHm Ostrów 75, p. 8. Frank. Resoconto del HSSPF Krüger sulla situazione nel GG nel 1943, BAB, NS 19/2664, pp. 6-10.tii Il 17 settembre 1942, l amministrazione interna di Ostrów Mazowiecka ordinò l arresto, eseguito dal capo locale della Gendarmerie Pfüttner, e la deportazione di Moses See mann al campo di Treblinka, avvenuta il 18 settembre, Doc 141 del 17 settembre 1942, in Friedrich, 2014, p. 440, nota 2. 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità Una volta iniziato a svolgere il proprio compito, si rese conto che il lascito dei suoi predecessori presentava problemi gravi: «I tempi sono così difficili che dob biamo tenere un comportamento esemplare nella nostra attività e nella nostra vita privata quotidiana, non solo verso noi stessi, ma di fronte al popolo, alla patria, al Führer e, nel nostro specifico caso, di fronte ai polacchi. Finora non è stato così»72. Valentin fece un appello per una maggiore cooperazione tra distret ti locali, invitando i colleghi ad abbandonare la linea di isolamento dal governo centrale e biasimando l’ennesimo caso di arresto di un funzionario, in quel caso dell’Organizzazione Todt, accusato di frode, appropriazione indebita e furto. Mi impegno ulteriormente per i miei compagni tedeschi e, in senso più ampio, an che per i laboriosi polacchi, poiché abbiamo bisogno di fare anche ciò che va oltre i nostri [rispettivi] ambiti. Non mi chiudo nei paragrafi dei libri di diritto. In nessuna maniera. Voglio sapere [come funzionano le cose nella realtà], in che modo io stesso potrei [cadere nella rete]. Al momento me ne occupo. Ho un piede e mezzo in un campo di concentramento. Ma me ne occupo73. Gli ammonimenti e le accuse, oltre che i timori di ritorsioni nei suoi confronti, si rivelarono parole vuote. Il mandato ricevuto da Valentin era in sostanza quello di riorganizzare e ripulire la struttura e l’immagine dell’amministrazione locale ma, per l’esecuzione del compito, l’incaricato spese una tale quantità di denaro per macchine, edifici, la sua nuova casa con relativa mobilia, mobili per gli uffici (progettati e realizzati appositamente), da rendere più gravoso l’onere rispetto al potenziale risultato; dovette approfittare delle confische dei beni e sui prodotti agricoli per trovare i fondi necessari. Infine l’alacre lavoro di “allestimento” di strutture adeguate e rispettabili distolse molte delle attenzioni necessarie al co ordinamento delle attività produttive del distretto. 73 Protocollo della riunione del 28 aprile 1942, in APW, KHm Ostrów 75, p. 23. 437 Tanti piccoli Führer? Inoltre, con parte dei soldi ricavati dalle ulteriori confische, dopo la rivendita dei prodotti agricoli al mercato nero, Valentin fece realizzare delle lussuose scar pe per la moglie. Il denaro raccolto non lo teneva nella sede istituzionale, ma in una cassaforte nel suo appartamento, trattenendone una cospicua parte per gli abbellimenti e le ristrutturazioni dell’abitazione e della sede del capo-distretto, mentre pochi erano destinati agli stipendi dei dipendenti. 74 Il capo della Sicherheitspolizei e del SD presso il Ministero della Giustizia del Reich, ottobre 1944, in IPN, Regierung des GG I/5238, p. 83; Cancelleria del capo del NSDAP, Ufficio per le domande di grazia presso il governo del GG, Dipartimento di Giustizia, in merito alla domanda di grazia di Karl Wilhelm Valentin, 3 giugno 1944, IPN, Regierung des GG I/5238, p. 63. Non ci sono documenti che testimoniano l’esito della procedura per la richiesta di grazia. 76 Bajohr, 2001, p. 76; cfr. Pohl, 1997, p. 302. 77 Gross, 1979, p. 159. 74 Il capo della Sicherheitspolizei e del SD presso il Ministero della Giustizia del Reich, ottobre 1944, in IPN, Regierung des GG I/5238, p. 83; Cancelleria del capo del NSDAP, Ufficio per le domande di grazia presso il governo del GG, Dipartimento di Giustizia, in merito alla domanda di grazia di Karl Wilhelm Valentin, 3 giugno 1944, IPN, Regierung des GG I/5238, p. 63. Non ci sono documenti che testimoniano l’esito della procedura per la richiesta di grazia. 75 Cfr. Bajohr, 2001, p. 76. 76 Bajohr, 2001, p. 76; cfr. Pohl, 1997, p. 302. 77 Gross, 1979, p. 159. Cfr. Bajohr, 2001, p. 76. 76 Bajohr, 2001, p. 76; cfr. Pohl, 1997, p. 302. 75 Cfr. Bajohr, 2001, p. 76. 76 Bajohr 2001 p 76; cfr Pohl 1997 p 302 78 DTB 19 gennaio 1940, BAB, R 52 II/225, p. 31. Riferimenti anche in Majer, 1981, pp. 487-96. 79 Cfr. du Prel, 1940, pp. 88, 99-100, 107, 111, 113, 115, 116, 120, 126, 128, 130, 133, 134, 139, 170-7, 181, 207-13 (i dati sono corrispondenti ai valori demografici e geografici dello stato nei primi 5-7 mesi circa di amministrazione). Con l’annessione della Galizia, i valori sono lievemente cresciuti, mantenendo comunque la distinzione col Reich sul piano delle proporzioni. 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità Arrestato e processato, la corte lo condannò a tre anni di detenzione e una multa di 5000 złoty. Ciono nostante, la sua attività come riorganizzatore dell’amministrazione locale venne addirittura lodata da alcuni esponenti delle SS, in particolare dalle Waffen-SS. Ernst Kaltenbrunner per esempio, dopo aver chiesto una «speciale riabilitazione» per Valentin nelle Waffen-SS (provvedimento che gli permise di uscire di galera nell’ottobre 1944 e usufruire di un permesso di «lavoro al fronte»), imputò la causa della condotta del funzionario all’influenza esercitata da alcuni elementi ambientali, come «in primo luogo […] le condizioni caotiche [della] vita economi ca del Governatorato generale»74. In esecuzione della sentenza della Corte spe ciale, Karl Valentin fu espulso dal partito il 18 marzo 1943.iti La distruzione e la politica predatoria attuata su larga scala dagli occupanti con sentirono l’arricchimento personale di molti gerarchi e funzionari a spese della popolazione, mediante il controllo sui commerci e sul fiorente mercato nero75. La commistione tra gerarchi e attività corruttive necessitava di un pretesto di na tura politica, o per meglio dire di dottrina razziale: una sorta di “comportamento della razza superiore” tedesca sui locali Untermenschen. Un fattore che consentì maggiori margini di guadagno nelle attività illegali fu il numero, relativamente ridotto, di funzionari di comando presenti sul territorio76: «La ricerca del profitto, la cooperazione economica reciprocamente vantaggiosa e gli interessi comuni erano le principali forze integratrici, insieme al terrore [esercitato] d[a]lla polizia, che tennero insieme il Generalgouvernement durante la guerra»77.iti In molti distretti locali si stabilirono i Landkommissariate e Stadtkommissariate, organi amministrativi non autonomi ma dipendenti dagli uffici centrali delle con tee. Nelle sedi locali delle amministrazioni c’era sempre un polacco o un ucrai no in regime di “autonomia amministrativa”: i tedeschi finsero di considerare le minoranze facendole partecipare alla gestione del territorio, sfruttando invece i locali che lavoravano sottopagati e spesso vessati dai “colleghi” occupanti; vi era 74 Il capo della Sicherheitspolizei e del SD presso il Ministero della Giustizia del Reich, ottobre 1944, in IPN, Regierung des GG I/5238, p. 83; Cancelleria del capo del NSDAP, Ufficio per le domande di grazia presso il governo del GG, Dipartimento di Giustizia, in merito alla domanda di grazia di Karl Wilhelm Valentin, 3 giugno 1944, IPN, Regierung des GG I/5238, p. 63. Non ci sono documenti che testimoniano l’esito della procedura per la richiesta di grazia. 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità 438 Emiliano Vitti comunque una certa autonomia per gli slavi, specialmente per gli ucraini. I Con sigli ebraici (Judenräte) erano al contrario solo gli esecutori degli ordini tedeschi, tanto che la popolazione del ghetto vedeva spesso il Presidente del Consiglio ebraico addirittura come un referente diretto dei nazisti; per questo molti presi denti venivano criticati, disprezzati e minacciati dai propri correligionari (l’esem pio più chiaro fu quello di Adam Cerniakow, presidente del Judenrat di Varsavia). L’amministrazione occupante avrebbe dovuto funzionare secondo il principio dell’unità amministrativa; il principio di competenza e le amministrazioni speciali dovevano essere abolite in tutti e tre i livelli dell’amministrazione statale, ossia governo centrale, distrettuale e di contea o distrettuale locale, e combinate sotto la guida unitaria di una sorta di figura manageriale pubblica, il Governatore (più chiaramente espressa col termine di Verwaltungsführer). Frank intendeva raffor zare il ruolo dei capidistretto, che costituivano secondo lui la spina dorsale dello stato e sui quali andava impostata l’efficienza burocratica e amministrativa, al fine di superare il dualismo stato/partito e velocizzare i processi decisionali, con la nomina di personalità forti dotate di grande assertività e capaci di iniziativa personale. Le inadeguatezze strutturali e personali dei dipendenti e dei dirigenti della pubblica amministrazione del GG determinarono il fallimento del tentativo di imporre l’unità tra le varie anime del settore78.itii L’apparato burocratico dei distretti e la sede centrale a Cracovia si gonfiavano sempre più mentre i Kreise minacciavano di collassare a causa della penuria di personale. La nuova suddivisione amministrativa limitò i danni al sistema ma non causò un miglioramento duraturo, poiché mancavano le condizioni strutturali per consolidarsi. Il governo di Cracovia ridusse le 72 contee polacche esistenti allo scoppio della guerra fino ai 40 distretti amministrativi locali più sei cittadini del 1° gennaio 1940. Il risultato fu una gestione della rete amministrativa strutturata su maglie molto più larghe rispetto al Reich. Rispetto ai Landkreisen tedesche, i Kreise del GG erano di regola svariate volte più grandi: la contea più piccola si estendeva su una superficie di 939 kmq, la più grande misurava 4506 kmq e la media era di 2551 kmq; la popolazione andava dai 95.685 ai 550.405 abitanti, con una media di 253.000 abitanti. 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità Nel Reich, la superficie media di una contea era di 600 kmq e la popolazione media si attestava sui 58.000 abitanti circa79.titi La suddivisione del personale delle contee e dei distretti cittadini fu modifica ta parecchie volte nel corso della guerra: come nel caso dell’ordinanza dell’apri le 1941, che determinò l’accorpamento delle contee di Skierniewice e Łowicz. 439 Tanti piccoli Führer? Dopo l’attacco all’Unione Sovietica e l’annessione del Distrikt Galizien una parte del personale del distretto di Cracovia fu trasferita e si applicarono le regole per l’accorpamento di distretti non più necessari e per la creazione di nuovi sogget ti amministrativi, come nel caso delle contee di Krosno e Przemyśl. Nel Distrikt Galizien vi furono dapprima quindici contee, poi ridotte a dodici. Anche lo status amministrativo delle città mutava velocemente: oltre alle quattro città capoluo go di distretto (Distrikthauptstädte – Varsavia, Radom, Cracovia, Lublino), anche Tschenstochau (Czestochowa) e Chełm erano guidate all’inizio dalle Stadthaupt mannschaften (di fatto le autorità amministrative “autonome” di ogni distretto cittadino).ti A metà del luglio 1940, in attuazione dell’accordo di spartizione germano-sovie tico sul territorio polacco, la cittadina di Przemyśl assunse lo status di Kreisstadt; il 2 settembre dello stesso anno Kielce, storica città capoluogo del voivodato della Santacroce, divenne la settima Stadthauptmannschaft, numero salito a otto con l’annessione del Distrikt Galizien. Questi movimenti, poveri di motivazioni prati che ma dettati dalla necessità di mantenere equilibri politici interni alle gerarchie naziste, arrivarono a stabilizzarsi verso la metà del 1943; dopo di che, fino alla fine del GG rimasero 53 Kreishauptmannschaften (gruppi di comando locali) e 6 Stadthauptmannschaften (gruppi di comando cittadini)80. Il potere delle Kreishauptleute era sostanziale e la loro competenza giurisdizio nale era ampia; per cercare di limitare i conflitti tra istituzioni diverse e all’interno della stessa istituzione, dall’estate 1940 la figura del capo partito sul territorio coincise con quella del Standortführer, rendendo quindi il funzionario locale di riferimento meno indipendente e più controllabile direttamente da Berlino. Il Kreishauptmann aveva la responsabilità dell’attuazione delle direttive e disposi zioni adottate dal governo centrale di Cracovia; egli doveva essere «l’unico rap presentante del Governatorato generale» nel distretto locale ed era subordinato al Governatore81. , , / , p 81 Cfr. Secondo ordine sulla struttura dell’amministrazione del GG (Ordinanza sull’unità dell’amministrazione) del 1° dicembre 1940, in Nolzen, 1997, pp. 247-75 (qui pp. 254-5). 82 Gollert, 1942, p. 88. 80 du Prel, 1940, pp. 88, 101, 147-8, 207; Relazione sulla struttura del GG fino al 1° luglio 1940, BAB, R 52 II/247, p. 40. 81 Cfr. Secondo ordine sulla struttura dell’amministrazione del GG (Ordinanza sull’unità dell’amministrazione) del 1° dicembre 1940, in Nolzen, 1997, pp. 247-75 (qui pp. 254-5). 82 Gollert, 1942, p. 88. 80 du Prel, 1940, pp. 88, 101, 147-8, 207; Relazione sulla struttura del GG fino al 1° luglio 1940, BAB, R 52 II/247, p. 40. 4. Una strana forma di “ordinaria amministrazione”: violenze e altre illegalità De facto però, date le circostanze spesso precarie in cui operava l’amministrazione centrale, «il governatore distrettuale locale [si trovò spesso ad] eseguire […] in modo spesso indipendente provvedimenti e disposizioni, in modo che corrispond[essero] agli orientamenti politici del Governatore generale e ga rantis[sero] il raggiungimento degli obiettivi riconoscibili dalla sua azione. Grandi dunque [furono] le aspettative nei confronti del governatore distrettuale locale, così come di ogni suo collaboratore»82. In generale, le sfere di competenza di Kreis- e Stadthauptleute dovevano essere regolate dall’alto, con una moltitudine di normative, vincoli e limitazioni (Verord nungsflut) che lasciavano all’Hauptmann la responsabilità di definire le priorità 80 du Prel, 1940, pp. 88, 101, 147-8, 207; Relazione sulla struttura del GG fino al 1° luglio 1940, BAB, R 52 II/247, p. 40.t 440 Emiliano Vitti per l’esecuzione83. Queste problematiche si presentarono anche nel nuovo Dis trikt Galizien, dove i Kreishauptleute conquistarono un ancor più ampio margine di manovra: «potevamo fare ciò che volevamo»84, dichiarò tempo dopo Wilhelm Rebay von Ehrenwiesen in una intervista85.ti Frank non mancò mai di elogiare le capacità di adattamento dei dirigenti locali, anche nei frequenti casi di abuso di potere e uso eccessivo di metodi repressi vi, poiché valutava le azioni rispetto al raggiungimento dell’obiettivo prestabilito, qualificando la violazione come «typisch östlich illegale Art» (tipica fattispecie illegale orientale)86. 84 Präg, Jacobmeyer, 1975, p. 415 (nota del 16 ottobre 1941). A questo proposito è in dicativa una vicenda che vide protagonista Friedrich Gollert. Il Dr. Gollert, capo del Di partimento di Giustizia dell’Ufficio del governatore di Varsavia, fece arrestare, tra il 10 e il 12 luglio 1940, 80 avvocati polacchi con l’accusa di aver sostenuto che anche gli avvocati ebrei dovevano essere ammessi all’esame di abilitazione alla professione, e li fece rinchiudere nella prigione di Pawiak. I prigionieri furono spediti ad Auschwitz nel settembre 1940. Cfr. Iranek-Osmecki, 2009, pp. 119-20, https://ipn.gov.pl/pl/publikacje/ ksiazki/12641,Kto-ratuje-jedno-zycie-Polacy-i-Zydzi-19391945.html (11-06-2021).t 83 DTB 19 gennaio 1940, in BAB, R 52 II/225, pp. 29-30; Präg, Jacobmeyer, 1975, p. 415 (nota del 16 ottobre 1941).t 85 Giurista e burocrate tedesco, Ehrenwiesen fu nominato Kreishauptmann del distretto locale di Kamionka Strumiɫowa, nel Distrikt Galizien; in Pohl, 1997, p. 419. 86 DTB 16 ottobre 1941, in BAB, R 52 II/239, p. 52. 87 Cfr. Primo decreto sull’istituzione dell’amministrazione dei territori polacchi occupa ti del 26 ottobre 1939, in Documenta Occupationis (Doc. Occ.) VI, p. 56, http://www. iz.poznan.pl/archiwum/documenta-occupationis/, (24-06-2021); Kreishauptmann Heinz Gustav Albrecht, esempi di amministrazione di una Kreishauptmannschaft, IPN, NTN 381, 83 DTB 19 gennaio 1940, in BAB, R 52 II/225, pp. 29-30; Präg, Jacobmeyer, 1975, p. 415 (nota del 16 ottobre 1941). 84 Präg, Jacobmeyer, 1975, p. 415 (nota del 16 ottobre 1941). A questo proposito è in dicativa una vicenda che vide protagonista Friedrich Gollert. Il Dr. Gollert, capo del Di partimento di Giustizia dell’Ufficio del governatore di Varsavia, fece arrestare, tra il 10 e il 12 luglio 1940, 80 avvocati polacchi con l’accusa di aver sostenuto che anche gli avvocati ebrei dovevano essere ammessi all’esame di abilitazione alla professione, e li fece rinchiudere nella prigione di Pawiak. I prigionieri furono spediti ad Auschwitz nel settembre 1940. Cfr. Iranek-Osmecki, 2009, pp. 119-20, https://ipn.gov.pl/pl/publikacje/ ksiazki/12641,Kto-ratuje-jedno-zycie-Polacy-i-Zydzi-19391945.html (11-06-2021). 85 Giurista e burocrate tedesco, Ehrenwiesen fu nominato Kreishauptmann del distretto locale di Kamionka Strumiɫowa, nel Distrikt Galizien; in Pohl, 1997, p. 419. 86 DTB 16 ottobre 1941, in BAB, R 52 II/239, p. 52. 87 Cfr. Primo decreto sull’istituzione dell’amministrazione dei territori polacchi occupa ti del 26 ottobre 1939, in Documenta Occupationis (Doc. Occ.) VI, p. 56, http://www. iz.poznan.pl/archiwum/documenta-occupationis/, (24-06-2021); Kreishauptmann Heinz Gustav Albrecht, esempi di amministrazione di una Kreishauptmannschaft, IPN, NTN 381, 86 DTB 16 ottobre 1941, in BAB, R 52 II/239, p. 52.i iurista e burocrate tedesco, Ehrenwiesen fu nominato Kreishauptmann del distretto ale di Kamionka Strumiɫowa, nel Distrikt Galizien; in Pohl, 1997, p. 419.t 5. Quando il “diritto” è allo sbando Le responsabilità dei capi locali, nell’esercizio delle loro funzioni, non venne re golata da una legge apposita, ma si applicarono in deroga le norme sulla discipli na dei singoli settori dell’attività amministrativa. I settori produttivi erano spesso per intero nella loro sfera di competenza: il rafforzamento del potere tedesco sul territorio, la riscossione di quote di raccolto in agricoltura, il mercato del la voro e il controllo diretto sulla manodopera locale, lo sfruttamento delle risorse minerarie ed energetiche, il controllo sul sistema economico e produttivo non ché la sorveglianza sulla Selbstverwaltung di polacchi, ebrei e ucraini. Inoltre i Kreishauptleute dovevano occuparsi di passaporti, notifiche, controllo dei prezzi, lotta al contrabbando, stato sociale per tedeschi e polacchi, trasporti, organizza zione e supervisione delle strutture sanitarie e del personale medico. Rispetto al settore agricolo, essi avevano la funzione di monitoraggio della fornitura e della distribuzione di sementi e fertilizzanti, gestione del magazzino delle quote agri cole. In più la Kreishauptmannschaft gestiva i controlli nelle scuole elementari e negli istituti tecnici e forniva l’assistenza culturale per tutti i Volksdeutsche87. 441 Tanti piccoli Führer? Inizialmente non c’era uno schema organizzativo unificato; ogni Kreishaupt mann poteva costruire la propria autorità a sua discrezione e in base alle con dizioni ambientali incontrate. Alla fine del marzo 1941 fu introdotta la regola mentazione che consentiva l’aggregazione o l’integrazione di autorità speciali nell’amministrazione fino al 1° maggio. Furono aggregati l’ufficiale sanitario, l’i spettorato della motorizzazione, l’ufficio di stato civile per cittadini del Reich, l’or ganismo di controllo sui prezzi, gli ispettori distrettuali locali nel settori agricolo e dell’istruzione, l’ispettorato finanziario (Finanzinspekteur), l’ispettore territoria le (Landinspektion) e quello addetto alla gestione delle risorse idriche (Wasser wirtschaftsinspektion), l’ufficio del lavoro (Arbeitsamt), l’ufficio di controllo sulla silvicoltura (Forstaufsichtsamt) e l’organismo di controllo sui cantieri in costruzio ne (Strombauinspektion). In più vennero subordinate al Kreishauptmann autorità guidate da “non tedeschi”: l’ufficio topografico della contea (Kreisvermessung samt), l’ambulatorio veterinario (Amtstierarzt), l’ufficio di verifica di pesi e misure (Eichamt), l’autorità dei terreni e delle acque. Nel caso di integrazione tra gli uffici era lo stesso Kreishauptmann il sovrintendente capo della sua contea; qualora si fosse proceduto ad un’azione di accorpamento, la responsabilità era in mano ad un ufficio indipendente al quale il Kreishauptmann aveva facoltà di dare istruzioni vincolanti sull’operato (Weisungsrecht) nella misura necessaria alla salvaguardia di una linea politica uniforme, o per bilanciare i punti di vista contrastanti di di versi rami dell’amministrazione. pp. 111-33. 88 Circolare amministrativa n. 4 del regolamento relativo all’unità dell’amministrazione, con efficacia a partire dal 1° dicembre 1940, 18 aprile 1941, AAN, Regierung des GG, 9c/3, pp. 54-6. 89. Cfr. Präg, Jacobmeyer, 1975, pp. 425-7 (nota del 17 ottobre 1941). 5. Quando il “diritto” è allo sbando Nell’aprile 1941 un’ordinanza amministrativa di sciplinò infine uniformemente il profilo dei servizi dei Kreishauptleute; di conse guenza l’autorità risultò divisa in quattro settori: amministrazione interna (Innere Verwaltung), economia (Wirtschaft), alimentazione e agricoltura (Ernährung und Landwirtschaft), istruzione (Schulamt)88.i t Nel luglio 1943 la normativa subì un ulteriore cambiamento. La polizia locale divenne l’ufficio per gli affari di polizia all’interno di Kreishauptmannschaften e Stadthauptmannschaften; così il capo della polizia locale si trovava sotto il con trollo del Kreishauptmann, come organo esecutivo di polizia. Per lo svolgimento dell’opera di amministrazione le autorità territoriali locali e cittadine si servirono di piccole Gendarmeriezüge (compagnie locali di pubblica sicurezza). In ogni contea una compagnia era di stanza in partenza con 20-25 uomini, per poi arrivare anche a 30-40 elementi. La Gendarmerie svolgeva ini zialmente una semplice attività di polizia, senza alcuna competenza sulle misure disciplinari. Il 17 ottobre 1941 divenne operativa la cosiddetta “autorità incon dizionata” dei capi cittadini e dei capi distrettuali locali sulla Gendarmerie89, che venne inoltre subordinata alle autorità polacche di polizia attive ancora sul ter pp 89. Cfr. Präg, Jacobmeyer, 1975, pp. 425-7 (nota del 17 ottobre 1941). 442 Emiliano Vitti ritorio (anche se la subordinazione era solo di facciata e determinata per ragioni corporative e politiche interne agli organismi di governo occupanti), con compiti di lotta alla criminalità comune e di pattugliamento principalmente dei quartieri cittadini. La polizia polacca venne sfruttata al pari dei lavoratori coatti polacchi dell’industria e alla manodopera contadina. In molti casi i tedeschi utilizzavano i poliziotti autoctoni per alleggerire il carico di lavoro delle SS e della polizia locale per la deportazione e l’uccisione degli ebrei dei ghetti.t Le contee e le amministrazioni cittadine avevano anche il controllo del Sonder dienst, il servizio speciale di protezione dei Volksdeutschen che, creato nell’au tunno 1939 reclutando i membri dalla minoranza tedesca, partecipò nei primi mesi alle uccisioni sommarie compiute dagli occupanti. Il Sonderdienst venne chiamato da Frank il 6 maggio 1940 in aiuto delle Kreishauptleute e renderle in dipendenti dalla Gendarmerie nell’esercizio delle loro funzioni. Grazie al Sonder dienst, la Zivilverwaltung avrebbe dovuto riprendersi il controllo di aree lascia te incustodite per carenza di personale e dissidi con la polizia (che non sempre era disposta ad assecondare gli amministratori locali andando a sopperire alle loro lacune normative e di organico). 90 DTB 24-25 febbraio 1940, BAB, R 52 II/230, p. 14; Präg, Jacobmeyer, 1975, p. 352 (nota del 8 aprile 1941); Broszat, 1961, p. 77. 91 Cfr. Chodakiewicz, 2004, pp. 105-32. Una menzione a parte va fatta per i Polizeibatail lone: composti da vari membri delle SS dalla non sempre spiccata appartenenza ideo logica, furono gli esecutori di non specificate «misure di polizia» (sicherheitspolizeiliche Maßnhmen) contro gli ebrei nei territori occupati. Vedi Browning, 2003, pp. 118-24. 5. Quando il “diritto” è allo sbando Ogni squadra, composta da 20-30 unità di Volksdeutschen in ogni Kreis, rispondeva al Kreishauptmann che ne faceva prin cipalmente uso per: la lotta al contrabbando (Bekämpfung des Schleichhandels), la riscossione delle quote di raccolto dei prodotti agricoli (Erfassung der Kontin gente), imposte (Eintreibung von Steuern), tasse e contravvenzioni (Gebühren und Strafgeldern), nonché il servizio di guardia della sede della Kreishauptmann schaft, del Kreishauptmann e dell’eventuale campo di detenzione locale90.ti Il numero di poliziotti ucraini variava da contea a contea e oscillava dai 100 ai 400 circa. Nelle contee con un distaccamento della Sicherheitspolizei (Sipo) c’erano almeno 20 agenti operativi, sui quali però il Kreishauptmann non aveva alcun controllo. Vi erano poi formazioni come il Werkschutz (sicurezza aziendale), Forstschutz (sicurezza forestale – settore della silvicoltura), Bahnschutz (sicurez za ferroviaria), con ruoli minori e circoscritti. Nel complesso si può stimare una presenza di 200-500 persone appartenenti ai servizi di sicurezza, suddivise nelle varie categorie e formazioni di appartenenza, escludendo la Wehrmacht, i Poli zeibataillone e le SS91. Fino alla fine dell’estate del 1940 le contee e le amministrazioni cittadine (ad esclusione dei centri principali) non erano formalmente in possesso di alcun po tere punitivo di natura giuridica; venivano attuate solo misure arbitrarie stabilite dagli Hauptmänner, come nei già citati casi di minacce, percosse, torture, ese 443 Tanti piccoli Führer? cuzioni capitali, danneggiamenti a proprietà private. Solo il 13 settembre 1940 venne emanato il regolamento sui procedimenti amministrativi che regolava le unità amministrative locali; i Kreishauptmänner poterono così imporre sanzioni pecuniarie fino ai 1000 zloty o in alternativa comminare pene fino ai tre mesi di reclusione92. All’inizio della primavera del 1940 alcuni capi contea del distretto di Lublino allestirono campi di detenzione e di lavoro per i polacchi e gli ebrei che avessero trasgredito le loro disposizioni o che non si fossero conformati adeguatamente ad esse. Tra i soggetti colpiti dal provvedimento, gli agricoltori che ottemperava no poco o per nulla alle richieste sulle quote di consegna; l’autorità civile spesso inseriva tra gli “ospiti” dei campi anche contrabbandieri e scansafatiche (Arbeit sscheue). I detenuti dovevano svolgere lavori pesanti, ritenuti un metodo ade guato di trattamento «per contribuire straordinariamente al naturale equilibrio dei polacchi, e dare la necessaria enfasi per obbedire agli ordini»93. 92 Ordinanza sulla procedura penale amministrativa nel Generalgouvernement del 12-13 settembre 1940, Verordnungsblatt des Generalgouvernements (VBGG) 1940, pp. 300-2, http://dlibra.umcs.lublin.pl/dlibra/publication?id=11980&tab=3 (11-06-2021), archivio online della UMCS Digital Library di Lublino. Vedi anche Musial, 1999, pp. 49-51; Majer D., 1981, passim. 94 DTB 13 aprile 1942, BAB, R 52 II/243, pp. 32-3; Musial, 1999, pp. 53-4. 95 Präg, Jacobmeyer, 1975, pp. 146-9. 93 Gollert, 1942, p. 95. 92 Ordinanza sulla procedura penale amministrativa nel Generalgouvernement del 12-13 settembre 1940, Verordnungsblatt des Generalgouvernements (VBGG) 1940, pp. 300-2 http://dlibra.umcs.lublin.pl/dlibra/publication?id=11980&tab=3 (11-06-2021), archivio online della UMCS Digital Library di Lublino. Vedi anche Musial, 1999, pp. 49-51; Maje D., 1981, passim. 93 Gollert, 1942, p. 95. 94 DTB 13 aprile 1942, BAB, R 52 II/243, pp. 32-3; Musial, 1999, pp. 53-4. 95 Präg, Jacobmeyer, 1975, pp. 146-9. 5. Quando il “diritto” è allo sbando Ogni azione venne svolta senza alcuna copertura normativa, elemento trascurato dal governo di Cracovia; anche i governatori di Varsavia e di Radom tollerarono e introdussero molte procedure non previste dalla normativa, come la creazione dei campi di lavoro non formalmente autorizzati. Il Governatore tentò in più occasioni di dare una base giuridica all’azione amministrativa dello Stato in tutti i suoi settori, per giustificarne e “nobilitarne” gli atti al fine di creare una sorta di “normalizzazio ne” dell’applicazione del sistema amministrativo nazista sul territorio94. Le finalità erano ovviamente di tipo pratico, con due esempi su tutti che ne spiegano la por tata: il primo riguardò l’intenzione di Frank di apparire un Governatore (apparen temente) morbido e dai modi “equilibrati” per gestire la popolazione polacca sul territorio, tramite l’utilizzo di una decretazione (rigorosamente di tipo personale) che disciplinasse un trattamento minimamente rispettoso, al fine di poter «di spo[rre] di [un adeguato] person[ale] per [la] costitu[zione] [e il funzionamento di] un apparato [amministrativo]»; il secondo invece mirò al mantenimento di una “buona reputazione” dei “governanti” tedeschi, così da far «desiderare [al]le altre nazioni di [finire] sotto la protezione tedesca»95.ti Nel distretto di Varsavia il Kreishauptmann Gramß istituì nel giugno 1941 un campo di lavoro a Treblinka nella contea di Sokołów (rinominato in seguito Treblinka I, da non confondere col successivo campo di sterminio, allestito l’anno successivo in un luogo differente). Gramß volle prevenire gli intoppi burocratici 444 Emiliano Vitti per i lavori di costruzione e assegnò il comando del campo a Theo van Eupen, un funzionario del suo ufficio. Poco più tardi fu lo stesso governatore distrettuale Fischer a stabilire l’apertura dei campi di detenzione penale nel distretto di Varsa via; oltre al governatore, SSPF, Kreis- e Stadthauptleute fecero internare opposi tori nei campi istituiti “illegalmente”; la costruzione del campo di lavoro era fina lizzata anche a scoraggiare la stampa clandestina e le campagne di affissione dei dissidenti polacchi. Il campo di Treblinka poteva contenere fino a 3000 prigionieri ma, dal momento che molti degli internati avrebbero potuto dare un contributo al settore tessile, specialmente per la produzione di divise per l’esercito, i Kreis- e Stadthauptleute furono costretti a limitarsi negli arresti, al fine di «poter dare ancora a Varsavia un numero sufficiente di asociali e fannulloni polacchi»96. 5. Quando il “diritto” è allo sbando Oltre al lavoro nelle officine, i prigionieri venivano utilizzati per la costruzione di strade, nelle cave di ghiaia e nella silvicoltura; fino alla chiusura del campo nell’agosto 1944 vi lavorarono 10.000 prigionieri97.i L’esempio del Kreisarbeitslager venne seguito da molti amministratori locali durante il 1941; solo il distretto di Cracovia costituì un’eccezione, nella quale i de tenuti polacchi finivano nelle carceri ordinarie o in altre strutture di detenzione. Nel Distrikt Galizien gli agricoltori che non versavano le quote venivano internati in strutture delle SS, diverse dai campi di lavoro98.iiti Molti gerarchi, impauriti dalle prospettive nel dopoguerra, Governatore com preso (il quale anzi fu precursore in questo cambio di rotta), iniziarono un pate tico e spesso insincero processo di riavvicinamento verso la popolazione polacca per cercare di ripulirsi la coscienza e (soprattutto) salvarsi la vita e per preservare una posizione all’interno dell’amministrazione anche dopo la guerra99. Da questi spesso impalpabili tentativi rimase piuttosto distante la condotta nelle attività economiche, che risultavano in ogni caso l’elemento più importante a livello am ministrativo della Zivilverwaltung. 96 Riferimenti su casi di internamento, come quello di Moses Seemann dalla contea di Ostrów, in Friedrich, 2014, p. 440. 97 Wojtczak, 1975, pp. 117-135, in http://iucat.iu.edu/catalog/2145349 (11-06-2021), ar chivio online dell’Indiana University. 98 DTB 13 luglio 1942, BAB, R 52 II/242, p. 65. 99 Kotula, 1999, p. 89. Riguardo al Governatore ricordo il suo Im Angesicht des Galgens…, passim, che riassume perfettamente la maturazione di un pensiero di (probabile) penti mento e paura per le azioni commesse. 99 Kotula, 1999, p. 89. Riguardo al Governatore ricordo il suo Im Angesicht des Galgens…, passim, che riassume perfettamente la maturazione di un pensiero di (probabile) penti mento e paura per le azioni commesse. 6. Conclusioni A proposito dell’applicazione della Verwaltungskunst (“arte” dell’amministrazio ne, intesa come insieme di grandi abilità di tipo “manageriale”), le ambizioni del Governatore uscirono ridimensionate da un quadro normativo incompleto e da modalità di azione basate più che altro sul potere personale dei singoli capi di stretto locali. Tanti piccoli Führer? 445 Tutti i Kreise e le amministrazioni cittadine del GG costituirono più di semplici cinghie di trasmissione nell’architettura dello stato. Esse infatti rappresentarono, ciascuna in misura diversa, le varie forme di personalizzazione del potere e abu so di metodi coercitivi all’interno della già “peculiare” macchina amministrativa guidata da Frank.titti La portata distruttiva con cui fu condotta l’attività di governo delle singole aree non fu solamente il risultato di carenze strutturali, ma venne costruita e “vissuta” volontariamente dai funzionari locali. Frank cancellò la visione centralista dello stato alla quale parve ispirarsi nei primi mesi di vita del GG, mostrando non poca incoerenza rispetto agli stessi principi guida della dottrina nazionalsocialista, da lui elaborati o comunque sottoscritti, sull’importanza di un indirizzo unico e de lineato, sul quale concentrare tutte le energie dello stato. Rimase la “sacralità” del Füherprinzip, ma la dispersione del potere tra i molti livelli amministrativi, di cui i vari signori locali approfittarono per fornire ulteriori interpretazioni sull’ap plicazione della «volontà del Führer» e per arricchirsi e fare carriera attraverso la malversazione e la carenza di scrupoli, determinò l’esito fallimentare dell’intera esperienza «governatorale”.ii Con l’esperienza del GG, risultò chiaro anche ai giuristi più rigorosi che la codifi cazione e la effettiva istituzionalizzazione del fenomeno nazionalsocialista erano obiettivi impossibili da raggiungere. Il richiamo alla «forza del diritto» di Frank può essere inteso, oltre che un modo con cui il despota tentò di deresponsabi lizzarsi di fronte al mondo rispetto alle atrocità commesse, anche come il for male riconoscimento dell’utopia del «diritto comunitario tedesco» da parte del giurista, del conseguente fallimento dell’esperienza di governo del Governatore, e della incapacità dell’uomo di raggiungere quel livello di autorevolezza e di cre dibilità per arginare e superare le proprie insicurezze personali. Il Governatorato generale amplificò dunque, nella sua peculiarità, tutti i difetti e le storture del sistema di stato e di teoria giuridica del nazionalsocialismo.ti La costruzione della struttura amministrativa fu decretata dall’alto, ma ebbe effettivamente luogo dal basso. Fonti Fonti AAN (Archiwum Państwowe w Warszawie), KHm Ostrów 75 AAN, Regierung des GG, 9c/3 BA (Bundesarchiv) R 43 II/1648 BA R 43 II/958 BAB (Bundesarchiv Berlin), R 43, II/141 BAB, NS 19/2664 BAB (ehem. BDC), SA, Valentin, Karl, 1° agosto 1895 BAB, R 52 II/225 BAB, R 52 II/247 BAB, R 52 II/239 BAB, R 52 II/230 BAB, R 52 II/243 BAB, R 52 II/242 BAL (Bundesarchiv Ludwigsburg), B 162/Dok-Slg. Polen, Ordn. 344 BAL, B 162/202 AR-Z 76/61, parte 2 Doc. Occ. (Documenta Occupationis) VI DTB (Diensttagebuch), IfZM, Fb 105/30 DTB, IfZ, Fb 105/1, Bl. 40 IPN (Instytut Pamięci Narodowej) BU-2586-329 IPN, NTN 381 IPN, Regierung des GG I/5238 RGBl. (Reichsgesetzblatt), 1934, I RGBl., 1943, I RGBl., 1933, I BAL (Bundesarchiv Ludwigsburg), B 162/Dok-Slg. Polen, Ordn. 344 BAL, B 162/202 AR-Z 76/61, parte 2 Doc. Occ. (Documenta Occupationis) VI DTB (Diensttagebuch), IfZM, Fb 105/30 DTB, IfZ, Fb 105/1, Bl. 40 IPN (Instytut Pamięci Narodowej) BU-2586-329 IPN, NTN 381 IPN, Regierung des GG I/5238 RGBl. (Reichsgesetzblatt), 1934, I RGBl., 1943, I RGBl., 1933, I 6. Conclusioni In molti distretti locali, specialmente nelle vaste zone rurali del paese, la popolazione slava si trovò a trattare quasi esclusivamen te con i livelli più bassi dell’amministrazione occupante, poiché proprio ai capi distretto locali, ai capi delle rispettive Gendarmerie, ai distaccamenti della polizia e del SD fu sostanzialmente demandato l’onere di tradurre le direttive generali e i regolamenti in azioni e comportamenti funzionali al raggiungimento degli obiet tivi dello stato. Hitler infatti non era interessato ai risvolti giuridici o istituzionali dei quali parlava Frank, ma vide il GG come la «pattumiera sociale del Reich», da sfruttare per ragioni razziali ed economiche. 446 Emiliano Vitti Bibliografia Baedeker K., 1945: Das Generalgouvernement: Reisehandbuch, Leipzig, Baedekerii Bajohr F., 2001: Parvenus und Profiteure: Korruption in der NS-Zeit, Frankfurt am Main, S. 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https://openalex.org/W2984259672	http://www.scielo.br/pdf/cebape/v17n4/en_1679-3951-cebape-17-04-1002.pdf	English	null	Innovation ecosystems articulation and shared value creation	Cadernos EBAPE.BR	2,019	cc-by	9,475	Abstract The changes in the perception about the role of organizations reinforce the notion that creating value for society is as important as generating profit for shareholders. Against this backdrop, this study assumes that innovation ecosystems, because they are based on the interconnection and interrelation of a network of actors to generate innovation, have an important role in balancing profit generation and the creation of value for society. The research question: how can the articulation of an innovation ecosystem generate shared value for all those involved? led to a participatory action research exploring an initiative gathering a German multinational company located in the southern region of Brazil, a public school, a university, the local government, and the community. The initiative producing this interaction was called project “Arcos.” It aimed to connect the company’s managers and university and school students, who formed an innovation ecosystem to create shared value and address local social problems. The study presents a framework with the main benefits perceived by the network of actors, as well as propositions about innovation ecosystems and shared value creation. Keywords: Ecosistema de innovación. Red de actores. Valor compartido. Resumo Frente a uma mudança de percepção acerca do papel das organizações, tão importante quanto gerar lucro para os acionistas é gerar valor para a sociedade. Para tanto, parte-se do pressuposto de que a formação de um ecossistema de inovação, por intermédio da interconexão e inter-relação de uma rede de atores para gerar inovação, pode contribuir neste processo. Assim, propõe-se a seguinte questão: como a articulação de um ecossistema de inovação pode gerar valor compartilhado a todos os envolvidos? Desse modo, realizou-se uma pesquisa- ação participante no sul do Brasil, com base na interação entre uma empresa multinacional alemã, uma escola pública, uma universidade, o governo municipal e a comunidade local. O projeto que resultou desta interação denominou-se Arcos e teve como finalidade a resolução de um problema social por parte de gestores, universitários e alunos da escola, formando um ecossistema que permitiu a criação de valor compartilhado a todos os envolvidos. Como resultados têm-se um framework com os principais benefícios e proposições acerca da temática. Palavras-chave: Ecossistema de inovação Rede de atores Valor compartilhado Palavras-chave: Ecossistema de inovação. Rede de atores. Valor compartilhado. Bruno Anicet Bittencourt ¹ ² Paola Schmitt Figueiró ³ Bruno Anicet Bittencourt ¹ ² Paola Schmitt Figueiró ³ ¹ Universidade Federal do Rio Grande do Sul (UFRGS) / Programa de Pós-Graduação em Administração, Porto Alegre, RS – Brazil ² Universidade do Vale dos Sinos (UNISINOS) / Escola de Gestão e Negócios, São Leopoldo, RS – Brazil ³ Universidade Feevale / Programa de Pós-Graduação em Administração, Novo Hamburgo, RS – Brazil Resumen Frente a un cambio de percepción acerca del papel de las organizaciones, tan importante como generar ganancias a los accionistas, es generar valor a la sociedad. Para ello, se parte del supuesto de que la formación de un ecosistema de innovación, a partir de la interconexión e interrelación de una red de actores para generar innovación, puede contribuir en este proceso. Así, se propone la siguiente cuestión: ¿cómo la articulación de un ecosistema de innovación puede generar valor compartido a todos los involucrados? Para ello, se realizó una investigación-acción participativa en el sur de Brasil, a partir de la interacción entre una empresa multinacional alemana, una escuela pública, una universidad, el gobierno municipal y la comunidad local. El proyecto que resultó de esta interacción se denominó Arcos y tuvo como finalidad la resolución de un problema social por parte de gestores, universitarios y alumnos de la escuela, formando un ecosistema que permitió la creación de valor compartido a todos los involucrados. Los resultados obtenidos fueron un marco de referencia con los principales beneficios percibidos por la red de actores y cuestionamientos acerca de la temática. Palabras clave: Difusión de innovaciones. Innovación en servicios públicos. Difusión de innovaciones en servicio Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. ISSN 1679-3951 Article submitted on March 30, 2018, and accepted for publication on May 13, 2019. [Translated version] Note: All quotes in English translated by this article’s translator. DOI: http://dx.doi.org/10.1590/1679-395174403x Article submitted on March 30, 2018, and accepted for publication on May 13, 2019. Article submitted on March 30, 2018, and accepted for publication on May 13, 2019. [Translated version] Note: All quotes in English translated by this article’s translator. [Translated version] Note: All quotes in English translated by this article’s translator. DOI: http://dx.doi.org/10.1590/1679-395174403x DOI: http://dx.doi.org/10.1590/1679-395174403x 1002-1015 Bruno Anicet Bittencourt Paola Schmitt Figueiró Innovation ecosystems articulation and shared value creation INTRODUCTION Critical insight into the interconnection of environmental, social, and economic issues emphasizes the need to rethink patterns grounded in a system that is not balanced. Therefore, the central perspective of this study is based on rethinking behaviors within the organizations, considering the importance of the companies in the environment they are operating, as well as their capacity to generate shared value for a network that goes beyond the company’s shareholders. Resumen This research recognizes that there is a shift in the field of innovation, from company-centered to network-centered innovation (NAMBISAN and SAWHNEY, 2011). However, for the company to be prepared for such a paradigm, it is essential that its managers and employees are motivated to face a very challenging process. In this context, innovation ecosystems, i.e., the interconnection and interrelation of a network of actors (GOMES, FACIN, SALERNO et al., 2018) that interact to foster innovation (REINOLDS and UYGUN, 2017), may act as a stimulating environment for shared value creation (PORTER and KRAMER, 2011). The central premise of shared value is that the company’s competitiveness and the health of the community are mutually dependent. The relation emphasized in this study, therefore, involves precisely the potential of an innovation ecosystem to createshared value through innovation. As for the issue of forming an ecosystem, the participation and interaction of organizations such as universities, companies, governments, and society are crucial, stressing that the element ‘society’ is the one that characterizes the quadruple helix approach proposed by Carayannis and Campbell (2009). The research question guiding this study addresses the interaction among these actors, askinghow can the articulation of an innovation ecosystem generate shared value for all those involved?The aim is to analyze the process of shared value creation from the perspective of the actors involved in an innovation ecosystem. The research adopted the participatory action research approach and observed a case in the South of Brazil. The case presents the interaction among a German multinational company, a public school, a university, the local government, and the community. The initiative was called project “Arcos,” and gathered the company’s managers, and university and school students, forming an ecosystem that allowed shared value creation and solving local social problems. This article is structured in six sections, including this introduction. The next section presents the theoretical framework, focusing on innovation ecosystems and shared value creation. The third section presents the methodology, followed by the analysis based on the participatory action research. The fifth section introduces the contributions of the project “Arcos,” and the sixth and last section presents the final considerations and suggestions for future research. Innovation ecosystems and shared value creation This section discusses the theoretical approach to innovation ecosystems and shared value creation. Based on a conceptual perspective, the intention is to show the ability of the connections in a network of actors to generate innovation, considering the actors’ willingness to cooperate and the importance of interactions in the process of shared value creation. One of the central elements to understand and to interpret the empiricalpart of the research involves the steps in the process of developing an innovation ecosystem (analysis, project, deployment, execution, and conclusion). Thus, the study considers these phases to analyze the interrelationship between the network of actors and the developments of the project “Arcos” with emphasis on the shared value creation. Innovation ecosystems The ecosystem approach emphasizes the connections and dynamics of evolution, competition, predation, and mutualism among the actors, which are precepts of natural systems that resemble what happens in business environments (SHAW and ALLEN, 2016). The term “ecosystem” is originally found in biology. It was first associated with business activities by Moore (1993) but wasobserved more often from the decade of 2010 onwards, mainly related to entrepreneurship (ISENBERG, 2010; STAM, 2015) and innovation (AUTIO and THOMAS, 2014). Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1003-1015 Innovation ecosystems articulation and shared value creation Bruno Anicet Bittencourt Paola Schmitt Figueiró Innovation ecosystems can be understood as open and dynamic business environments (RABELO and BERNUS, 2015), permeated by cyclical flows of tangible resources (e.g.,financial resources) and intangible resources (e.g.,information). In such ecosystems, a network of interconnected and interrelated actors (GOMES, FACIN, SALERNO et al., 2018) interact in a complex way, competing but also cooperating to foster innovation (REYNOLDS and UYGUN, 2017). Innovation ecosystems can be understood as open and dynamic business environments (RABELO and BERNUS, 2015), permeated by cyclical flows of tangible resources (e.g.,financial resources) and intangible resources (e.g.,information). In such ecosystems, a network of interconnected and interrelated actors (GOMES, FACIN, SALERNO et al., 2018) interact in a complex way, competing but also cooperating to foster innovation (REYNOLDS and UYGUN, 2017). Innovation ecosystems count on elements such as infrastructure, regulations, financial capital, knowledge, ideas, the interface between actors, architecture principles (RABELO and BERNUS, 2015), companies, consumers, suppliers, and regulatory agents (GOMES, FACIN, SALERNO et al., 2018). Spigel (2017) adds to these elements: entrepreneurs, workers, investors, mentors, favorable governmental policies, universities, and other sources of innovative knowledge, as well as a culture that encourages risk-taking. Moreover, beyond these tangible and intangible elements, it is crucial to consider the processes that occur within innovation ecosystems. Therefore, it is necessary to understand, from an evolutionary and dynamic perspective, how innovation ecosystems develop over time and adapt to the contexts (SPIGEL and HARRISON, 2018). A thriving innovation ecosystem is the result of a process of continuous evolution, that is usually long, complex, and slow (RABELO and BERNUS, 2015). The ecosystem’s development may present different stages of maturity (GOMES, FACIN, SALERNO et al., 2018), and possibly these different stages influence innovation in different ways. Few studies propose to describe how innovation ecosystems emergence and develop. Innovation ecosystems For Rabelo and Bernus (2015), the steps in the process of forming innovation ecosystems are: analysis (the decision to create the ecosystem is made); project (the architecture of the ecosystem is defined); deployment (the phase of taking the necessary actions for its operation, such as recruitment of actors, publicizing the actions, and establishing the infrastructure conditions); execution (ecosystem management activities); and conclusion (the ecosystem undergoes a metamorphosis to survive and continue to develop, or it is discontinued. Ecosystems derive from an exogenous process (RABELO and BERNUS, 2015). Therefore, it is possible to understand that, for the formation and sustenance of an innovation ecosystem, promoting the interaction among different actors, it is necessary for all the stakeholders to perceive value in the cluster. The next topic emphasizes the value creation that connects the stakeholders within the ecosystem. Creating Shared Value The shared value creation is a perspective introduced as a management model by Michael Porter and Mark Kramer (2006, 2011). The authors assume that, for the long-term success of the business, creating value for society is as important as generating profit for shareholders. Value generation is an evolved form of capitalism, understanding that increasing the purchasing power of the population is not enough to generate long-term growth. It is necessary to engage in people development and add value (PORTER and KRAMER, 2011). The authors assume that shared value can be responsible for the next great transformation of administrative thinking, considering that it is a distinct, powerful and transformative model embedded in the central purpose of corporations (PORTER and KRAMER, 2014). In this sense, Hart and Prahalad (2002) affirm that multinational companies should look for globalization strategies through the lens of inclusive capitalism. In other words, it is a matter of prospering without harming the communities. But for this change in business to take place, leadership roles need to develop new knowledge and skills, such as: more significant understanding of social problems, a broader view of the impacts of social problems on the enterprise’s value chain, and capacity to transpose the discussions throughout profit or non-profit spheres (PORTER and KRAMER, 2011). Morais Neto, Neis, and Pereira (2016), studying the field of organizational environment, describe how the progressive growth of a multinational company can occur, based on the notion of creating shared value. According to the authors, this is a management model that connects concepts of productivity and competition to the commitment toward environmental and social demands. This perspective was validated by analyzing the multinational studied, taking into account the following elements – aligned to the perspective of Porter and Kramer (2006, 2011): (i) organization’s core business and the positioning of each business unit; (ii) points of intersection between business units and social issues; (iii) social issues chosen for strategic intervention; and (iv) the existence of social dimensions for the value proposition. The company researched presented concrete Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1004-1015 Innovation ecosystems articulation and shared value creation Bruno Anicet Bittencourt Paola Schmitt Figueiró Innovation ecosystems articulation and actions related to all elements, a scenario that helps to strengthen the regional cluster where the firm is inserted, in Brazil (MORAIS NETO, NEIS and PEREIRA, 2016). Innovation ecosystem and shared value: conceptual model Innovation ecosystem and shared value: conceptual model As mentioned before, this study adopts the perspective of Morais Neto, Neis and Pereira (2016), who indicate the inclusion of shared value in organizations through a management model that connects concepts of productivity and competition to the commitment to environmental and social demands. This perspective implies that people development and adding value can stimulate the adoption of shared value in the organization’s purpose. The connection of shared value creation and the innovation ecosystem occurs as the interaction and cooperation in a network formed by different actors – in this case, coming from the company, university, society, and government foster the creation of value to all stakeholders. Following the perspective of Porter and Kramer (2011), this connection involves individuals who, through complementary understandings, can at the same time generate competitiveness and opportunities related to social and environmental values for the company. Figure 1 shows the conceptual model that demonstrates such relation. Creating Shared Value It should be noted that partnerships between companies and other institutions with projects that may aggregate and facilitate the execution of shared value strategies are fundamental for the success of the actions. Often, approximations are necessary in order to complement the skills and knowledge of the actors involved. On the one hand, there is a scenario with managers without a deeper understanding of social and environmental issues. On the other, practitioners working in the social sector, in general, have limited knowledge on administrative matters, little entrepreneurial attitude and vision of the opportunities to generate shared value (PORTER and KRAMER, 2011). Considering the growing academic interest in creating shared value, Dembek, Singh, and Bhakoo (2016) performed a systematic analysis on the issue, offering a better ontological and epistemological comprehension. The authors realized that the approach of creating shared value was still considered vague, and there was a discrepancy between the approach’s definition and operationalization. This evidence reinforced the need for empirical studies on creating shared value (DEMBEK, SINGH and BHAKOO, 2016). Given the above, the next section presents the conceptual model used as the basis for the analysis in the empiricalpart of the research. METHODOLOGY The method adopted was participatory action research, an approach that includes the implementation of a transformative action (KEMMIS and MCTAGGART, 2007). In this method, there is no separation between the subject and the object since the subjects researched are also subjects of the research and collaboratively participate in its construction. In addition, the approach unites theory and practice. It takes theory to the field, acting together with the subjects studied (BRANDÃO, 1984; THIOLLENT, 2003; KEMMIS and MCTAGGART, 2007). There is an educational and social transformation nature since all those involved in the research learn together. In addition, it is emancipatory, since the participants become aware of the situation, and can act more critically about their place in the environment (KEMMIS and MCTAGGART, 2007). This type of research has a strong social commitment on the part of the researcher, who feels connected to the subjects studied and acts according to the values shared with them. Therefore, the researcher has the concern of making the study accessible to the public researched. Also, the researcher seeks to put their scientific curiosity in the service of a social benefit for the community (BRANDÃO, 1984). The study followed the steps proposed by Kemmis and McTaggart (2007): (1) planning – collecting the necessary data and planning together the action to be taken; (2) acting and observing – the moment of action, which must be observed to generate data that will feed the reflections; and (3) reflecting – together with the subjects researched, this step involves reflection on the action taken, to understand what has emerged from the previous step and, if necessary, to provide inputs for planning a new action. It is important to briefly present the project “Arcos,” object of this study, before introducing the procedures of data collection and analysis. Project “Arcos” was designed and implemented by the authors of this research, in partnership with a German multinational company with operations in Brazil in the steel industry. The project counted on the initial participation of seven managers working in the company, 25 students from a public school near the company’s facilities, and fifteen undergraduate students of the course of administration offered by a local private university. In a second moment, the local government and the community (parents of students and residents) joined the process. Figure 1 Conceptual model of an innovation ecosystem based on shared value Source: Elaborated by the authors. Conceptual model of an innovation ecosystem based on shared value Conceptual model of an innovation ecosystem based on shared value Source: Elaborated by the authors. 1005-1015 Bruno Anicet Bittencourt Paola Schmitt Figueiró Innovation ecosystems articulation and shared value creation The assumption is that the development and sustenance of an innovation ecosystem take place through the creation of shared value, that is, it is necessary that the different actors of the network perceive value in their participation in order to guarantee the ecosystem’s growth and sustainability. In this case, this study adopts the quadruple helix approach, with the presence of a company, university, society, and government (CARAYANNIS and CAMPBELL, 2009). This construction takes place through the steps brought by Rabelo and Bernus (2015): analysis, project, deployment, execution, and conclusion. In this perspective, the company assumes the role of pushing the movement forward, evidencing its capacity to generate not only financial but also social results (PORTER and KRAMER, 2011). In the sequence, the other actors are connected, cooperating, and generating innovation (GOMES, FACIN, SALERNO et al., 2018). In short, the shift from a competition between individual companies to a competition between networks in the business arena has been discussed in the organizational literature for more than two decades (HATANI and MCGAUGHEY, 2013). However, in the last years, social welfare (PORTER and KRAMER, 2011) and the relationship between different actors such as university, society, and government (CARAYANNIS and CAMPBELL, 2009) have been identified as crucial factors for the generation of innovation and companies’ survival. The next section presents the methodology adopted in this study to empirically understand these relationships. METHODOLOGY The purpose of the project was to form an ecosystem of local innovation with the engagement of different actors in the quadruple helix approach, as proposed by Carayannis and Campbell (2009): company, university, school, society, and government. Project “Arcos” was designed and adapted into five steps, based on the work by Rabelo and Bernus (2015): (1) analysis; (2) project; (3) deployment; (4) execution and (5) conclusion. The first step consisted of identification (by the authors of this research in partnership with the German multinational company) of the need to create a local innovation ecosystem. After assessing this need, these same actors designed the project, focused on mobilizing players to solve a local problem. In the Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1006-1015 Innovation ecosystems articulation and shared value creation Bruno Anicet Bittencourt Paola Schmitt Figueiró third step, the managers who held leadership positions in the company and the public school and university students were invited to join the group. The actors gathered in meetings and organized the project’s schedule. The fourth step consisted of weekly meetings held to identify a local problem to be addressed and to develop and execute a possible solution. In the fifth and last step, the results were analyzed, generating reflections to guide the continuity of the project. third step, the managers who held leadership positions in the company and the public school and university students were invited to join the group. The actors gathered in meetings and organized the project’s schedule. The fourth step consisted of weekly meetings held to identify a local problem to be addressed and to develop and execute a possible solution. In the fifth and last step, the results were analyzed, generating reflections to guide the continuity of the project. The weekly meetings held during the execution were conducted with the company’s managers and the school students to identify the local problem to be addressed in the context of project “Arcos.” University students engaged in the project in the same step, in the moment of proposing possible solutions. Finally, the local government and community also joined the project during execution. The meetings took place in different locations (school, company, university, and in the local park). The meetings occurred over six months. Data collection and research follow-up were conducted using the approach of participant observation since the researchers facilitated all the project meetings. METHODOLOGY The researchers used a field diary to examine the planning process and the materials elaborated during the meetings. Finally, nine semi-structured interviews were carried out with the following actors: the director of human resources of the company; one of the managers that participated in the project; a professor of the undergraduate program in administration, responsible for the course the university students were attending when engaged in the project; a university student; the public school’s principal and one school student; two residents in the local community; and one civil servant working for the local government. The questions of the interview script were elaborated based on the perception of the actors involved regarding the benefits obtained and the role played, considering that each actor collaborates and benefits from the process of creating shared value (PORTER and KRAMMER, 2011). The data were analyzed and grouped following the steps proposed by Kemmis and McTaggart (2007) previously mentioned: (1) planning; (2) acting and observing; and (3) reflecting. The process of analysis considered the proposed objective, observing how the process of forming an innovation ecosystem can generate shared value. The analysis was guided by the conceptual model shown in Figure 1. ANALYSIS BASED ON PARTICIPATORY ACTION RESEARCH In this section, the results are presented and analyzed based on the steps mentioned in the methodology. Acting and observing The first meetings were structured with a focus on self-knowledge, team building, and the identification of a collective goal. In the first three meetings, company managers and school and university students did not know each other. The first activity with managers focused on listening, identifying the purpose of the individuals, and stimulating exchange among colleagues. The group then had the opportunity to learn about new business models and global changes for an era of collaboration and social responsibility. After these first activities, the weekly meetings (at school or in the company) gathering managers and the school students started. The meetings were an opportunity for the actors to experience each other’s routine. During these sessions, the actors were able to get to know each other, interact, and explore their relations with the neighborhood they were all connected to. Subsequent meetings were marked by brainstorming around possible problems in the region, and by identifying ways to understand these problems more deeply. The group decided to conduct exploratory research with the residents in the community, in order to assess the most prominent local problems. Both managers and school students were involved in the elaboration and application of an instrument to explore the perceptions of the residents regarding the local problems. The group interviewed 103 people, who answered ten questions about their relationship with the community and their opinion about the context they were living in, exposing positive or negative aspects and things they wish to see in the region. The results of these interviews were presented to the university students who were engaged in contributingproposing solutions to the problems identified and to assist in the construction of a collective project. The meetings that followed brought together the three publics: corporate managers, school students, and university students. The three groups came up with the same problem: the revitalization of a park near the company and the school. The argument was that, when solving this problem, other issues mentioned in the interviews would be tackled indirectly, such as public safety, income generation, leisure spaces, and community self-esteem. After much interaction and debate, it was concluded that the solution to this problem would be the reform and occupation of the park, organizing community events. The idea was to prepare an event with cultural activities, sports, food, and activities related to entrepreneurship. The design of this solution was prepared collaboratively and organically. Planning As observed before, the project “Arcos” was organized based on the steps outlined by Rabelo and Bernus (2015), with some adaptation. The steps used were: (1) analysis; (2) project; (3) deployment;(4) execution, and (5) conclusion. In the step‘analysis,’ the researchers, together with the German multinational company, identified the need to form a local innovation ecosystem. The company triggered the process, presenting a demand for more effective social responsibility and involvement with its surroundings. In addition, the company was in a period of restructuring and sought to develop its leaders and generate innovation. On the part of the researchers, the conception of the project emerged as an opportunity to broaden, in an empirical way, the understanding around forming an innovation ecosystem and this ecosystem’s contribution to creating value for all stakeholders. In the step‘project,’ after the analysis of the needs, a methodological proposal was designed involving the study of learning practices that promote stakeholder engagement and generation of innovation. The approaches used were problem-based learning, design thinking, and theory U. The project was then structured in three main phases: problem identification, the proposal of solutions, and execution of the proposal. In the third step (deployment), the participants were invited. The profile of the company’s employees to be invited was established together with the human resources department. The managers of the company’s areas were chosen because of their capacity to impact more people within the organization. Seven managers were appointed as potential participants. Then, the university was approached through contact with a professor of the undergraduate program in administration of the largest higher education institution in the region. The intention was to verify the possibility of including a group of university students in the project. The professor was receptive, and 15 students of the course on social responsibility Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1007-1015 Innovation ecosystems articulation and shared value creation wereincluded in the project. For the professor, the project was an opportunity to teach elements of the course in a practical and contextualized way. The next actors approached were the students of the local public school, located next to the company’s facilities. In the design of the project, the school would be a representative of the link with the community, since the residents are somehow involved in the school’s activities. Planning Another element that led to the school’s engagement was the fact that several of the company’s employees’ children studied there. Also, mixing the vision of adults, children, and young people would give potential to the project. Thus, the school’s administration appointed 25 youngsters to participate in the initiative, aging from 10 to 15 years old. The project schedule was structured with the participation of the company’s human resources department, the school’s administration, and the professor of the university – the actors engaged in weekly meetings lasting three hours each. In the first meetings, only the company’s managers and the school students participated, due to time availability. During the activity of proposing solutions, the university students participated, contributing to a collective construction. After designing the proposal, the local government was contacted, in order to assist in the execution of the solution proposed. F ll i th t f R b l d B (2015) li d ith th t ib ti b K i d M T t (2007) th ti Following the steps of Rabelo and Bernus (2015), aligned with the contributions byKemmis and McTaggart (2007), theexecution and conclusion steps are described below. Reflecting The research examined the benefits and the role played by the participants of the projectto reflect on the process of forming a local ecosystem and creating shared value. This part of the study relied on interviews conducted with nine representatives of the different groups involved, as detailed in the methodology. The interview focused on the role of each actor, their involvement with the project, and the perceived individual and collective benefits. “An analysis of each of the actors allows inferring that the company was the actor pushing project “Arcos.” The firm was committed by encouraging the involvement of its managers, providing financial resources to acquire materials and supply other possible demands, and taking the risk of developing an innovative project. It represents, therefore, the cyclical flows of tangible (e.g., financial resources), and intangible resources (e.g., information), which, according to Gomes, Facin, Salerno, et al. (2018), are present in the interconnection of a network of actors. According to the human resources manager, the company was looking for a more assertive and meaningful way to develop leadership and found in this proposal a practical way to leverage leadership skills and, at the same time, to promote community benefits. It was a unique and impressive experience,” said one of the participating managers. The interviewee also mentioned that the project took him totally out of his comfort zone, generating learning beyond his professional life. “It is much more difficult to lead a group of children and young people than a group of workers,” said the manager, considering the difficulties of adapting to the scenario posed by the project. The group reported that during the process, employees from different sectors became closer, establishing stronger relations with the community. The atmosphere around the project also generated positive publicity for the company, evidence that the relationship was mutually beneficial. As for the public school, the principal appointed 25 students aging from 10 to 15 years old to participate in the initiative, with the condition that the students would not be requested to miss their regular classes. The school also offered space to host meetings. The school’s principal mentioned that the experience promoted several benefits. In her interview, the principal pointed out that the students were able to access different experiences that were not offered in the regular school activities. Acting and observing The participants mapped possible partner initiatives that would contribute to the solution proposed. The students at the school presented the proposal to their colleagues and involved parents and teachers in the activities. The same happened with the university students, who brought companies and volunteers to contribute to organizing the event. In the company, the mobilization was intense. The managers presented the project to the employees and other stakeholders, who became actively involved in the initiative. This mobilization reached the city administration and local civil servants, such as those responsible for managing the park. The mayor and several civil servants attended meetings, expressing their support, and validated the group’s proposal. Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1008-1015 Bruno Anicet Bittencourt Paola Schmitt Figueiró Innovation ecosystems articulation and shared value creation Other meetings held afterward focused on planning actions, practices, and control. The students met weekly with the managers and exchanged information online with the university students. With the support of a network of partners, it was possible to carry out a revitalization of the park and publicize the action in several channels, which helped to increase mobilization. The event involved approximately 500 people and featured musical and artistic attractions, sports tournaments, play areas for children, food court, handicraft fairs, and exhibitions of local businesses. All stakeholder had clear responsibilities and, in order to avoid excessive group segmentation, they worked in teams and collective alignment in order to value this collective learning process. Other meetings held afterward focused on planning actions, practices, and control. The students met weekly with the managers and exchanged information online with the university students. With the support of a network of partners, it was possible to carry out a revitalization of the park and publicize the action in several channels, which helped to increase mobilization. The event involved approximately 500 people and featured musical and artistic attractions, sports tournaments, play areas for children, food court, handicraft fairs, and exhibitions of local businesses. All stakeholder had clear responsibilities and, in order to avoid excessive group segmentation, they worked in teams and collective alignment in order to value this collective learning process. Given the success of the event planned by the group, the next challenge wasto continue the project. Thus, there were two more meetings to evaluate the positive and negative points, collect feedback of the event, and celebrate the achievements of the group. Reflecting She added that the relationship with the other actors participating in the project made it possible for young people to gain visibility. In the words of a school student interviewed: “It was cool, we managed to change the park!” The feeling of pride was also present in the speech of a parent (and resident) interviewee “[...] there are plenty of problems. Thus, if these kids get to know how to address them from an early age, we will have a better world.” This theoretical-practical connection is one of the motivations for the university professor that facilitated the participation of the undergraduate students in the initiative. According to her, the contextualization of teaching allows students to easily assimilate learning. For the professor, the initiative was extremely beneficial: “My students were offered to live the content we studied in class, being exposed to the community’s real problems. Certainly, they will be more prepared for the labor market after this experience.” “We managed to assist more in the communication part of the project, as well as to identify activities to be conducted during the event [...] I had never imagined working with children and experienced managers together, it was a unique opportunity!”said one of the university students, who also highlighted teamwork and collaboration as main points for the success of the project. The local community participated in different actions, from the identification of the problem to the activities performed in the event at the park. “We cannot wait for someone to solve the problems, we have to do it ourselves,” said a resident interviewed. The resident also highlighted the fact that the project brought the feeling that it is possible for the residents to act to improve Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1009-1015 Innovation ecosystems articulation and shared value creation Bruno Anicet Bittencourt Paola Schmitt Figueiró Bruno Anicet Bittencourt Paola Schmitt Figueiró Bruno Anicet Bittencourt Paola Schmitt Figueiró their neighborhood. The discourse of the residents showed the community revitalization as agreat benefit to all. “The park had not had so much life for some time,” said one resident who took advantage of the event to exhibit her handicrafts. Finally, when the city council learned about the initiative, it also volunteered to participate. Reflecting The local government action involved the validation of the proposals, the availability of the staff responsible for the park’s management and public environment, and they participated by publicizing the activity. As a return, the municipality observed that the community is more mature and engaged with local problem-solving. The local public administration managed to establish a closer relationship with the people and organizations involved, also enjoying the benefits of the visibility brought by project “Arcos.” “It is exciting to see so many people gathered to improve a common space,” said a civil servant interviewed. Finally, when the city council learned about the initiative, it also volunteered to participate. The local government action involved the validation of the proposals, the availability of the staff responsible for the park’s management and public environment, and they participated by publicizing the activity. As a return, the municipality observed that the community is more mature and engaged with local problem-solving. The local public administration managed to establish a closer relationship with the people and organizations involved, also enjoying the benefits of the visibility brought by project “Arcos.” “It is exciting to see so many people gathered to improve a common space,” said a civil servant interviewed. In short, it is possible to affirm that during the process of interconnection and interrelationship of this network, the actors identified their role and contributed according to their competence. It is also worth mentioning that the different actors of the ecosystem perceived benefits with this articulation and collective construction. Thus, it can be inferred that the initiative allowed shared value creation. The nexttopic discusses the contributions of project “Arcos” and its developments. From the ecosystem to value generation: contributions of project “arcos” From the ecosystem to value generation: contributions of project “arcos” Following the conceptual model proposed for this research, it was possible to observe that the project, as an initiative forming an innovation ecosystem (RABELO and BERNUS, 2015), resulted in both collective and individual benefits for all stakeholder. It was clear the cooperation among the groups to foster innovation (REYNOLDS and UYGUN, 2017), creating shared value for the community, in line with Porter and Krammer’s (2011) perspective. Among the innovations generated, it is worth highlighting the opportunity offered for school and university students to learn curricular contents at the same time as gaining social-emotional competencies. Along the same lines, the process of leadership development within the company stood out, since the project allowed managers to use different tools and develop various behavioral skills when acting in practical tasks. In addition, the company performed a new model of social responsibility, focused on local development rather than welfare. For the government, a new form of partnership was established, with a relationship centered on the society as a whole, extrapolating the regular public-private dynamic. As for the residents and small businesses, the initiative created a market opportunity, since the businesses could offer their products in an environment not yet explored. The project evidenced a value common to all stakeholders: the community engagement and strengthening, which is a structuring action for future innovations. Thus, considering that the innovation ecosystem is the result of a dynamic and continuous evolution process (RABELO and BERNUS, 2015), Figure 2 shows the main benefits perceived by the actors participating in the ecosystem analyzed. Figure 2 Ecosystem of project “Arcos” Source: Elaborated by the authors. Figure 2 Ecosystem of project “Arcos” Source: Elaborated by the authors. Based on the relations among the conceptual elements present in Figure 1, forming an innovation ecosystem from the project “Arcos,” and on the benefits generated for the actors involved in the ecosystem (Figure 2), the research drafts three propositions, detailed in this section. First, it was possible to identify the interconnection and interrelation of the network of actors (GOMES, FACIN, SALERNO et al., 2018). They interact in a complex way to foster innovation (REYNOLDS and UYGUN, 2017), indicating the formation of an ecosystem. In addition, it was verified the existence of social dimensions favoring value proposition (MORAIS NETO, NEIS and PEREIRA, 2016), as well as activities of people development aiming at adding value (PORTER and KRAMER, 2011), which are premises for creating shared value. Contributions of actors for the consolidation of the innovation ecosystem Source: Elaborated by the authors. Source: Elaborated by the authors. It is possible to say that the participation of actors in these four dimensions has enhanced the value created by the innovation ecosystem. Each actor played a fundamental role in the development of the ecosystem, and their participation increased the benefits generated. Thus, the second proposition is that the innovation generated by the ecosystem is proportional to the heterogeneity of its actors (Proposition 2). It is possible to say that the participation of actors in these four dimensions has enhanced the value created by the innovation ecosystem. Each actor played a fundamental role in the development of the ecosystem, and their participation increased the benefits generated. Thus, the second proposition is that the innovation generated by the ecosystem is proportional to the heterogeneity of its actors (Proposition 2). Finally, the development of the project “Arcos” and the benefits perceived by its actors (Figure 2) led to understand these benefits at different levels, classified as micro, meso, and macro. The micro level is related to personal benefits; the meso level reflects organizational benefits; and the macro refers to local/community benefits. Box 1 shows the benefits and levels observed. Box 1 Level of benefits Level Micro Level Meso Level Macro Company Leadership development Marketing and social responsibility Community approximation University Contextualized learning/ teaching Research/extension opportunity Approximation between academy and labor market School / Society Contextualized learning/ teaching; Business opportunity and income generation Engagement of the school community Leisure areas Government Visibility Public-private partnership and community approximation Revitalization of public spaces Source: Elaborated by the authors. Box 1 Level of benefits Source: Elaborated by the authors. There is an explicit relation between Figure 3 and Box 1. For the company, the micro and macro levels are provided through human resources, while the meso level is linked to the financial resource. For the university, all levels are directly related to the technical knowledge and ability to articulate connections in the environment. Regarding the school and society, engagement becomes one of the main drivers of the benefits generated at all three levels. Finally, the government played an important role in legitimizing the process. Starting from the macro level, the government demonstrated interest and granted permission for the revitalization of the public space, which generated a meso-level partnership and consequent visibility at the micro level. There is an explicit relation between Figure 3 and Box 1. From the ecosystem to value generation: contributions of project “arcos” Thus, it can be inferred that the experience studied indicates that an ecosystem of articulated innovation creates value for the different actors involved. The innovation ecosystem presented contemplates the quadruple helix approach (CARAYANNIS and CAMPBELL, 2009), and it is not focused on business, but on creating social value. The benefits generated with the emerged innovation contributed to improving the environment where the actors live, particularly by promoting community engagement and revitalization of the public spaces of interaction. Therefore, the first proposition is that the efforts to create shared value consolidate the formation of an innovation ecosystem (Proposition 1). The participation of different actors was essential for the development of the ecosystem and to generate innovation. Each of them contributed with skills at a particular step, as evidenced in Figure 1. The company was the actor that started the process, responsible for investing human and financial resources to carry out the analysis and the project itself. The university assisted in the initiative’s deployment, offering technical knowledge and articulating to connect the actors forming the ecosystem. The society, represented by the school and the local community, played a fundamental role, being responsible for engaging people in the execution of the project. Finally, the government’s involvement legitimized and validated the process, concluding the project. Such contributions are shown in Figure 3, which represents a spiral viewed from above. In addition, it is possible to observe that such contributions have emerged with the evidence listed in Figure 2. Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1011-1015 Bruno Anicet Bittencourt Paola Schmitt Figueiró Figure 3 Contributions of actors for the consolidation of the innovation ecosystem FINAL CONSIDERATIONS Project “Arcos” was an organic process, centered on the value generation through the interconnection and interrelationship of a network of actors. The main objective of the research was to analyze how the interaction in an innovation ecosystem formed by university, company, government, and society can create shared value. It is important to mention that there is no empirical research adopting the same format as presented here, connecting different organizations as observed in project “Arcos,” as well as using the research method and the same combination of theoretical approaches. The process of forming the innovation ecosystem was based on the steps developed by Rabelo and Bernus (2015): analysis, project, deployment, execution, and conclusion. The conceptual model adopted the notion that shared value creation is connected to the innovation ecosystem as an alternative for the formation and sustenance of such agglomerations. The analysis showed the importance of defining clearly the roles of each participant since the beginning of the initiative. In the experience studied, this measure helped to focus on the proposed objectives. Regarding the interaction between the actors, greater stimulus and support was needed for planning and publicizing information among the stakeholders, making better use of the available communication channels. Finally, it was observed that internal communication could have been better explored. Communication deficiencies have occurred, for example, between managers and university students, and with the parents of the students in the school. It was clear that the parents needed to understand what the project was providing for their children, because some expressed concern, fearing that the initiative was not constructive in the context of the children’s education. Given this, three propositions emerged as theoretical contributions. The first proposition concerns the fact that the search for creating shared value consolidates the formation of an innovation ecosystem. The essence of creating shared value, which includes the participation of society in the network of organizational relationships and people development (PORTER and KRAMMER, 2006, 2011), was crucial in forming the innovation ecosystem. The engagement of the company’s leadership, in this case, was particularly important to help these actors to understand the impacts of social problems in the company’s value chain (PORTER and KRAMER, 2011; MORAIS NETO, NEIS and PEREIRA, 2016). As the different actors approached to bring benefits to the community, an open and dynamic business environment was formed (RABELO and BERNUS, 2015). Contributions of actors for the consolidation of the innovation ecosystem For the company, the micro and macro levels are provided through human resources, while the meso level is linked to the financial resource. For the university, all levels are directly related to the technical knowledge and ability to articulate connections in the environment. Regarding the school and society, engagement becomes one of the main drivers of the benefits generated at all three levels. Finally, the government played an important role in legitimizing the process. Starting from the macro level, the government demonstrated interest and granted permission for the revitalization of the public space, which generated a meso-level partnership and consequent visibility at the micro level. Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1012-1015 Bruno Anicet Bittencourt Paola Schmitt Figueiró Innovation ecosystems articulation and shared value creation Thus, the generation of benefits at different levels may be considered as a stimulating factor for the engagement of actors and value creation in innovation ecosystems. Thus, the third proposition is that creating shared value in an ecosystem is related to the benefits generated at micro, meso, and macro levels for each actor (Proposition 3). The propositions built on the reflections over the empirical case studied in this research intend to inspire theoretical and practical discussions and further contributions. The next and final section presents the research’s conclusions lessons learned. Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. FINAL CONSIDERATIONS The environment fostered innovation, mainly through the sharing of knowledge and ideas. Therefore, it was necessary to understand the value generated for the different audiences in order to guarantee the formation and sustainability of an innovation network. Thus, the second proposition indicates that the innovation generated by the ecosystem is proportional to the heterogeneity of its actors. The theoretical contribution, therefore, revolves around the importance of interconnection and interrelation (GOMES, FACIN, SALERNO et al., 2018), as well as the cooperation among those involved in fostering innovation (REYNOLDS and UYGUN, 2017). This reinforces that, corroborating the literature, the connection between different knowledge and competencies of an ecosystem contributes to an effective opportunity to generate innovation. The third proposition relates to the creation of shared value in an ecosystem to the benefits generated at micro, meso, and macro levels for each of the actors. This proposition shows the need to think ofinstitutions in a broader way, including the impact they have on people and the environment. This proposition contributes notably to the theoretical perspective that points to the understanding of how innovation ecosystems develop and adapt to new contexts(SPIGEL and HARRISON, 2018), considering that new projects and new contexts demand different connections and, consequently, bring different results for each level of engagement. It is necessary, therefore, to identify the value perceived by and generated for each actor, considering the use of approximations to complement the actors’ skills and knowledge (PORTER and KRAMER, 2011). Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1013-1015 Innovation ecosystems articulation and shared value creation Bruno Anicet Bittencourt Paola Schmitt Figueiró In general, the project “Arcos” came about with a proposition of forming an innovation ecosystem in which there is the creation of shared value, i.e., generation of benefits to all stakeholders. Such an innovative proposition is inspiring in the current time when there is an economical, political, and social crisis at the national level in Brazil. The notion of a company that assumes co-responsibility in transforming the region in which it operates (principle of creating shared value) through participatory processes, is an alternative to promote the emergence and sustainability of innovation ecosystems, a theme still little approached in the literature (RABELO and BERNUS, 2015). This work contributes to the literature on ecosystems and shared value creation, seeking to understand the construction of a network of innovation and the values it generates. Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. FINAL CONSIDERATIONS In this way, the research brings an approximation of the existing theoretical approaches to the practice, as well as using a method in which the researcher is directly inserted in the field research. It was possible to rapidly observe the social change and the adoption of new values in the region, as a result of the interactions and exchanges that led to a collaborative proposal to address local problems. In addition to the concrete results, the experience showed an increasing awareness about the individuals’ responsibility for the environment and their role as change makers, capable of inspiring and involving more people and organizations. This research offers a relevant contribution to decision making in the public and private spheres. It offersa reflection on the importance of the articulation between different actors, and the project “Arcos” is an example of how to stimulate other actions that provide shared value creation. The participatory action research provides a direction for the planning and the accomplishment of projects aligned to the perspective presented in this article. The lack of time to execute the research may be pointed out as the most critical limitation of the study. It was not possible to explore the project’s medium and long-term results. A suggestion for future research would be to expand the research to other contexts, comparing different scenarios and groups of actors involved. Another suggestion is to study the process of articulation and the role of an actor coordinating the emergence and formation of innovation ecosystems. Finally, future research could explore elements such as the concept of the social innovation ecosystem, the links between shared value creation and public policies, and the relationship between forming innovation ecosystems and dependence on multinational companies. 1014-1015 Bruno Anicet Bittencourt Paola Schmitt Figueiró Bruno Anicet Bittencourt Paola Schmitt Figueiró Innovation ecosystems articulation and shared value creation Bruno Anicet Bittencourt Paola Schmitt Figueiró REFERENCES AUTIO, E.; THOMAS, L. 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International Journal of Technology Management, v. 46, n. 3/4, p. 201, 2009. Journal of Technology Management, v. 46, n. 3/4, p. 201, 2009. PORTER, M. E.; KRAMER, M. R. Criação de Valor Compartilhado. 2011. Available at: <http://www.hbrbr.com.br/materia/criacao-de- valor-compartilhado>. Accessed on: Jan. 28, 2016. DEMBEK, K.; SINGH, P.; BHAKOO, V. Literature review of shared value: a theoretical conceptor a management buzzword? Journal of Business Ethics, v. 137, p. 231-267, 2016. RABELO, R. J.; BERNUS, P. A holistic model of building innovation ecosystems. IFAC-PapersOnLine, v. 48, n. 3, p. 2250-2257, 2015. GOMES, L. A. de V. et al. Unpacking the innovation ecosystem construct: evolution, gaps and trends. Technological Forecasting and GOMES, L. A. de V. et al. Unpacking the innovation ecosystem construct: evolution, gaps and trends. Technological Forecasting and Social Change, v. 136, p. 30-48, 2018. Available at: <http://dx.doi. org/10.1016/j.techfore.2016.11.009>. Accessed on: June 08, 2019. REYNOLDS, E. B.; UYGUN, Y. Strengthening advanced manufacturing innovation ecosystems: The case of Massachusetts. Technological Social Change, v. 136, p. 30-48, 2018. Available at: <http://dx.doi. org/10.1016/j.techfore.2016.11.009>. Accessed on: June 08, 2019. Forecasting and Social Change, v. 136, p. 178-191, Nov. 2018. Available at: <http://dx.doi.org/10.1016/j.techfore.2017.06.003>. Accessed on: June 08, 2019. HART, S.; PRAHALAD, C. K. The fortune at the bottom of the pyramid. Strategy Business, v. 26, p. 54-67, 2002. HATANI, F.; MCGAUGHEY, S. L. Network Cohesion in Global Expansion: An Evolutionary View. Journal of World Business, v. 48, n. 4, p. 455- 465, 2013. SHAW, D. R.; ALLEN, T. Studying innovation ecosystems using ecology theory. Technological Forecasting and Social Change, v. 136, p. 1-362, Nov. 2018. ISENBERG, D. J. How to start an entrepreneurial revolution. Harvard business review, v. 88, n. 6, p. 40-50, 2010. SPIGEL, B. The relational organization of entrepreneurial ecosystems. Entrepreneurship Theory and Practice, v. 41, n. 1, p. 49-72, 2017. KEMMIS, S.; MCTAGGART, R. Participatory Action Research. In: DENZIN, N. K.; LINCOLN, Y. Handbook of qualitative research. 3. ed. London: Sage, 2007. SPIGEL, B.; HARRISON, R. Toward a process theory of entrepreneurial ecosystems. Strategic Entrepreneurship Journal, v. 12, n. 1, p. 151- 168, 2018. MOORE, J. F. FINAL CONSIDERATIONS Predators and prey: a new ecology of competition. Harvard Business Review, v. 71, n. 3, p. 75-83, 1993. STAM, E. Entrepreneurial ecosystems and regional policy: a sympathetic critique. European Planning Studies, v. 23, n. 9, p. 1759-1769, 2015. MORAIS NETO, S.; NEIS, D.; PEREIRA, M. F. O processo de criação de valor compartilhado. Revista de Administração FACES Journal, Belo Horizonte, v. 14, n. 4, p. 148-166, 2015. THIOLLENT, M. Metodologia da Pesquisa-Ação. São Paulo: Cortez, 2003. THIOLLENT, M. Metodologia da Pesquisa-Ação. São Paulo: Cortez, 2003. Bruno Anicet Bittencourt ORCID: https://orcid.org/0000-0002-6499-3588 PhD Student and Master in administration from the Federal University of Rio Grande do Sul (UFRGS); Professor at the University of Vale dos Sinos (UNISINOS), São Leopoldo – RS, Brazil. E-mail:banicet@unisinos.br Paola Schmitt Figueiró ORCID: https://orcid.org/0000-0002-5160-9831 PhD in administration from the Federal University of Rio Grande do Sul (UFRGS); Professor at the Feevale University, Novo Hamburgo – RS, Brazil. E-mail: paolafigueiro@feevale.br Bruno Anicet Bittencourt ORCID: https://orcid.org/0000-0002-6499-3588 Innovation ecosystems articulation and shared value creation ORCID: https://orcid.org/0000-0002-6499-3588 PhD Student and Master in administration from the Federal University of Rio Grande do Sul (UFRGS); Professor at the University of Vale dos Sinos (UNISINOS), São Leopoldo – RS, Brazil. E-mail:banicet@unisinos.br Paola Schmitt Figueiró ORCID: https://orcid.org/0000-0002-5160-9831 PhD in administration from the Federal University of Rio Grande do Sul (UFRGS); Professor at the Feevale University, Novo Hamburgo – RS, Brazil. E-mail: paolafigueiro@feevale.br Cad. EBAPE.BR, v. 17, nº 4, Rio de Janeiro, Oct./Dec. 2019. 1015-1015
https://openalex.org/W2133309118	https://dash.harvard.edu/bitstream/1/33078210/1/unofficial_economy_brookings_2008.pdf	English	null	The Unofficial Economy and Economic Development	null	2,008	cc-by	29,614	Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33078210 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Published Version https://www.brookings.edu/bpea-articles/the-unofficial-economy-and-economic-development/ Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility RAFAEL LA PORTA Dartmouth College ANDREI SHLEIFER Harvard University The Unofﬁcial Economy and Economic Development ABSTRACT In developing countries, informal ﬁrms account for up to about half of all economic activity. Using data from World Bank ﬁrm-level surveys, we ﬁnd that informal ﬁrms are small and extremely unproductive compared with even the small formal ﬁrms in the sample, and especially relative to the larger formal firms. Formal firms are run by much better educated managers than informal ones and use more capital, have different customers, market their products, and use more external ﬁnance. Few formal ﬁrms have ever oper- ated informally. This evidence supports the dual economy (“Wal-Mart”) theory of development, in which growth comes about from the creation of highly pro- ductive formal ﬁrms. Informal ﬁrms keep millions of people alive but disappear as the economy develops. I I n many developing countries, unofficial economic activity—that con- ducted by unregistered firms or by registered firms but hidden from taxation—accounts for between a third and a half of the total. This share declines sharply as the economy develops. Despite the sheer magnitude of unofﬁcial activity, little is understood about its role in economic develop- ment, and in particular about how important “officializing” this hidden activity and the resources devoted to it might be for economic growth. In this paper we attempt to shed some light on these issues by presenting some new facts about the unofﬁcial (also called “informal”) economy and interpreting them in light of various theories. We begin by reviewing the basic stylized facts: that the unofficial economy is huge, that it shrinks sharply in relative terms as the economy develops, and that various policy variables that determine the costs and beneﬁts of becoming and staying ofﬁcial inﬂuence its size. This evidence is consistent with the generally 275 276 Brookings Papers on Economic Activity, Fall 2008 accepted view that unofﬁcial ﬁrms avoid paying taxes and adhering to reg- ulations, but lose access to public goods and other beneﬁts of ofﬁcial status, such as external ﬁnance. Much of the existing literature on the unofﬁcial economy emphasizes these public policy aspects of the problem.1 Yet crucial as this perspective might be, it says little about the role of unofficial firms in development. There are three broad views of this role, which we refer to as the romantic view, the parasite view, and the dual economy (“dual” for short) view. 1. This literature includes de Soto (1989), Loayza (1996), Johnson, Kaufmann, and Shleifer (1997), Friedman and others (2001), Djankov and others (2002), Almeida and Carneiro (2006), Dabla-Norris, Gradstein, and Inchauste (2008), and Russo (2008), as well as the recent work on Brazil by De Paula and Scheinkman (2008), Monteiro and Assunção (2006), and Fajnzylber, Maloney, and Montes Rojas (2006). 2. De Soto (1989, 2000). 3. United Nations (2008, p. 1). j y 2. De Soto (1989, 2000). 3. United Nations (2008, p. 1). The Unofﬁcial Economy and Economic Development According to the romantic view, which we associate with the work of Hernando de Soto,2 unofﬁcial ﬁrms are either actually or potentially extremely productive but are held back by govern- ment taxes and regulations, as well as by lack of secure property rights and access to ﬁnance. Pending the necessary legal reforms, “four billion people around the world are robbed of the chance to better their lives and climb out of poverty, because they are excluded from the rule of law.”3 If the barriers to ofﬁcial status were lowered and capital supplied through microﬁnance, unofﬁcial ﬁrms would register, borrow, and take advantage of other beneﬁts of ofﬁcial status, and by doing so expand and spark economic growth. The key aspect of this optimistic view is that unofﬁcial ﬁrms are fundamentally similar to ofﬁcial ones but are kept down by policy. In particular, unofﬁcial ﬁrms should look similar to ofﬁcial ﬁrms with respect to characteristics not affected by government policies, such as the characteristics of their entre- preneurs (for example, their education). The other two views are more skeptical about unofﬁcial ﬁrms and in par- ticular see them as quite unproductive, not just because they are deprived of the beneﬁts of ofﬁcial status, but also because they are run by entrepreneurs with lower human capital. In these alternative views, development comes about not so much from the unleashing of informal ﬁrms as from their dis- placement by efﬁcient formal ﬁrms, usually run by totally different people. This is the “Wal-Mart” theory of development. The latter two views differ in what they see as the benefits and the harms of the unofﬁcial sector. The parasite view, associated primarily with the excellent empirical studies by the McKinsey Global Institute, sees unofficial firms primarily from the perspective of their illegality. These 277 RAFAEL LA PORTA and ANDREI SHLEIFER ﬁrms need to stay small to avoid detection and therefore lack the necessary scale to produce efﬁciently. However, the “substantial cost advantage that informal companies gain by avoiding taxes and regulations more than off- sets their low productivity and small scale.”4 This cost advantage allows unofﬁcial ﬁrms to undercut the prices of ofﬁcial ﬁrms. Informal ﬁrms, then, hurt growth both because their small scale makes them unproductive and because they take away market share from bigger, more productive formal competitors. 10. Lucas (1978). 11. See also Amaral and Quintin (2006) and de Paula and Scheinkman (2008). 5. Baily, Farrell, and Remes (2005, p. 18). 4. Farrell (2004, p. 28). 4. Farrell (2004, p. 28). 5. Baily, Farrell, and Remes (2005, p. 18). 6. Farrell (2004, p. 34). 7. Harris and Todaro (1970). 8. For example, Rosenstein-Rodan (1943); Rostow (1960); Murphy, Shleifer, and Vishny (1989). 9. Rauch (1991). 10. Lucas (1978). 11. See also Amaral and Quintin (2006) and de Paula and Scheinkman (2008). 9. Rauch (1991). 7. Harris and Todaro (1970). 8. For example, Rosenstein-Rodan (1943); Rostow (1960); Murphy, Shleifer, and ishny (1989). 6. Farrell (2004, p. 34). The Unofﬁcial Economy and Economic Development According to one McKinsey report, “The high proportion of small ﬁrms in service industries makes them particularly likely to operate informally, ignoring tax requirements, employee beneﬁts, and other regu- lations. This is a much larger barrier to growth than most policymakers in emerging—and developed—economies acknowledge. Steps to reduce informality in local service sectors will be rewarded by rapid increases in their productivity, growth, and employment.”5 The ﬁrst step in redressing the problems created by informal ﬁrms is to “add resources and beef up a government’s audit capabilities.”6 More broadly, government policy should aim to eradicate informal ﬁrms by reducing tax evasion and increasing the enforcement of government regulations. The dual view, associated in our minds with traditional development economics,7 likewise emphasizes the inherent inefficiency of unofficial ﬁrms. This view is intimately related to the “big push” models of develop- ment economics, which see the coordinated transition from the informal, preindustrial economy to the formal, industrial one as the crucial strategy of economic development.8 The earliest formal model of the unofficial economy is that of James Rauch,9 who uses the framework of Robert Lucas to consider the allocation of talent between the unofﬁcial and the ofﬁcial sectors.10 In Rauch’s framework, workers with lower human capital work in informal and smaller ﬁrms and receive lower wages, whereas those with higher human capital are allocated to the larger and more productive ﬁrms and receive higher wages.11 Unlike the romantic view, the dual view predicts that unofﬁcial ﬁrms should look very different from ofﬁcial ﬁrms in their characteristics not 9. Rauch (1991). 278 Brookings Papers on Economic Activity, Fall 2008 affected by government policies. Productive entrepreneurs are willing to pay taxes and bear the cost of government regulation in order to advertise their products, raise outside capital, and access public goods. Such entre- preneurs ﬁnd it more proﬁtable to run the bigger, ofﬁcial ﬁrms than the smaller, unofﬁcial ones. In contrast, the increase in ﬁrm value that less able entrepreneurs or managers could generate by operating formally is not large enough to offset the additional costs from taxes and regulations. The strong prediction of the dual view is that managers and assets are matched through a sorting process that results in low-ability managers being paired with low-quality assets. 13. The sharp distinction we have drawn between the parasite and the dual views is too extreme. For example, informal ﬁrms may compete with formal ones in some industries and not in others, and they might pose a greater competitive threat at higher levels of economic development, when they perhaps become more similar to formal ﬁrms. We will return to the discussion of the relevance of the two views after presenting some of the data. 12. Tokman (1992). The Unofﬁcial Economy and Economic Development Unlike the parasite view, the dual view does not see the unofﬁcial ﬁrms as threatening the official ones, because they are hugely inefficient and hence unlikely to be able to charge lower prices for the same products. Indeed, official and unofficial firms operate largely in different markets and have different customers. The dual view sees the unofficial firms as providers of a livelihood to millions, perhaps billions, of extremely poor people,12 and it cautions against any policies that would raise the costs of these ﬁrms. This view sees the hope of economic development in policies, such as human capital, tax, and regulatory policies, that promote the creation of ofﬁcial ﬁrms, letting the unofﬁcial ones die as the economy develops. The ofﬁcial ﬁrms thus created will be new ﬁrms run by new people, not previously unofﬁcial ﬁrms.13 To shed light on these alternative views, this paper follows the presen- tation of basic correlations with a comparative analysis of the characteris- tics and productivity of ofﬁcial and unofﬁcial ﬁrms in several developing countries. We use three sets of surveys of both ofﬁcial and unofﬁcial ﬁrms conducted recently by the World Bank. The ﬁrst set, known as Enterprise Surveys, covers small, medium-size, and large registered ﬁrms in nearly 100 countries. We use these surveys largely for comparison. The second set, known as Informal Surveys, covers primarily unregistered, but also some registered, small ﬁrms in about a dozen countries. The third set, known as Micro Surveys, covers primarily registered, but also some unregistered, small ﬁrms in about a dozen countries (mostly different from those covered by the Informal Surveys). These surveys enable us to make comparative RAFAEL LA PORTA and ANDREI SHLEIFER 279 statements about the size, inputs, management characteristics, and—in a rough way—productivity of both ofﬁcial and unofﬁcial ﬁrms. We note from the start that the data we use have many problems, not least because we focus on firms that are by definition avoiding the gov- ernment’s notice. Nonetheless, our ﬁndings tend to favor the dual view over the romantic and the parasite views. The unofﬁcial ﬁrms in the sur- veys tend to be small and unproductive compared even with the small but registered ﬁrms (which themselves are much less productive than larger registered firms). The unofficial firms also use lower-quality inputs and have less access to public goods and ﬁnance. 14. Rauch (1991). The Unofﬁcial Economy and Economic Development Extremely few of the regis- tered ﬁrms have ever operated as unregistered, again suggesting, as argued by Rauch,14 that the two groups are very separate animals. The evidence points to a substantial difference between the registered and the unregis- tered ﬁrms in the human capital of their managers and suggests that this gap in human capital drives many other differences, including the quality of inputs and access to ﬁnance. The unregistered ﬁrms pay sharply lower wages to their employees, again consistent with the dual model. As a final step, we consider how firms perceive their obstacles to doing business as reported in the three surveys. Informal firms see lack of access to markets and finance as their biggest problems. Formal firms also emphasize those, but taxes, tax administration, and problems with electricity supply as well. The legal system, regulations, and registration procedures rank lower as obstacles to doing business among both formal and informal firms. Finally, the surveys offer little evidence that the unregistered ﬁrms pose much of a competitive threat to the registered ones: the latter do not treat such competition (or unfair competition more gen- erally) as a serious problem. This last result does not support the parasite view of the unofficial economy, which focuses on price undercutting by informal firms. Over all, the evidence paints a relatively consistent picture. There is very little support for the romantic view, and indeed the differences in pro- ductivity between formal and informal ﬁrms are so large that it is hard to believe that simply registering unregistered ﬁrms would eliminate the gap. On the other hand, there is little support for the parasite view either, and the evidence suggests that subjecting unofﬁcial ﬁrms to stronger enforcement would devastate the livelihood of millions of people surviving near sub- sistence. The evidence rather points to the dual view, with the fairly stan- dard implication that the hope of economic development lies in the creation 280 Brookings Papers on Economic Activity, Fall 2008 of large registered ﬁrms, run by educated managers and utilizing modern practices, including modern technology, marketing, and ﬁnance. 15. World Economic Forum (2007). The Size of the Informal Economy and Its Determinants Measuring the informal economy is inherently difﬁcult. To start with, the informal economy encompasses very different phenomena. One is hidden ﬁrms. Such ﬁrms hide all of their output from the police, the tax authorities, or the regulators. Another phenomenon is hidden output. Output may be hidden even by registered ﬁrms to reduce their tax liability. Both phenom- ena occur in all developing countries. Indeed, the face of informality may change as the economy develops, from near-universal informality at earlier stages to mere tax avoidance as the economy grows richer. Beyond these conceptual issues, there are serious practical problems in measuring hidden ﬁrms and output. Nevertheless, a variety of methods have been proposed. Since each method has its strengths and weaknesses, we gathered data on seven measures of the informal economy based on alternative methodologies and sources. All these measures of the informal economy are, if anything, likely to understate its true size. Surveys are the most direct, although necessarily subjective, measure. We assembled data on two survey measures. The first is an indicator of unofficial or unregistered business activity from the World Economic Forum’s Global Competitiveness Report 2006–2007.15 Top business leaders from 125 countries were asked to estimate the size of the infor- mal sector using a 1-to-7 scale, where 1 indicates that more than 50 per- cent of economic activity is unrecorded and 7 that all of it is registered. For comparability with the other measures, we rescaled this index on a scale from 0 to 50 percent of GDP. The 50 percent cutoff adopted by the Global Competitiveness Report is arbitrary and introduces a downward bias in this measure. The second survey measure is the percentage of total sales that a typical establishment reports for tax purposes, from the World Bank Enterprise Surveys. The respondents are the top managers of registered businesses in (mostly) developing countries. Accordingly, this measure of tax evasion likely understates the size of the informal economy, as entrepreneurs in the informal sector are not surveyed. This measure of tax evasion is available for 95 countries. Most countries have been surveyed twice, and we average the available observations between 2002 and 2006. 16. International Labour Ofﬁce (2007). 17. Loayza and Rigolini (2006). 18. Mondragón-Vélez and Peña-Parga (2008). 19. There are two known biases in the self-employment data. First, OECD statistics relate to civilian employment and, as such, leave out the armed forces. Second, self- employment statistics in most Latin American countries relate to urban areas only. Both biases tend to understate the true size of self-employment. 20. Johnson and others (1997); Ernste and Schneider (1998). 20. Johnson and others (1997); Ernste and Schneider (1998). 18. Mondragón-Vélez and Peña-Parga (2008). 16. International Labour Ofﬁce (2007). 17. Loayza and Rigolini (2006). 19. There are two known biases in the self-employment data. First, OECD statistics relate to civilian employment and, as such, leave out the armed forces. Second, self- employment statistics in most Latin American countries relate to urban areas only. Both biases tend to understate the true size of self-employment. The Size of the Informal Economy and Its Determinants RAFAEL LA PORTA and ANDREI SHLEIFER 281 An alternative method infers the size of the informal economy from observable variables, such as the incidence of micro- and small enterprises, the male participation rate in the labor force, the fraction of workers con- tributing to social security, electricity consumption, and currency in circu- lation. We gathered data on three such indicators. The ﬁrst is the percentage of the active labor force that is self-employed, where self-employment is defined by the International Labour Office to include “jobs where the remuneration is directly dependent upon the proﬁts derived from the goods and services produced,”16 but not work by unpaid family workers, although the incidence of informality among the latter is probably high. This is admittedly a crude measure. In most developing countries there is a strong association between self-employment and infor- mal activity, as most self-employed tend to be low-skilled, unregistered workers.17 Of course, self-employment in developing countries may be high not only because informality is prevalent, but also because self-employment is common in agriculture. For this reason our second objective indicator is the percentage of workers in the nonagricultural sector who are self- employed. Other interpretations of self-employment are also possible. In particular, self-employment has been used as an indicator of entrepreneurial activity in the United States. However, the vast majority of self-employed workers in our data are, in fact, “own-account” workers who do not hire per- sons to work for them. Camilo Mondragón-Vélez and Ximena Peña-Parga show along these lines that the self-employed are rarely business owners in Colombia.18 Data on self-employment are collected through population censuses as well as through household or labor force surveys.19 Data on total and nonagricultural self-employment are available for 133 countries and 96 countries, respectively, from the International Labour Organization. The third objective indicator is based on electricity consumption. For each country the ratio of electricity consumption to GDP for a base period is calculated and then extrapolated to the present, assuming that the elastic- ity of electricity consumption to GDP is one.20 The size of the informal sector is then computed as the difference between GDP as estimated from 282 Brookings Papers on Economic Activity, Fall 2008 this ratio and ofﬁcial GDP. 21. Friedman and others (2001). 22. Schneider (2007). 23. Schneider (2007). 24. World Bank (2007); Djankov and others (2002). 24. World Bank (2007); Djankov and others (2002). 23. Schneider (2007). The Size of the Informal Economy and Its Determinants First, we use two measures of the cost of RAFAEL LA PORTA and ANDREI SHLEIFER 283 paying taxes, from a 2008 paper by Djankov and coauthors:25 total taxes (except for labor taxes) payable by businesses after accounting for deduc- tions and exemptions; and the time it takes to prepare, ﬁle, and pay (or withhold) corporate income tax, value-added tax, and social security con- tributions, in hours per year. Second, we capture the cost of complying with labor laws with three variables: an index of the difﬁculty of hiring a new worker; an index of the difﬁculty and expense of ﬁring a redundant worker; and the nonwage labor costs (payroll taxes and social security pay- ments) associated with hiring a new worker as a percentage of the worker’s salary. Data on complying with labor laws are from Juan Botero and coau- thors and Doing Business 2008.26 Third, we capture the cost of red tape using the percentage of senior management’s time spent in dealing with requirements imposed by government regulations (such as taxes, cus- toms, labor regulations, licensing, and registration); this includes time spent interacting with ofﬁcials, completing forms, and other tasks. This variable is from the World Bank’s Enterprise Surveys. The beneﬁts of being formal include expanded access to both public goods and ﬁnance. Regarding public goods, registered business may ﬁnd it easier than unregistered ones to use the courts to enforce property rights and adjudicate disputes. We use two proxies for the efﬁciency of courts: the log of the number of steps required to collect on a bounced check, from the 2003 paper by Djankov and coauthors and Doing Business 2008;27 and the efficiency of the bankruptcy procedure, from a recent paper by Djankov and coauthors.28 We measure the quality of property rights using indices of corruption and the rule of law from Daniel Kaufmann, Aart Kraay, and Massimo Mastruzzi.29 In addition, we use the density of the paved road network from World Development Indicators as a rough proxy for the scope of the domestic market. Finally, we measure the beneﬁts of access to finance using three indicators of the size of financial markets. The first two indicators are standard: private credit and the market capitalization of domestic ﬁrms, both as a ratio to GDP. These two variables are also from the World Development Indicators. 25. Djankov and others (2008b). 26. Botero and others (2004). 27. Djankov and others (2003). 28. Djankov and others (2008a). 29. Kaufmann, Kraay, and Mastruzzi (2005). The Size of the Informal Economy and Its Determinants This measure of the informal economy under- states its size to the extent that informal activities are less electricity inten- sive than formal activities, and to the extent that technological progress allows for increased output per unit of electricity. This indicator is avail- able for 57 countries from Eric Friedman and coauthors.21 Still another approach to measuring the informal economy models hid- den output as a latent variable, using several indicator and causal variables. This is the approach followed by Friedrich Schneider to estimate a multi- ple indicators, multiple causes (MIMIC) model.22 The indicator variables include the labor force participation rate among persons aged 18–64, annual GDP growth, and the change in local currency in circulation per capita. The causal variables are the tax-to-GDP ratio, the Heritage Foundation index of economic freedom, the unemployment rate, GDP per capita, and lagged values of the latent variable. This measure of the informal economy, which is available for 145 countries,23 is only as good as the model that supports it. Later in this section we present evidence that the correlation between the size of the informal economy and variables such as tax rates is not particularly robust. As a ﬁnal robustness check, we gathered data on a direct measure of the formal economy: the number of registered businesses per 1,000 inhabitants. This measure, too, has problems. The number of firms per capita may increase with development, for example, as product variety expands. It may also be affected by cross-country differences in entrepreneurship. Finally, the data on total registered ﬁrms may be biased upward, especially in developing countries, because of underreporting of ﬁrms that have closed or exited. Data on the number of registered businesses are available for 83 countries from the World Bank’s World Development Indicators dataset. We group the determinants of the size of the unofficial economy into three broad categories: the cost of becoming formal, the cost of staying formal, and the beneﬁts of being formal. As a proxy for the cost of becom- ing formal, we use the logarithm of the number of procedures required to legally start a business, from the 2002 paper by Simeon Djankov and coauthors and the World Bank’s Doing Business 2008.24 The costs of stay- ing formal include paying taxes and obeying government regulations; we use six proxies for these costs. 25. Djankov and others (2008b). 26. Botero and others (2004). 29. Kaufmann, Kraay, and Mastruzzi (2005). The Size of the Informal Economy and Its Determinants The third measure of the size of ﬁnan- cial markets is a subjective indicator of the ease of access to credit, from the World Economic Forum’s Global Competitiveness Report 2006–2007. The index ranges from 1 (impossible) to 7 (easy). 284 Brookings Papers on Economic Activity, Fall 2008 Table 1 presents our measures of the size of the informal economy. Countries are grouped into quartiles based on average income per capita at purchasing power parity (PPP) over the period 1996–2006. In practice, measures of the informal sector based on multiple indicators, energy con- sumption, self-employment, and the World Economic Forum survey are highly correlated with each other (see the correlation table in the appendix). In contrast, tax evasion and the number of registered businesses are less correlated with these other four indicators. Two facts stand out. First, the informal economy in the average country in the sample is large, ranging from 22.5 percent of the total economy according to the tax evasion measure to 34.5 percent according to the mul- tiple indicators approach. These numbers are especially large in light of the fact that our measures are likely biased down. About 26.5 percent of a country’s workers, on average, are self-employed. That figure rises to 30.8 percent in the nonagricultural sector. Respondents to the World Economic Forum survey estimate that 27.6 percent of output is informal. Estimates based on electricity consumption suggest that 29.0 percent of output is informal. The various estimates thus suggest that, in an average country, roughly 30 percent of the economy is informal. Second, the size of the informal economy is strongly negatively corre- lated with income per capita. Figure 1 illustrates this relationship, using the multiple indicators variable to measure the informal economy. The other measures also show the informal economy to be very large in poor countries, ranging from 29.0 percent according to the tax evasion measure to 57.3 percent according to the nonagricultural self-employment measure. The measure from the World Economic Forum survey suggests that the informal economy is 18 percentage points larger in poor countries than in rich ones. Estimates based on electricity consumption and multiple indica- tors suggest that the informal economy is between 21 and 24 percentage points larger, respectively, in poor countries than in rich ones. The Size of the Informal Economy and Its Determinants Size of the Informal Economy and GDP per Capita AGO ALB ARE ARG ARM AUS AUT AZE BDI BEL BEN BFA BGD BGR BIH BLR BOL BRA BTN BWA CAF CAN CHE CHL CHN CIV CMR COG COL CRI CZE DEU DNK DOM DZA ECU EGY ESP EST ETH FIN FJI FRA FSM GBR GEO GHA GIN GRC GTM HKG HND HRV HTI HUN IDN IND IRL IRN ISRITA JAM JOR JPN KAZ KEN KGZ KHM KIR KOR KWT LAO LBN LKA LSO LTU LVA MAR MDA MDG MDV MEX MHL MKD MLI MNG MOZ MRT MWI MYS NAM NER NGA NIC NLD NOR NPL NZL OMN PAK PAN PER PHL PLW PNG POL PRI PRT PRY ROM RUS RWA SAU SEN SGP SLB SLE SLV SVK SVN SWE SYR TCD TGO THA TON TUN TUR TWN TZA UGA UKR URY USA UZB VEN VNM VUT WSM YEM YUG ZAF ZARZMB ZWE 70 60 50 40 30 20 10 7 8 9 10 11 Log of GDP per capita at purchasing power parityb Informal share of GDP, multiple indicators measure (percent)a Sources: Schneider (2007); World Bank, World Development Indicators. a. Average of the observations available for 1999–2004. b. Average for 1996–2006. Figure 1. Size of the Informal Economy and GDP per Capita The Size of the Informal Economy and Its Determinants Even tax evasion by registered businesses—which is likely to understate tax evasion in poor countries—is 21 percentage points higher in poor countries than in rich ones. The self-employment statistics show that the fraction of self- employed workers rises from 13.3 percent in rich countries to 46.4 percent in poor ones. (Figure 2 illustrates the striking relationship between self- employment and income per capita.) The pattern for nonagricultural self-employment is even more extreme: self-employment as a share of nonagricultural employment rises by 44.8 percentage points as one moves from rich countries to poor ones. Consistent with this pattern, the number of registered businesses rises from 3.2 to 41.8 per thousand inhabitants as Second, the size of the informal economy is strongly negatively corre- lated with income per capita. Figure 1 illustrates this relationship, using the multiple indicators variable to measure the informal economy. The other measures also show the informal economy to be very large in poor countries, ranging from 29.0 percent according to the tax evasion measure to 57.3 percent according to the nonagricultural self-employment measure. Table 1. Size of the Informal Economy by Alternative Measuresa Percent except where stated otherwise Measure of informality Informal share No. of GDP per of GDP as Self-employed registered capita at estimated by Self-employed as share of ﬁrms per PPP business leaders Tax as share of nonagricultural Electricity Multiple 1,000 Income quartilea (dollars) (WEF survey) evasionb labor force labor force consumption indicators population Bottom 429 35.4 29.0 46.4 57.3 38.9 42.3 3.2 Second 1,362 33.7 23.3 35.7 37.1 42.7 39.8 8.2 Third 4,002 27.6 19.7 23.1 24.6 31.3 34.1 28.7 Top 20,348 17.3 8.2 13.3 12.5 17.6 18.3 41.8 Sample mean 10,015 27.6 22.5 26.5 30.8 29.0 34.5 24.7 Difference −19,919* −18.1* −20.8* −33.1* −44.8* −21.4* −23.9* 38.7* between top and bottom quartiles No. of observations 185 125 95 133 96 57 145 83 Sources: World Bank, World Development Indicators; World Economic Forum (WEF; 2007); World Bank Enterprise Surveys; International Labour Organization; Friedman and others (2001); Schneider (2007). a. Countries are grouped into quartiles by GDP per capita at purchasing power parity (PPP). Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. b. Calculated as 1 minus the share of sales reported for tax purposes. Informal share of GDP as estimated from 286 Brookings Papers on Economic Activity, Fall 2008 Figure 1. Informal share of GDP, multiple indicators measure (percent)a Informal share of GDP, multiple indicators measure (percent)a AGO ALB ARE ARG ARM AUS AUT AZE BDI BEL BEN BFA BGD BGR BIH BLR BOL BRA BTN BWA CAF CAN CHE CHL CHN CIV CMR COG COL CRI CZE DEU DNK DOM DZA ECU EGY ESP EST ETH FIN FJI FRA FSM GBR GEO GHA GIN GRC GTM HKG HND HRV HTI HUN IDN IND IRL IRN ISRITA JAM JOR JPN KAZ KEN KGZ KHM KIR KOR KWT LAO LBN LKA LSO LTU LVA MAR MDA MDG MDV MEX MHL MKD MLI MNG MOZ MRT MWI MYS NAM NER NGA NIC NLD NOR NPL NZL OMN PAK PAN PER PHL PLW PNG POL PRI PRT PRY ROM RUS RWA SAU SEN SGP SLB SLE SLV SVK SVN SWE SYR TCD TGO THA TON TUN TUR TWN TZA UGA UKR URY USA UZB VEN VNM VUT WSM YEM YUG ZAF ZARZMB ZWE 70 60 50 40 30 20 10 7 8 9 10 11 Log of GDP per capita at purchasing power parityb Sources: Schneider (2007); World Bank, World Development Indicators. a. Average of the observations available for 1999–2004. b. Average for 1996–2006. one moves from poor to rich countries. These ﬁndings suggest that under- standing the decline of informal firms as countries grow richer may be central to development economics. Table 2 examines the determinants of the size of the informal sector. We present results ﬁrst without (top panel) and then with GDP per capita (bottom panel) in the regression. The dependent variables are ﬁve of the above proxies for the size of the informal economy as well as the number of registered businesses per capita. (We omit the results using nonagricul- tural self-employment as they are qualitatively similar to those for total self-employment.) The independent variables are proxies for the cost of becoming formal and the costs and beneﬁts of operating in the formal sec- tor. Each cell in each panel presents the results from a single univariate regression (we do not report the constant). The results in the top panel show the influence of policy variables. First, our proxy for the cost of becoming formal—the number of proce- dures necessary to start a business—is consistently associated with a larger 287 RAFAEL LA PORTA and ANDREI SHLEIFER Figure 2. Self-Employment and GDP per Capita ABW ANT ARG ARM ATG AUS AUT BEL BFA BGD BGR BHS BLZ BOL BRA BRB BWA CAN CHE CHL CMR COL CRI CUB CYP CZE DEU DJI DMA DNK DOM DZA ECU EGY ESP EST ETH FIN FRA GAB GBR GEO GRC GRD GTM GUY HKG HND HRV HUN IDN IMY IRL IRN ISL ISR ITA JAM JPN KAZ KGZ KHM KNA KOR LAO LCA LKA LSO LTU LUX LVA MAC MAR MDA MDG MDV MEX MKD MLI MLT MNG MUS MWI MYS NAM NCL NIC NLD NOR NZL OMN PAK PAN PER PHL POL PRI PRT PRY QAT ROM RUS SEN SGP SLV SMR SUR SVK SVN SWE SWZ SYC SYR TCD THA TTO TUN TUR TWN TZA UGA UKR URY USA VCT VEN VNM WBG WBG YEM ZAF ZMB ZWE 70 80 60 50 40 30 20 10 6 7 8 9 10 11 Log of GDP per capita at purchasing power parityb Percent of active labor force that is self-employeda Sources: International Labour Office (2007); World Bank, World Development Indicators. a. Data are as of the most recent year available. b. Average for 1996–2006. Figure 2. Self-Employment and GDP per Capita Sources: International Labour Office (2007); World Bank, World Development Indicators. a. Data are as of the most recent year available. b. Average for 1996–2006. informal sector as well as with fewer registered firms. However, the economic effect is modest in size. For example, a 1-standard-deviation (equal to 0.4) increase in the log of the number of procedures is associated with a 4.8-percentage-point rise in the multiple indicators measure of the informal economy. Second, the results for proxies for the cost of staying formal are mixed. All six proxies are statistically signiﬁcant when the dependent variable is the measure from the World Economic Forum survey (ﬁrst data column). On the other hand, none of the explanatory variables is signiﬁcant when using the tax evasion proxy. Results for the other dependent variables are in between these two extremes. Among the explanatory variables in this category, the most consistently signiﬁcant one is the time required to com- ply with taxes, which is signiﬁcant for all dependent variables except tax evasion. Even then, increasing the time required to comply with taxes by 1 standard deviation (0.75) is associated with an increase of only 4.8 per- centage points in the multiple indicators measure. Table 2. Figure 2. Self-Employment and GDP per Capita Regressions Explaining the Size of the Informal Sectora Dependent variable Informal share of GDP as estimated Self-employed No. of registered by business leaders as share of Electricity Multiple ﬁrms per 1,000 Independent variable (WEF survey) Tax evasionb labor force consumption indicators population Regressions not controlling for GDP per capita Log of no. of 10.1815* 14.7558* 13.9296* 11.3482* 11.9328* −23.8551* procedures required (1.2958) (3.3801) (2.6355) (3.3149) (2.1122) (4.4736) to register a business Total taxes as percent 0.0426 0.0599 −0.0408 −0.1107 0.0579* −0.2745 of proﬁts (0.0173) (0.0524) (0.0995) (0.0774) (0.0182) (0.1361) Hours per year needed 5.9038* −0.4877 7.4048* 8.4290* 6.4492* −13.2818* to comply with taxes (0.8215) (2.3969) (1.8772) (2.5841) (1.4057) (3.4070) Percent of management 0.5087* 0.3931 0.6275* 0.4452 0.3928 −0.2511 time spent dealing (0.1069) (0.2941) (0.2175) (0.3513) (0.2411) (0.5630) with regulations Index of difﬁculty of 0.1096* 0.0274 0.0614 0.0809 0.1638* −0.0859 hiring a new worker (0.0280) (0.0660) (0.0526) (0.0716) (0.0387) (0.0875) Index of difﬁculty 0.0942 0.0079 0.0996* 0.0558 0.1147 −0.2565 and expense of (0.0371) (0.0599) (0.0574) (0.0971) (0.0530) (0.1206) ﬁring a worker Nonwage costs as 0.1583* −0.2358 0.0880 0.0273 −0.1008 −0.2031 percent of salary (0.0494) (0.1553) (0.0794) (0.1159) (0.0980) (0.1359) Log of no. of steps 3.2047* 1.3864 3.3703* 4.4214* 5.4614* −5.0812 required to collect (0.8214) (1.5126) (1.1305) (1.5261) (1.3257) (3.2976) on a bounced check Efﬁciency of bankruptcy −0.2207* −0.1832 −0.2537* −0.2686* −0.2981* 0.3117 procedure (0.0252) (0.0757) (0.0347) (0.0677) (0.0352) (0.1175) Informal share of GDP as estimated from Log of paved −0.0090* −0.0334 −0.0112* −0.0137* −0.0163* 0.0297* roads per km2 (0.0019) (0.0130) (0.0020) (0.0023) (0.0018) (0.0109) Corruption index −7.4778* −7.1143* −10.8238* −8.5364* −9.4626* 11.9029* (0.3604) (1.8722) (0.9804) (1.6594) (0.5970) (1.9284) Rule of law index −8.0286* −6.0779* −11.6845* −9.1701* −9.9850* 13.3112* (0.3697) (1.7659) (0.9440) (2.0373) (0.6170) (2.0744) Private credit as −14.3709* −14.8612* −19.2173* −11.7983* −19.7457* 25.4301* percent of GDP (1.5940) (5.2608) (2.8056) (3.5882) (2.3164) (8.9047) Stock market −9.3152* −6.9204 −10.5142* −9.4449* −13.8049* 10.2845 capitalization as (1.7191) (5.8906) (2.5057) (2.5483) (3.0358) (4.2383) percent of GDP Access to credit 51.5059* 32.8132* 60.4548* 71.4457* 62.0713* −13.1594 (1.6596) (5.3360) (4.8082) (7.8939) (3.3155) (9.7251) Regressions controlling for GDP per capita Log of no. Figure 2. Self-Employment and GDP per Capita of 3.9083* 12.1526*** 1.4158 1.7380 3.6917* −13.1812 procedures required (1.1217) (3.5141) (2.0266) (3.2263) (1.8926) (5.2796) to register a business Total taxes as percent −0.0039 0.0283 0.0389 −0.2306 −0.0049 −0.3029* of proﬁts (0.0170) (0.0563) (0.0648) (0.0866) (0.0196) (0.1019) Hours per year needed 3.1399* −0.5181 2.6988* 2.5281 3.3539* −6.9528 to comply with taxes (0.6296) (2.3814) (1.5122) (2.6397) (1.2687) (3.2654) Percent of management 0.3950* 0.2007 0.2610 0.2312 0.2400 0.3767 time spent dealing (0.0725) (0.3011) (0.1829) (0.3418) (0.1962) (0.4977) with regulations Index of difﬁculty of 0.0485* −0.0033 −0.0388 −0.0201 0.0777 0.0537 hiring a new worker (0.0182) (0.0631) (0.0397) (0.0526) (0.0342) (0.0682) Index of difﬁculty 0.0311 −0.0294 0.0170 −0.0469 0.0274 −0.1100 and expense of (0.0275) (0.0547) (0.0420) (0.0752) (0.0470) (0.1007) ﬁring a worker Nonwage costs as 0.0806 −0.1075 −0.0405 −0.0966 0.0917 −0.0579 percent of salary (0.0311) (0.1649) (0.0571) (0.0714) (0.0769) (0.1090) (continued) (continued) Log of no. of steps 1.7096* 2.2855 0.5051 −1.9308 3.3370* −2.0003 required to collect (0.5891) (1.5024) (0.8129) (1.3614) (1.0474) (2.8724) on a bounced check Efﬁciency of bankruptcy −0.0560 −0.0491 −0.0252 0.2044** −0.0356 0.0752 procedure (0.0395) (0.0863) (0.0545) (0.0895) (0.0417) (0.1736) Log of paved −0.0025 −0.0168* −0.0029* −0.0044 −0.0051* 0.0175 roads per km2 (0.0016) (0.0088) (0.0010) (0.0018) (0.0016) (0.0119) Corruption index −5.6768* −3.4956 −1.8315 0.3169 −6.9316* 3.8139 (0.6844) (3.0779) (1.9129) (3.1140) (1.1440) (3.7512) Rule of law index −6.4793* −2.1094 −2.5183 1.6005 −7.7984* 4.9209 (0.7095) (3.0715) (2.2886) (3.9196) (1.3920) (3.3668) Private credit as −5.6811* −6.1499 2.7131 9.3552 −8.1961* 6.2312 percent of GDP (1.6812) (6.1198) (2.3110) (4.5279) (3.1009) (12.5764) Stock market −3.3739* −1.1785 0.5142 0.6396 −5.3196* −3.5914 capitalization as (1.0313) (5.7787) (1.4782) (2.0652) (1.8352) (8.0838) percent of GDP Access to credit 68.5488* −0.3639 125.7942* 164.0424* 99.0417* −82.9238*** (4.2760) (1.7380) (17.0908) (21.0187) (7.5245) (14.1297) Source: Authors’ regressions. a. Each cell reports the estimated regression coefﬁcient for a single univariate regression. All regressions include a constant (not reported). Robust standard errors are in parenthe- ses. Asterisks indicate statistical signiﬁcance at the 10 percent, 5 percent, and 1 percent level. b. Calculated as 1 minus the share of sales reported for tax purposes. Table 2. Regressions Explaining the Size of the Informal Sectora (Continued) Dependent variable Informal share of GDP as estimated Self-employed No. Figure 2. Self-Employment and GDP per Capita of registered by business leaders as share of Electricity Multiple ﬁrms per 1,000 Independent variable (WEF survey) Tax evasionb labor force consumption indicators population Informal share of GDP as estimated from RAFAEL LA PORTA and ANDREI SHLEIFER 291 Third, the proxies for the beneﬁts of being formal are consistently asso- ciated with the size of the informal sector and the number of registered ﬁrms: the only two exceptions are court formalism (the number of steps necessary to collect on a bounced check) in the regressions for tax evasion and registered ﬁrms. The economic impact, in terms of the multiple indica- tors measure, of increasing these variables by 1 standard deviation ranges from 5.8 percentage points for court formalism to 9.6 percentage points for the rule of law. In sum, without controlling for income per capita, both the cost of becoming formal and the beneﬁts of operating in the formal sector have a reliable but modest impact on the size of the informal economy. Our proxies for the cost of operating in the formal sector also have a modest effect but are less often signiﬁcant. Next, we rerun the previous regressions adding GDP per capita as an independent variable. The motivation is that the extent of the informal economy may be correlated with a country’s development level. In poor countries the informal economy may provide subsistence income for work- ers unable to ﬁnd formal employment. To the extent that informal ﬁrms avoid labor laws, the beneﬁts of informality may be larger in the labor- intensive activities common in poor countries than in the capital-intensive activities common in rich countries. Along the same lines, informality may decline as more transactions are intermediated through the ﬁnancial system. Finally, tax compliance may rise with income per capita as governments become more efficient at collecting taxes. The bottom panel of table 2 shows the coefﬁcients for the variables of interest when we control for GDP per capita. (As in the top panel, we do not report the constant. Nor do we report the coefﬁcient for GDP per capita, but it is strongly signiﬁcant in all regressions.) Most of the estimated coefﬁcients fall in value and lose signiﬁcance compared with the regressions without GDP per capita. Indeed, the coefficients remain consistently significant (11 of the 15 regressions) only for the World Economic Forum survey. Results for the other dependent variables are mostly insigniﬁcant. Figure 2. Self-Employment and GDP per Capita Our proxy for the cost of becoming formal remains signiﬁcant in four of the six regressions (World Economic Forum survey, tax evasion, multiple indica- tors, and registered ﬁrms). Among the proxies for the cost of operating in the formal sector, the strongest variable is the time to comply with taxes, which is signiﬁcant in four of the six regressions. Yet in contrast to the results on tax rates, nonwage costs are signiﬁcant in only one regression. Finally, among the proxies for the beneﬁts of operating in the formal sector, the strongest variables are road density (signiﬁcant in four regressions) and the subjective assessment of access to credit (signiﬁcant in ﬁve regressions). 292 Brookings Papers on Economic Activity, Fall 2008 The results using objective measures of the development of ﬁnancial mar- kets are mixed: private credit remains signiﬁcant in three regressions, but market capitalization does so in only two regressions. In sum, GDP per capita is the most robust predictor of the size of the informal economy. The most straightforward interpretation of the results in this section is that the informal economy is a manifestation of under- development. It recedes as the economy develops, perhaps because public goods become better and financial markets larger, or because avoiding detection becomes harder. It remains a crucial, and open, question whether this decline of the informal sector results from the conversion of informal ﬁrms to ofﬁcial status, or from their death and replacement by formal ﬁrms. An alternative interpretation is that we are overcorrecting by includ- ing GDP per capita. In particular, GDP per capita is strongly correlated (70 percent or better) with the efficiency of bankruptcy procedures, pri- vate credit, corruption, and the rule of law (see the correlation table in the appendix). Interestingly, variables that explicitly capture a country’s economic structure (such as the share of agriculture in GDP; results not reported) leave much of the explanatory power of GDP per capita unchanged. Although GDP per capita is strongly correlated with some of the determi- nants of the size of the informal economy, multicollinearity is unlikely to explain why tax rates, nonwage costs, and labor laws work so poorly when we control for GDP per capita. We return to this issue below when we examine the productivity of informal ﬁrms, using micro data. Figure 2. Self-Employment and GDP per Capita Although the cross-country evidence reveals some interesting patterns, it is merely suggestive and does not discriminate among the three views of the role of the informal economy. For this we need micro data, which we analyze next. Accordingly, the remainder of the paper is organized as follows. The next section describes our data on informal and formal ﬁrms. We ask such questions as: Are informal firms engines of growth as the romantic view would hold? For example, do informal ﬁrms grow quickly and over time join the formal sector? Is there evidence that—consistent with the parasite view—formal and informal firms operate in the same markets or that formal ﬁrms fear competition from informal ﬁrms? What evidence is there that—as predicted by the dual view—informal ﬁrms have inferior assets and management? The third section is the heart of the paper. It presents evidence on the relative productivity of formal and informal ﬁrms. We ask ﬁve questions. First, are our data on productivity reliable? Second, how big are the differ- ences in productivity between formal and informal ﬁrms? We want to know whether the prediction of the parasite view that informal ﬁrms have a cost RAFAEL LA PORTA and ANDREI SHLEIFER 293 advantage is borne out by the data. Third, what views of the informal econ- omy are consistent with the observed differences in productivity? We want to examine whether it is plausible to believe—as in the romantic view—that all that is holding back informal ﬁrms are high taxes and bad government regulation. Fourth, what accounts for the difference in the productivity of formal and informal ﬁrms? The goal is to see whether differences in pro- ductivity can be traced to differences in inputs. Finally, what evidence is there that more-able managers run ﬁrms with better assets? Evidence of a strong selection effect would support the dual view and cast doubt on the prediction of the romantic view that relieving informal ﬁrms from oppres- sive taxes and regulation would put an end to poverty as we know it. The fourth section focuses on obstacles to doing business, as reported by ﬁrms in all three surveys. We ask which of several problems, such as market access, ﬁnancing, taxes, and regulations, but also unfair competi- tion, are perceived as principal obstacles to doing business. These results shed light on the alternative theories but perhaps bear most directly on the parasite theory. Figure 2. Self-Employment and GDP per Capita The ﬁnal section concludes with some implications of the evidence. 30. The World Bank also carried out an Informal Survey of Cameroon in 2006. How- ever, data on sales are missing from that survey. Characteristics of Informal Firms In this section we describe our data and present simple descriptive statistics. Our basic approach is to compare, country by country, the relative perfor- mance of formal and informal ﬁrms. To do so, we combine data from three World Bank surveys of individual ﬁrms. The ﬁrst survey—the Enterprise Survey—covers formal ﬁrms and is available for 105 countries. The other two surveys—the Informal and Micro Surveys—contain information on both informal and formal firms in a few poor countries. The Informal Survey is available for 13 countries: Bangladesh, Brazil, Cambodia, Cape Verde, Guatemala, India, Indonesia, Kenya, Niger, Pakistan, Senegal, Tanzania, and Uganda.30 With the exception of Brazil, all these countries were below the world median income per capita in 2003 (equal to $5,322), and 7 out of 13 were below the 25th percentile (equal to $1,682). The Micro Survey is available for 14 mostly African countries: Angola, Botswana, Burundi, Democratic Republic of the Congo, The Gambia, Guinea, Guinea- Bissau, India, Mauritania, Namibia, Rwanda, Swaziland, Tanzania, and Uganda. With the exception of Botswana, all were below the world median 294 Brookings Papers on Economic Activity, Fall 2008 income per capita in 2006 (equal to $6,224), and 9 out of 14 were below the 25th percentile (equal to $1,965). The concept of informality used in the Informal and Micro Surveys focuses on registration (as we discuss below, there are several possible kinds of registration). Although questions about tax avoidance are asked, they are indirect. As discussed in the preceding section, this deﬁnition has both advantages and conceptual limitations. Before describing the data in detail, we need to preempt a possible mis- conception about the nature of the ﬁrms in our data. In the context of poor countries, the term “informal ﬁrm” evokes the image of street hawkers selling goods out of baskets, or of eateries in front of homes. In fact, such an image is a good description of how the very poor make a living.31 However, the informal ﬁrms in our sample do not ﬁt that image. For example, ﬁrms accounting for roughly 85 percent of the observations in the Informal and Micro Surveys have, in addition to the entrepreneur, two employees or more. The informal ﬁrms in our sample are likely to be substantially more productive than the own-account workers described by Abhijit Banerjee and Esther Duﬂo. 31. Banerjee and Duﬂo (2007). Data All three World Bank surveys have a similar structure and differ mainly in the ﬁrms that they sample. It is easiest to start by describing the Enter- prise Survey, the source for our control group of registered or formal ﬁrms. It covers mainly manufacturing and certain services ﬁrms with ﬁve or more employees in 105 countries. The earliest available data are from 2002 and the latest from 2007. The initial step in carrying out an Enterprise Survey involves contacting the government statistical ofﬁce of the relevant country to request a list of registered establishments. In some instances the World Bank supplements the government’s list with ﬁrms registered with the chamber of commerce of the relevant country or listed by Dun & Bradstreet or by similar private vendors of business directories. Thus, although ﬁrms in the Enterprise Survey may hide some of their output, the government typically knows of their existence. We refer to these ﬁrms as “registered” and deﬁne the term below. The next step involves contacting the ﬁrms that will be sampled. Enterprise Surveys use either simple random sampling or random stratiﬁed sampling. A local World Bank contractor telephones each ﬁrm to set up an interview with the person who most often deals with banks or government agencies. At that stage, ﬁrms with fewer than ﬁve employees are dropped from the sample, as are government-owned establishments, RAFAEL LA PORTA and ANDREI SHLEIFER 295 cooperatives, and community-owned establishments. Typical ﬁnal sample sizes range between 250 and 1,500 businesses per country. As described on the Enterprise Surveys website, “The core questionnaire is organized in two parts. The ﬁrst part seeks managers’ opinions on the . . . business environ- ment. The second part focuses on productivity measures and is often com- pleted with the help of the chief accountant or human resource manager.” The World Bank has also conducted separate surveys of informal ﬁrms to complement the Enterprise Survey in countries with large informal economies. Initially, data on the unofﬁcial sector were collected through the “Informal Sector” questionnaire. Starting in 2005, the World Bank switched to the “Micro Sector” questionnaire while phasing out the Infor- mal Sector questionnaire. Institutional amnesia makes it hard to ascertain the precise methodology followed with the Informal Sector questionnaire. Nevertheless, the basic outlines of what was done are clear. World Bank contractors identiﬁed neighborhoods perceived to have a large number of informal ﬁrms. 33. We lack detailed data on nonparticipation rates. In Mali, the only country for which we have data on nonparticipation, the refusal rate is 9 percent. 32. Jorge Rodriguez Meza, World Bank, personal correspondence with the authors, June 27, 2008. Data In the Micro Survey, we rely on the respondent’s answer to whether ﬁrms have either “registered with the Ofﬁce of the Registrar . . . or other government institutions responsible for commercial registration” or “obtained a tax identiﬁcation number from the tax administration or other agency responsible for tax registration.”34 Both Critically, the Informal and Micro Surveys cover registered ﬁrms as well as ﬁrms that exist without the government’s knowledge (“unregistered” ﬁrms). In the remainder of this paper, we focus on informality understood in terms of hidden ﬁrms rather than hidden output. To compare the perfor- mance of registered and unregistered ﬁrms, we need to deﬁne what it means to be registered. The questions regarding the legal status of the ﬁrm are worded differently in the Informal and the Micro questionnaires. In the Informal Survey we rely on the respondent’s answer to whether ﬁrms are “registered with any agency of the central government.” In practical terms, ﬁrms are registered with an agency of the central government if they have obtained a tax identification number. In the Micro Survey, we rely on the respondent’s answer to whether ﬁrms have either “registered with the Ofﬁce of the Registrar . . . or other government institutions responsible for commercial registration” or “obtained a tax identiﬁcation number from the tax administration or other agency responsible for tax registration.”34 Both surveys also keep track of whether firms are registered with “any local government agency” or with any “industry board or agency.” We focus on registration with the central government because this form of registration is more directly relevant to avoiding taxes, enforcing contracts, and raising ﬁnance. We will also present statistics on municipal and industry board registration. In sum, the Informal and Micro Surveys allow us to examine the productivity of (small) registered and unregistered ﬁrms, whereas the Enterprise Survey provides information on the productivity of registered ﬁrms that have at least ﬁve employees. 34. We obtain very similar results if the deﬁnition of “registered” ﬁrms in the Micro Survey includes only ﬁrms that have a tax identiﬁcation number. Data These neighborhoods were then divided into enumeration blocks, which were then surveyed on foot.32 A similar methodology was followed to implement the Micro Sector questionnaire. A local contractor selected districts and zones within each district where, based on national information sources, there was a high concentration of establishments with fewer than ﬁve employees (“micro” establishments). The contractor then created a comprehensive list of all establishments in these zones. Finally, the contractor selected randomly from that list and went door to door to set up interviews with the top man- agers of the selected establishments. Although the Micro Survey targets establishments with fewer than ﬁve employees, larger establishments are not dropped from the sample. In fact, establishments with fewer than ﬁve employees account for only 50 percent of the Micro Survey sample. Participation in the surveys is voluntary, and respondents are not paid to participate.33 Respondents are asked sequentially about the business environment, infrastructure, government relations, employment, ﬁnancing, and ﬁrm productivity. There is some variation in the response rate across questions. To illustrate, out of 6,466 Informal and Micro ﬁrms surveyed, we have the age of 6,412 firms, the number of employees of 6,416 firms, the sales of 6,136 firms, the fraction of investment financed internally of 5,689 ﬁrms, assessments of the fraction of taxes typically evaded by ﬁrms 296 Brookings Papers on Economic Activity, Fall 2008 in their industry of 4,670 firms, and capacity utilization of 3,083 firms. Since Informal and Micro ﬁrms typically do not keep detailed records of their operations, some respondents may simply not have the information being asked. Unfortunately, we have no way of quantifying the biases, if any, from missing data. Critically, the Informal and Micro Surveys cover registered ﬁrms as well as ﬁrms that exist without the government’s knowledge (“unregistered” ﬁrms). In the remainder of this paper, we focus on informality understood in terms of hidden ﬁrms rather than hidden output. To compare the perfor- mance of registered and unregistered ﬁrms, we need to deﬁne what it means to be registered. The questions regarding the legal status of the ﬁrm are worded differently in the Informal and the Micro questionnaires. In the Informal Survey we rely on the respondent’s answer to whether ﬁrms are “registered with any agency of the central government.” In practical terms, ﬁrms are registered with an agency of the central government if they have obtained a tax identification number. Descriptive Statistics Tables 3 and 4 list the countries surveyed and present the number of observations and average sales for the Informal and Micro samples, respec- tively. Most of the surveys (19 out of a total of 27) were carried out in African countries, but 6 surveys were done in Asia and 2 in Latin America. India, Uganda, and Tanzania were surveyed with both the Informal and the Micro questionnaires. As indicated earlier, most countries covered by the RAFAEL LA PORTA and ANDREI SHLEIFER 297 Informal and Micro Surveys are poor. The average income per capita in current purchasing power terms is roughly $2,400 and ranges from $281 in Congo to $12,744 in Botswana. Informal and Micro Surveys are poor. The average income per capita in current purchasing power terms is roughly $2,400 and ranges from $281 in Congo to $12,744 in Botswana. The Informal Survey covered 13 countries. The surveys were typically carried out in 2003 and, on average, have 223 ﬁrms with nonmissing sales in each country. The Micro Surveys were carried out in 14 countries in 2006 and, on average, have 214 ﬁrms with nonmissing sales per country. The World Bank also carried out Enterprise Surveys in parallel with the relevant Informal and Micro Surveys. We use ﬁrms from the Enterprise Survey as the control group. The average number of ﬁrms in the control group with available sales data is 474 for the Informal sample and 554 for the Micro sample and ranges from 53 in Niger (table 3) to 3,860 in India (table 4). Throughout the paper we emphasize productivity differences between registered and unregistered ﬁrms and between small and big ﬁrms. Criti- cally, whereas ﬁrms in the Informal Survey are typically unregistered, ﬁrms in the Micro Survey are typically registered. The average Informal Survey has 31 registered firms out of a total of 223 firms, whereas the average Micro Survey has 137 registered firms out of a total of 214 firms. To examine differences in size, we group Enterprise Survey ﬁrms into three categories according to the number of employees: fewer than 20 (“small”), between 20 and 99 (“medium”), and 100 or more (“big”). When assessing some of our results on productivity, it is worth keeping in mind that the distribution of firms across these three categories is fairly uneven. Descriptive Statistics For example, there is 1 big ﬁrm with nonmissing sales data (out of 93) in the control group for ﬁrms in Cape Verde, but there are 337 (out of 640) in the control group for ﬁrms in Indonesia (table 3). Perhaps because of the small number of observations, there are few extreme outliers in the data; these most likely result from errors in currency units. To mitigate the role of out- liers, we cap at the 95th percentile the value of sales, sales per employee, and value added per employee in each country and in each survey. Capping does not qualitatively change the results we present. The most striking fact in tables 3 and 4 is that the average annual sales of ﬁrms in the Informal and Micro Surveys are tiny even in comparison with those of small ﬁrms in the Enterprise Survey. Speciﬁcally, average sales are $24,671 for Informal Survey ﬁrms but $948,805 for small ﬁrms in the Enterprise Survey control group for those countries. Similarly, average sales are $50,853 for Micro Survey ﬁrms but $354,318 for small ﬁrms in that control group. Unregistered ﬁrms are even smaller than the average ﬁrm in the Informal and Micro Surveys. For example, in the Informal Survey p g p p y p Informal Survey sample Unregistered ﬁrms Registered ﬁrms All ﬁrms No. of No. of No. of untry Year Sales observations Sales observations Sales observations ngladesh 2003 19,794 195 48,856 2 20,089 197 azil 2003 32,528 218 51,227 126 39,377 344 mbodia 2003 25,710 209 75,165 6 27,090 215 pe Verde 2006 29,917 85 18,922 18 27,996 103 uatemala 2003 16,339 183 23,604 10 16,716 193 dia 2002 31,956 419 69,237 30 34,447 449 donesia 2003 29,237 276 . . . . . . 29,237 276 nya 2003 20,297 149 30,712 36 22,323 185 ger 2005 15,169 48 14,927 58 15,037 106 kistan 2003 15,435 210 7,805 3 15,327 213 negal 2004 24,944 153 29,827 41 25,976 194 nzania 2003 9,212 285 19,260 23 9,963 308 ganda 2003 35,082 91 45,341 23 37,152 114 Average 23,509 194 36,240 31 24,671 223 Table 3. Sales of the Informal Survey Sample and Its Control Group Dollars at purchasing power parity except where stated otherwise Informal Survey sample Unregistered ﬁrms Registered ﬁrms All ﬁrms No. of No. of No. Descriptive Statistics of Country Year Sales observations Sales observations Sales observations Bangladesh 2003 19,794 195 48,856 2 20,089 197 Brazil 2003 32,528 218 51,227 126 39,377 344 Cambodia 2003 25,710 209 75,165 6 27,090 215 Cape Verde 2006 29,917 85 18,922 18 27,996 103 Guatemala 2003 16,339 183 23,604 10 16,716 193 India 2002 31,956 419 69,237 30 34,447 449 Indonesia 2003 29,237 276 . . . . . . 29,237 276 Kenya 2003 20,297 149 30,712 36 22,323 185 Niger 2005 15,169 48 14,927 58 15,037 106 Pakistan 2003 15,435 210 7,805 3 15,327 213 Senegal 2004 24,944 153 29,827 41 25,976 194 Tanzania 2003 9,212 285 19,260 23 9,963 308 Uganda 2003 35,082 91 45,341 23 37,152 114 Average 23,509 194 36,240 31 24,671 223 Enterprise Survey control group Small ﬁrms Medium-size ﬁrms Big ﬁrms (<20 employees) (20–99 employees) (>99 employees) All ﬁrms No. of No. of No. of No. of Country Sales observations Sales observations Sales observations Sales observations Bangladesh 321,193 64 2,360,761 259 8,367,846 642 6,221,918 965 Brazil 767,484 252 3,419,992 811 24,100,000 406 8,683,195 1,469 Cambodia 167,574 193 979,849 40 3,260,287 26 603,488 259 Cape Verde 374,308 69 1,738,857 23 4,149,963 1 752,375 93 Guatemala 460,772 163 1,782,770 131 9,557,032 83 2,922,765 377 India 459,165 749 2,804,990 485 17,200,000 230 3,871,384 1,464 Indonesia 34,244 2 4,608,116 301 41,500,000 337 24,000,000 640 Kenya 1,675,268 49 6,070,552 65 31,800,000 41 11,500,000 155 Niger 4,999,650 34 4,416,983 16 14,700,000 3 5,371,892 53 Pakistan 2,066,015 7 4,316,266 66 9,332,258 33 5,729,247 106 Senegal 433,291 86 4,542,087 90 18,400,000 35 5,169,733 211 Tanzania 278,088 77 3,754,425 62 15,700,000 38 4,796,542 177 Uganda 297,418 107 3,222,021 58 10,700,000 28 2,681,279 193 Average 948,805 142 3,385,975 185 16,059,030 146 6,331,063 474 Sources: World Bank Informal and Enterprise Surveys; authors’ calculations. Table 4. Sales of the Micro Survey Sample and Its Control Group Dollars at purchasing power parity except where stated otherwise Micro Survey sample Unregistered ﬁrms Registered ﬁrms All ﬁrms No. of No. of No. of Country Year Sales observations Sales observations Sales observations Angola 2006 22,524 8 46,153 107 44,509 115 Botswana 2006 27,192 27 105,688 73 84,494 100 Burundi 2006 31,950 16 44,336 121 42,889 137 Congo, Dem. Rep. Descriptive Statistics 2006 20,150 40 32,891 64 27,991 104 Gambia, The 2006 12,955 47 20,307 76 17,498 123 Guinea 2006 93,345 27 129,568 77 120,164 104 Guinea-Bissau 2006 22,532 29 48,451 108 42,965 137 India 2006 40,179 643 92,382 906 70,713 1,549 Mauritania 2006 56,070 69 38,977 53 48,644 122 Namibia 2006 5,392 49 31,419 47 18,134 96 Rwanda 2006 8,295 22 46,821 106 40,199 128 Swaziland 2006 5,658 34 52,230 83 38,696 117 Tanzania 2006 30,093 25 48,327 40 41,314 65 Uganda 2006 43,584 38 93,144 59 73,729 97 Average 29,994 77 59,335 137 50,853 214 Enterprise Survey control group Small ﬁrms Medium-size ﬁrms Big ﬁrms (<20 employees) (20–99 employees) (>99 employees) All ﬁrms No. of No. of No. of No. of Country Sales observations Sales observations Sales observations Sales observation Angola 219,543 353 440,131 64 826,909 6 261,533 423 Botswana 1,054,364 212 4,027,974 86 9,497,498 39 2,790,306 337 Burundi 262,566 219 1,313,305 43 2,923,213 8 508,740 270 Congo, Dem. Rep. 156,191 258 779,580 71 1,675,336 11 335,518 340 Gambia, The 191,976 118 975,985 47 3,564,678 7 543,472 172 Guinea 180,759 194 979,018 19 2,246,573 7 315,430 220 Guinea-Bissau 155,735 97 441,720 16 . . . . . . 196,228 113 India 391,872 2,839 2,121,049 714 8,301,780 307 1,340,829 3,860 Mauritania 258,159 181 2,287,588 44 8,216,648 5 819,408 230 Namibia 665,167 225 2,917,353 82 9,329,198 17 1,689,759 324 Rwanda 344,204 143 2,071,016 53 7,671,968 16 1,328,946 212 Swaziland 391,593 207 2,418,694 55 6,982,505 32 1,488,191 294 Tanzania 326,825 259 3,430,273 111 16,400,000 44 2,866,305 414 Uganda 361,505 367 1,609,611 149 5,885,212 36 1,058,645 552 Average 354,318 405 1,843,807 111 6,424,732 41 1,110,236 554 Sources: World Bank Micro and Enterprise Surveys; authors’ calculations. 302 Brookings Papers on Economic Activity, Fall 2008 sample, average sales for unregistered Brazilian ﬁrms are $32,528, com- pared with $51,227 for registered ﬁrms. Looking across countries, unregis- tered ﬁrms in the Informal Survey sample have average sales of $23,509, compared with $36,240 for registered ﬁrms. Similarly, unregistered ﬁrms in the Micro Survey sample have average sales of $29,994, compared with $59,335 for registered ﬁrms. It is natural to worry that the reported sales of unregistered ﬁrms may be low because respondents lie about their output. We address this issue in the third section of the paper. What do unregistered ﬁrms do? Descriptive Statistics In contrast, large ﬁrms are the main client of 13.5 percent of registered firms—a percentage comparable to the average firm in the Enterprise Survey (15.1 percent). The next block of variables describes the employees and their human capital in the Informal Survey. Unsurprisingly, unregistered ﬁrms have the smallest average number of employees (3.9). More interestingly, registered ﬁrms in the Informal Survey and small ﬁrms in the Enterprise Survey have very similar employment levels (9.8 and 10.3 employees, respectively). The key fact regarding informal ﬁrms is that—consistent with the dual view but not with the other two views—their top managers have low human capital. For example, the probability that the top manager of a ﬁrm has some college education is only 6.1 percent in the Informal Survey if the ﬁrm is unregistered, compared with 15.9 percent for registered ﬁrms in the same survey and 63.9 percent for all ﬁrms in the Enterprise Survey. To summarize the differences in human capital, we created an index ranging from 1 to 4 according to whether the top manager’s highest level of educa- tion attended was primary school, secondary school, vocational school, or college. This index equals 1.6 for managers of unregistered ﬁrms and 3.3 for managers of Enterprise Survey ﬁrms. We constructed a similar index for the employees, with strikingly different results. Employees of Informal Survey ﬁrms have levels of education very similar to those of Enterprise Survey ﬁrms (indexes of 2.4 and 2.3, respectively). Next, we turn to how ﬁrms are ﬁnanced. All views of informality agree that greater access to finance is an important benefit of operating in the formal sector. In fact, roughly 75.1 percent of the unregistered Informal Survey ﬁrms have never even had a commercial loan. Instead, they ﬁnance 74.9 percent of investment with internal funds and 10.5 percent with help from the owner’s family. The most striking fact about ﬁnancing is that all small ﬁrms—not just unregistered ones—lack access to ﬁnance. In fact, small ﬁrms in the Enterprise Survey ﬁnance 67.8 percent of their invest- ment with internal funds and 6.3 percent with family funds. Big ﬁrms in the Enterprise Survey have more access to external ﬁnance than small ones. For example, internal funds pay for 50.4 percent of the investment of big ﬁrms. Descriptive Statistics Tables 5 and 6 shed light on some of the basic characteristics of ﬁrms in the Informal and Micro Surveys, respec- tively. The two tables have a similar—but not identical—structure, since there are only small differences between the two questionnaires. For each variable we present the mean for each group (for example, unregistered, registered, small, medium, and big) as well as the differences between the means for selected groups of interest (for example, small versus unregis- tered) and their statistical signiﬁcance. So that the results are not driven by the countries with the most observations, we ﬁrst average all observations within a country and then compute means and t statistics across countries. The ﬁrst block of variables in table 5 shows some general characteristics of the ﬁrms. Unregistered ﬁrms, although younger (9.9 years on average) than the average ﬁrm in the control group (17.8 years), have been operating for quite a long time. By deﬁnition, unregistered ﬁrms are not registered with the central government. Yet 34 percent of them are registered with a local government agency, and 7.2 percent are registered with an industry board or agency. g y The next four variables describe the assets owned by ﬁrms in the Infor- mal Survey. Unregistered ﬁrms own, on average, 52.3 percent of the land and 45.1 percent of the buildings that they occupy. Registered ﬁrms have comparable ﬁgures (55.5 percent and 48.1 percent). In contrast, ﬁrms in the Enterprise Survey control group own a significantly larger fraction of the land and buildings that they occupy (on average, 67.4 percent and 71.2 percent, respectively). The ownership of electric generators—a key asset in poor countries—shows a similar pattern. Few ﬁrms, unregistered or registered, in the Informal Survey own a generator (5.5 percent and 5.1 percent, respectively). In contrast, 20.1 percent of small firms and 77.0 percent of big ﬁrms in the Enterprise Survey own a generator. Capac- ity utilization rates vary little between unregistered Informal Survey ﬁrms and Enterprise Survey ﬁrms (61.9 percent versus 68.2 percent, respectively). The evidence also suggests that unregistered and registered ﬁrms may not share the same clients. In the Informal Survey, only 1.2 percent of the RAFAEL LA PORTA and ANDREI SHLEIFER 303 unregistered firms make the largest fraction of their sales to large firms. Descriptive Statistics Yet the fact that all small ﬁrms lack access to ﬁnance suggests that it may be misguided to put access to finance for unregistered firms at the center of the development agenda. Finally, contrary to the romantic view, there is no evidence in the Informal Survey that unregistered ﬁrms are dynamic engines of employ- ment creation. Two-year growth rates of employment are 5.2 percent for Table 5. Attributes of Firms in the Informal Survey Sample Percent except where stated otherwise Informal Survey sample Enterprise Survey control group Differencea Enterprise v. Registered v. Small v. Big v. Attribute Unregistered Registered All Small Medium Big All informal unregistered unregistered small General characteristics Age of the ﬁrm (years) 9.9 11.6 9.9 14.4 18.8 22.6 17.8 7.9* 1.7 4.5* 8.1* Share of ﬁrms registered with a 0.0 100.0 14.9 . . . . . . . . . . . . . . . 100.0 . . . . . . central government agency Share of ﬁrms registered with a 34.0 47.2 37.0 . . . . . . . . . . . . . . . 13.2 . . . . . . local government agency Share of ﬁrms registered with 7.2 14.8 8.9 . . . . . . . . . . . . . . . 7.6 . . . . . . Descriptive Statistics an industry board or agency Share of occupied land owned 52.3 55.5 53.9 59.0 70.9 70.8 67.4 13.5 3.2 6.7 11.8 by the ﬁrm Share of occupied buildings 45.1 48.1 44.9 60.8 74.8 79.3 71.2 26.2 3.0 15.7 18.4 owned by the ﬁrm Share of ﬁrms that own 5.5 5.1 5.6 20.1 53.9 77.0 45.9 40.3* −0.4 14.6* 56.8* a generator Average capacity utilization 61.9 65.8 62.4 66.5 68.0 71.2 68.2 5.8 3.9 4.5 4.7 Share of ﬁrms for which main 1.2 13.5 1.6 9.2 17.8 16.1 15.1 13.5* 12.3 8.0* 6.9 customers are large ﬁrms Employment and human capital Average number of employees 3.9 9.8 4.1 10.3 43.1 487.8 151.0 146.9* 5.9 6.4* 477.5* Median number of employees 3.8 4.6 4.1 10.2 42.9 426.7 100.6 96.5* 0.9 6.4* 416.5* Index of education of top 1.6 2.0 1.6 2.8 3.3 3.8 3.3 1.7* 0.4 1.2* 1.0* manager (4 = attended college) indicated highest educational attendance: Primary 64.8 47.8 64.2 13.0 7.0 2.1 8.9 −55.3* −17.0* −51.8* −10.9* Secondary 19.3 20.8 18.6 34.8 19.5 5.9 18.1 −0.5 1.4 15.4* −28.9*** Vocational 9.8 15.6 10.4 10.9 8.2 4.6 9.1 −1.3 5.8 1.0 −6.3* College 6.1 15.9 6.8 41.4 65.2 87.5 63.9 57.0* 9.8 35.3* 46.1* Index of education of average 2.4 2.5 2.4 2.3 2.3 2.4 2.3 −0.1 0.1 −0.1 0.2 employee (4 = attended college) Share of employees with indicated highest educational attendance: Primary 59.0 50.0 58.2 52.3 51.5 45.2 47.9 −10.3 −9.1 −6.8 −7.1 Secondary 34.3 40.2 34.9 24.4 27.7 33.5 32.5 −2.4 5.9 −9.9 9.0 College 6.7 9.8 6.9 21.8 18.3 17.7 17.4 10.5* 3.2 15.1 −4.0 Finance Share of ﬁrms that have ever 24.9 35.6 26.0 . . . . . . . . . . . . . . . 10.7 . . . . . . Descriptive Statistics had a commercial loan Share of ﬁnancing from: Internal funds 74.9 71.0 75.7 67.8 56.3 50.4 58.2 −17.5* −3.9 −7.2 −17.4 Trade 4.6 6.2 4.7 4.9 6.2 7.2 5.9 1.2 1.7 0.4 2.2 Owner’s family 10.5 9.1 9.5 6.3 6.3 3.7 6.6 −2.9 −1.4 −4.2 −2.6 Banks 3.0 4.0 3.0 9.6 16.0 20.9 15.0 12.0* 1.1 6.6 11.4* Duration of last loan (months) 14.6 13.3 14.1 29.0 32.7 35.1 32.2 18.1 −1.2 14.5** 6.1 Growth Annual growth in employment 5.2 7.1 5.4 8.1 11.1 11.6 10.0 4.6* 1.9 2.9 3.5 over previous two years Sources: World Bank Informal and Enterprise Surveys; authors’ calculations. a. Asterisks indicate signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. cent except where stated otherwise Micro Survey sample Enterprise Survey control group Differencea Enterprise v. Registered v. Small v. Big v. ribute Unregistered Registered All Small Medium Big All micro unregistered unregistered small neral characteristics e of the ﬁrm (years) 7.0 8.2 7.8 9.2 14.3 18.3 10.7 3.0* 1.2 2.2* 9.1* re of ﬁrms registered with a 0.0 100.0 68.4 . . . . . . . . . . . . . . . 100.0 . . . . . . entral government agency re of ﬁrms registered with a 39.4 81.1 68.7 . . . . . . . . . . . . . . . 41.7* . . . . . . ocal government agency re of ﬁrms registered with 5.0 30.7 20.0 . . . . . . . . . . . . . . . 25.7 . . . . . . n industry board or agency re of ﬁrms located in the 17.2 13.4 13.8 . . . . . . . . . . . . . . . −3.9 . . . . . . wner’s home re of ﬁrms located in a 71.4 80.4 77.0 . . . . . . . . . . . . . . . 9.0 . . . . . . ermanent structure re of occupied land owned 21.7 20.1 20.1 28.4 54.3 71.0 36.2 16.1* −1.6 6.7 42.7* y the ﬁrm re of ﬁrms forced to move 11.3 8.8 9.8 . . . . . . . . . . . . . . . −2.5 . . . . . . Descriptive Statistics ast year because of lack of ecure title re of ﬁrms that own 12.7 23.6 20.2 32.5 52.1 75.8 43.0 22.8* 10.9* 19.8* 43.3* generator re of ﬁrms with an 60.0 79.2 73.6 . . . . . . . . . . . . . . . 19.2* . . . . . . lectrical connection re of ﬁrms that use their own 6.6 22.9 18.2 . . . . . . . . . . . . . . . 16.3* . . . . . . ransportation equipment Hours per week that the ﬁrm 64.8 64.6 64.9 59.4 60.9 79.8 62.2 −2.7 −0.2 −5.4 20.4* operates Share of ﬁrms for which main 0.4 2.6 1.8 21.1 36.1 44.7 29.0 27.2 2.2* 20.7 23.6 customers are large ﬁrms Exports as share of sales 0.1 0.7 0.5 0.9 4.4 19.9 2.8 2.3* 0.7* 0.8* 19.0* Share of ﬁrms that use e-mail to 3.2 9.1 7.1 29.5 57.8 78.7 39.0 31.9* 5.9* 26.3* 49.1* connect with clients Share of ﬁrms that use a webpage 0.9 2.8 2.2 8.9 22.2 42.2 14.1 11.8* 2.0 8.0* 33.3* to connect with clients Employment and human capital Average number of employees 2.9 4.5 3.9 8.7 38.7 290.4 32.7 28.8* 1.5 5.8* 281.6* Median number of employees 2.7 3.7 3.5 8.7 39.4 253.2 29.1 25.6* 1.0 5.9* 244.5* Index of education of top 1.8 2.3 2.1 2.7 3.2 3.8 2.8 0.7* 0.4* 0.8* 1.1* manager (4 = attended college) Share of top managers with indicated highest educational attendance: Primary 49.8 35.9 40.2 22.1 0.1 2.2 19.6 −20.6* −13.9* −27.7* −19.9* Secondary 27.8 26.2 26.2 25.3 0.1 5.5 21.6 −4.6 −1.7 −2.6 −19.8* Vocational 10.2 13.4 12.4 17.0 0.1 6.9 15.7 3.3 3.2 6.8 −10.1* College 12.2 24.6 21.2 35.7 0.6 85.3 43.1 21.9* 12.4* 23.5* 49.7* Index of education of average 2.3 2.3 2.3 2.5 2.5 2.8 2.5 0.2 0.0 0.1 0.3 employee (4 = attended college) Share of employees with indicated highest educational attendance: Primary 48.7 46.1 46.4 47.8 0.4 31.2 44.8 −1.6 −2.7 −0.9 −16.5 Secondary 40.2 41.2 41.3 42.9 0.5 52.8 45.8 4.5 1.0 2.7 9.9 College 4.0 5.7 5.3 9.3 0.1 16.0 9.4 4.1 1.7 5.3 6.6 (continued) Finance Share of ﬁrms that have ever had a commercial loan 7.3 12.5 10.9 . . . . . . . . . . . . 5.1 . . . . . . Descriptive Statistics Share of ﬁnancing from: Internal funds 81.9 76.9 78.9 75.5 64.4 59.1 72.4 −6.5* −5.1 −6.4* −16.4*** Trade 8.3 11.5 10.6 13.3 17.3 16.5 14.2 3.6 3.3 5.0* 3.2 Owner’s family 6.6 6.7 6.2 4.6 3.1 0.9 4.1 −2.1 0.1 −2.0 −3.7* Banks 0.4 2.0 1.5 4.1 11.3 18.5 6.4 4.9* 1.6* 3.7* 14.4 Duration of last loan (months) 13.2 29.9 26.8 30.5 39.3 55.5 37.6 10.8* 16.8 17.4 25.0 Growth Annual growth in employment 24.3 27.1 25.9 17.5 18.9 14.6 17.6 −8.3* 2.8 −6.8** −2.9 over previous two years Sources: World Bank Micro and Enterprise Surveys; authors’ calculations. a. Asterisks indicate signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. Table 6. Attributes of Firms in the Micro Survey Sample (Continued) Percent except where stated otherwise Micro Survey sample Enterprise Survey control group Differencea Enterprise v. Registered v. Small v. Big v. Attribute Unregistered Registered All Small Medium Big All micro unregistered unregistered small RAFAEL LA PORTA and ANDREI SHLEIFER 309 unregistered ﬁrms, 7.1 percent for registered ﬁrms, and 10.0 percent for all Enterprise Survey ﬁrms. Firms in the Micro Survey sample show patterns very similar to those in the Informal Survey sample (table 6). We therefore discuss them only brieﬂy, focusing on the questions that are available only on the Micro Sur- vey questionnaire and on the few results that are different between the two questionnaires. The Micro questionnaire provides a bit more insight into the ﬁrms’ assets. Only 17.2 percent of the unregistered ﬁrms and 13.4 percent of the registered ones are located in the owner’s house. Most unregistered (71.4 percent) and registered (80.4 percent) firms occupy a permanent structure. However, there is evidence of hardship resulting from the lack of secure title:35 11.3 percent of unregistered ﬁrms and 8.8 percent of regis- tered ﬁrms were forced to move in the previous year for this reason. Much like their counterparts in the Informal Survey, unregistered ﬁrms in the Micro Survey sample are significantly less likely to own a genera- tor than all other firms. This lack of generators is suggestive of insuffi- cient capital, since unregistered ﬁrms are signiﬁcantly less likely to have an electrical connection than registered ones (60 percent versus 79.2 per- cent). Furthermore, unregistered firms are much less likely to use their own transportation equipment than registered ﬁrms (6.6 percent versus 22.9 percent). 35. De Soto (2000). Descriptive Statistics Consistent with the view that unregistered ﬁrms and Enter- prise Survey ﬁrms may serve different clients, big Enterprise Survey ﬁrms export 19.9 percent of their sales, whereas unregistered ﬁrms export only 0.1 percent of their sales. Finally, there is evidence that unregistered ﬁrms have less access to computers than do other ﬁrms. In particular, unregis- tered ﬁrms are less likely to use e-mail to communicate with their clients than either registered firms or Enterprise Survey firms (3.2, 9.1, and 39.0 percent, respectively). Similarly, unregistered ﬁrms are less likely to use a webpage to connect with clients than either registered ﬁrms or Enter- prise Survey ﬁrms (0.9, 2.8, and 14.1 percent, respectively). Consistent with the dual view, unregistered ﬁrms tend to own low-quality assets. Unregistered ﬁrms in the Micro sample—unlike their counterparts in the Informal sample—have a faster growth rate of employment than ﬁrms in the Enterprise Survey. Average annual employment growth among unregistered firms (24.3 percent), although not quite matching that of registered firms (27.1 percent), exceeds that of Enterprise Survey firms (17.6 percent). The fast employment growth rate of unregistered Micro Survey firms is consistent with the romantic view. However, this finding 310 Brookings Papers on Economic Activity, Fall 2008 Brookings Papers on Economic Activity, Fall 2008 Table 7. Legal Status of Enterprise Survey Firms in Latin America Percent of ﬁrms Percent not No. of that registered knowing when ﬁrm Firm age Country observations upon formation was registered (years) Argentina 1,051 92.8 1.1 28.6 Bolivia 609 85.7 0.7 21.8 Chile 1,007 98.0 1.0 26.6 Colombia 995 89.0 0.5 17.0 Ecuador 652 91.6 0.9 21.3 El Salvador 683 77.7 1.4 21.4 Guatemala 511 90.4 2.1 20.9 Honduras 424 89.4 2.8 20.5 Mexico 1,439 94.9 2.8 18.5 Nicaragua 474 80.4 0.8 22.9 Panama 601 97.8 0.5 24.5 Paraguay 608 94.4 0.8 21.3 Peru 630 96.8 0.3 19.8 Uruguay 607 97.5 2.3 28.8 Average 91.2 1.3 22.4 Source: World Bank Enterprise Survey 2006. Table 7. Legal Status of Enterprise Survey Firms in Latin America Source: World Bank Enterprise Survey 2006. needs to be interpreted cautiously, since these firms remain very small despite having been around for an average of 7 years. needs to be interpreted cautiously, since these firms remain very small despite having been around for an average of 7 years. Descriptive Statistics To complement the evidence on growth rates, we examine, for a few countries, how often registered ﬁrms initially started operating as unregis- tered. The Enterprise Survey ﬁles for 14 Latin American countries include a question on whether ﬁrms were registered when they started operations and, if not, on whether they have since registered. As it turns out, all ﬁrms in this sample of 14 countries are registered. Table 7 shows the available data regarding the initial legal status of these ﬁrms. The fraction of ﬁrms that were registered from the outset ranges from 77.7 percent in El Salvador to 98 percent in Chile and averages 91.2 percent. Since 1.3 percent of the respondents did not answer the question, we estimate that only 7.5 percent of the ﬁrms registered after starting operations. Firms that start operations without being registered often register relatively quickly: 36.5 percent of the initially unregistered ﬁrms had registered by the end of the second year of operations (table 8). It is unclear whether those ﬁrms spent two years hiding from the government or, alternatively, started operations while their request for a permit was pending. Either way, ﬁrms rarely start as unregis- tered and later change their status. This is not the pattern that one would expect to see if the informal sector were a reservoir of entrepreneurial talent, as predicted by the romantic view. Nor is it the pattern that one would 311 RAFAEL LA PORTA and ANDREI SHLEIFER Table 8. Delays in Registering by Enterprise Survey Firms in Latin America Yearsa Frequency Percent of total Cumulative percent 1 129 17.9 17.9 2 134 18.6 36.5 3 79 11.0 47.5 4 52 7.2 54.7 5 58 8.1 62.8 6 26 3.6 66.4 7 28 3.9 70.3 8 19 2.6 72.9 9 22 3.1 76.0 10 23 3.2 79.2 Source: World Bank Enterprise Survey 2006. a. Year of operations in which the ﬁrm registered. Table 8. 36. Bennett and Estrin (2007). Descriptive Statistics Delays in Registering by Enterprise Survey Firms in Latin America expect to see if entrepreneurs used entry into the informal sector as a way of acquiring information (for example, about demand for the ﬁrm’s products) at a lower cost than entry into the formal sector.36 expect to see if entrepreneurs used entry into the informal sector as a way of acquiring information (for example, about demand for the ﬁrm’s products) at a lower cost than entry into the formal sector.36 We conclude this section by presenting some data on the institutional environment in which ﬁrms operate. All views of informality agree on the basic trade-off faced by ﬁrms (the tax and regulatory burden versus access to public goods and ﬁnance). The previous literature has emphasized access to public goods as one of the main attractions of operating in the formal sector. Tables 9 and 10 present data on the institutional environment faced by ﬁrms in the Informal and the Micro Surveys, respectively, and how they operate in it. Three facts stand out. First, consistent with all views of informality, unregistered ﬁrms enjoy tangible advantages. Managers of unregistered firms in the Informal sample estimate that a typical firm in their sector evades 74.8 percent of its tax liability. Tax evasion sharply decreases with ﬁrm size. For example, managers of small ﬁrms in the control group estimate that a typical ﬁrm in their sector evades 35.5 percent of its liability; tax evasion drops to 22.9 percent for big ﬁrms in the control group. Tax evasion by unregistered Micro Survey ﬁrms and by small ﬁrms in their control group follows a similar pattern (67.7 percent versus 44.4 percent, respectively). Likewise, the regulatory burden increases rapidly with ﬁrm size. Whereas managers of unregistered firms in the Informal Survey sample report spending 5.6 percent of their time dealing with government regulations, that task requires 14.5 percent of the time of managers of big ﬁrms in the control group; the corresponding ﬁgures for the Micro Survey sample and Table 9. Indicators of the Institutional Environment Facing Informal Survey Firms Percent except where stated otherwise Enterprise Survey Informal Survey sample control group Differencea Enterprise v. Registered v. Small v. Big v. Descriptive Statistics Indicator Unregistered Registered All Small Medium Big All informal unregistered unregistered small Compliance with government regulations Share of tax liability evaded 74.8 53.5 72.2 35.5 28.6 22.9 30.3 −41.9* −21.4* −39.3* −12.6 by “typical” ﬁrm Share of management’s 5.6 6.8 5.3 9.8 15.4 14.5 12.9 7.6* 1.2 4.2* 4.7 time spent dealing with government regulations Share of sales a “typical” 3.6 4.8 3.9 4.6 4.1 3.8 4.6 0.7 1.1 1.0 −0.8 ﬁrm pays in informal gifts or payments to get things done Public goods Days last year with power 50.0 56.3 50.6 44.9 52.1 53.5 48.0 −2.6 6.3 −5.0 8.6 outages 8.6 Days last year with water 33.6 31.3 34.3 22.5 24.4 24.2 23.5 −10.8 −2.4 −11.1 1.6 outages Days last year with 4.1 19.3 14.2 13.1 10.6 11.8 11.7 −2.5 15.2 9.0 −1.3 telephone outages Days last year with 33.6 22.0 32.7 7.1 9.2 10.9 9.1 −23.6 −11.6 −26.5 3.8 transportation outages Property rights Share of sales lost last year 2.9 3.5 3.1 3.8 1.7 0.8 2.2 −0.9 0.6 0.9 −3.0 owing to theft Share of sales spent on 1.8 1.2 1.6 2.2 2.3 2.5 2.3 0.6 −0.6 0.4 0.3 security expenses Share of sales spent on 1.0 0.5 1.0 0.5 0.8 0.7 0.7 −0.3 −0.5 −0.5 0.3 “protection payments” Share of incidents reported 14.1 26.2 19.1 36.0 38.0 54.0 42.6 23.5 12.1 21.9** 18.1 to police Share of ﬁrms that had a 21.5 0.0 21.3 . . . . . . . . . . . . . . . −21.5 . . . . . . payment dispute in last two years Days it took a typical court 60.9 90.3 66.2 67.9 56.1 68.3 61.9 −4.3 29.4 7.0 0.4 case to be resolved Sources: World Bank Informal and Enterprise Surveys; authors’ calculations. a. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. Table 10. Indicators of the Institutional Environment Facing Micro Survey Firms Percent except where stated otherwise Micro Survey sample Enterprise Survey control group Differencea Enterprise v. Registered v. Small v. Big v. Descriptive Statistics Indicator Unregistered Registered All Small Medium Big All micro unregistered unregistered small Compliance with government regulations Share of tax liability evaded 67.7 54.2 58.3 44.4 33.7 32.5 41.6 −16.6 −13.5 −23.3*** −11.9* by “typical” ﬁrm Share of management’s 1.5 4.2 3.5 8.2 9.3 10.5 8.5 5.0* 2.7* 6.6*** 2.3* time spent dealing with government regulations Share of sales a “typical” 4.0 3.5 3.3 6.6 7.1 5.6 6.6 3.2* −0.5 2.6 −1.0 ﬁrm pays in informal gifts or payments to get things done Public goods No. of power outages in 167.1 134.4 138.8 138.3 151.7 157.9 143.7 4.9 −32.7 −28.8 19.6 last year Days last year with water . . . . . . . . . 57.9 56.4 51.8 51.9 . . . . . . . . . −6.1 outages Days last year with . . . . . . . . . 3.7 4.8 3.5 4.0 . . . . . . . . . −0.2 telephone outages Property rights Share of sales lost last year 0.5 0.5 0.5 2.6 1.8 1.6 2.4 1.8* 0.0 2.1*** −1.0 owing to theft Share of sales spent on 3.4 2.8 2.9 2.3 2.1 1.2 2.1 −0.8* −0.6 −1.1 −1.1*** security expenses Share of sales spent on . . . . . . . . . 0.4 2.9 0.2 1.2 . . . . . . . . . −0.1 “protection payments” Share of ﬁrms that had a 6.0 8.4 7.5 9.5 16.6 19.4 11.4 4.0* 2.3 3.5 9.9* payment dispute in last two years Share of ﬁrms with payment 29.2 33.2 30.1 51.3 67.6 81.8 58.3 28.3* 4 22* 31.0*** dispute that used courts to resolve it Days it took a typical court . . . 44.3 44.3 45.4 74.1 64.4 51.9 7.7 . . . . . . 19.1 case to be resolved Sources: World Bank Micro and Enterprise Surveys; authors’ calculations. a. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. 316 Brookings Papers on Economic Activity, Fall 2008 its control group are 1.5 and 10.5 percent, respectively. Finally, unregistered ﬁrms pay a smaller fraction of their sales in bribes than do ﬁrms in the con- trol group. Descriptive Statistics Managers of unregistered ﬁrms in the Informal Survey estimate that ﬁrms in their sectors pay 3.6 percent of their sales to “get things done.” In contrast, managers of registered ﬁrms in the Informal Survey report that bribes equal 4.8 percent of sales, a percentage similar to that reported by ﬁrms in the control group (4.6 percent). Similarly, managers of unregistered ﬁrms in the Micro Survey estimate that ﬁrms in their sector pay 4.0 percent of their sales to “get things done”; the comparable ﬁgures are 3.5 percent for registered Micro Survey ﬁrms and 6.6 percent for ﬁrms in the control group. In sum, lower taxes and less regulation confer a clear cost advantage on unregistered ﬁrms. Second, the quality of public goods in our sample is very low. In the Informal Survey, unregistered ﬁrms report that they experienced power outages on 50 days of the previous year. Firms in the Enterprise Survey fare only slightly better (48 days on average). On many days, ﬁrms experi- ence multiple power outages. For this reason the number of power outages for the Micro Survey is dramatically higher than the number of days with- out power in the Informal Survey: unregistered ﬁrms in the Micro survey experienced 167.1 power outages in the previous year. Once again, Enter- prise Survey ﬁrms do only marginally better (143.7 outages). In such an environment, only ﬁrms large enough to afford a generator can be produc- tive. Outages of water, phones, and transportation are less frequent than power outages but nevertheless very common by the standards of devel- oped countries. As a result, the performance of ﬁrms that are too small to provide substitutes for these public goods (their own transportation equip- ment, for example) may be severely impaired. Third, outright theft is very prevalent in our sample, but small ﬁrms do not make much use of police or the courts. Theft affects all small ﬁrms, not just unregistered ones. Speciﬁcally, unregistered ﬁrms in the Informal Sur- vey report that, in a typical year, losses from theft amount to 2.9 percent of annual sales. Registered ﬁrms in the same survey and small ﬁrms in the Enterprise Survey report even higher losses (3.5 percent and 3.8 percent, respectively). Somewhat surprisingly, losses as a result of theft appear to be lower for Micro Survey ﬁrms (0.5 percent) than for small ﬁrms in the con- trol group (2.6 percent). Descriptive Statistics To put these ﬁgures in context, note that Enterprise Survey respondents estimate losses as a result of theft equal to 0.54 percent of sales in Germany, 0.26 percent in Ireland, and 0.22 percent in Spain. In response to theft, firms in our sample spend heavily on security and make “protection” payments to gangsters. For example, security and pro- 317 RAFAEL LA PORTA and ANDREI SHLEIFER tection payments equal, respectively, 1.8 and 1.0 percent of the sales of unregistered ﬁrms in the Informal Survey sample. Firms in their control group spend a bit more on security and a bit less on protection, but their total expenditure is similar (3 percent). The police do not appear to play a central role in addressing theft. In fact, most theft is not even reported to the police. Only 14.1 percent of incidents suffered by unregistered firms in the Informal Survey were reported to the police. Registered ﬁrms in the same survey reported 26.2 percent of incidents—still a low ﬁgure. This pattern is consistent with the view that unregistered ﬁrms may have trouble protecting their property rights. Alternatively, the absolute value of the losses suffered by unregistered firms may be too low to justify filing a police complaint. Firm size does play a role in reporting theft to the police. However, even big ﬁrms in the control group for the Informal Survey sam- ple report to the police only about half of theft incidents (54.0 percent). Interestingly, small firms do not make much use of the courts to adju- dicate disputes either. Only 29.2 percent of unregistered and 33.2 percent of registered ﬁrms in the Micro Survey sample used the courts to resolve commercial disputes during the previous year. In the control group, the use of the courts to solve commercial disputes rises quickly with ﬁrm size, from 51.3 percent for small ﬁrms to 81.8 percent for big ﬁrms. Surprisingly, the courts appear to work in a reasonably efﬁcient manner. It takes roughly 62 days to resolve a commercial dispute in the Informal Survey countries and approximately 52 days in the Micro Survey countries. These ﬁgures are in line with the average length of court proceedings in Germany (35 days), Ireland (79 days), and Spain (91 days). 37. Data on wages are unavailable for most countries in the Informal sample. For this reason we are unable to remove labor costs from our measure of value added. 38. Klette and Griliches (1996). 39. These include Bernard and others (2003); Katayama, Lu, and Tybout (2006); Foster, Haltiwanger, and Syverson (2008); Hsieh and Klenow (2007). Descriptive Statistics The fact that unregistered ﬁrms and small firms in the control group behave similarly in solving commercial disputes suggests that inadequate access to courts is unlikely to explain differences in productivity between the two groups of firms. The same argument applies to lack of police protection. The tentative picture that emerges from this section is inconsistent with the romantic view. Unregistered firms have been around for a long time (7 to 10 years on average), but their sales are still trivially small. Moreover, few registered ﬁrms started out unregistered. The small size of unregistered firms is symptomatic of uneducated management and low- quality assets. When public goods are unreliable, unregistered firms are too small to afford substitutes such as generators, computers, or trans- portation equipment. They do not have large ﬁrms as clients. They do not export. Despite de Soto’s emphasis on access to credit as the key to ignit- ing the growth of unregistered ﬁrms, lack of external ﬁnance appears to be an attribute of all small ﬁrms in poor countries, not just of unregistered 318 Brookings Papers on Economic Activity, Fall 2008 ﬁrms. In sum, the limitations of unregistered ﬁrms appear to be far more severe than acknowledged by proponents of the romantic view. 39. These include Bernard and others (2003); Katayama, Lu, and Tybout (2006); Foster, Haltiwanger, and Syverson (2008); Hsieh and Klenow (2007). 37. Data on wages are unavailable for most countries in the Informal sample. For this reason we are unable to remove labor costs from our measure of value added. 38. Klette and Griliches (1996). Productivity of Unregistered Firms In this section we examine the productivity of unregistered ﬁrms and pre- sent the key ﬁndings of the paper. In measuring the productivity of these ﬁrms, we face severe data limitations. In particular, we lack information on how much capital these ﬁrms have. The Informal and Micro questionnaires do not collect such information, since unregistered entrepreneurs typically lack detailed records to estimate the value of their assets. We thus have to measure productivity without capital. To this end we use two crude measures of productivity: sales per employee and (gross) value added per employee, the latter deﬁned as sales net of expenditure on raw materials and energy.37 Thus, we deﬁne value added per employee for ﬁrm i in industry s as VA P Y P M P E L si si si m si E si si = − − , where PsiYsi is the level of sales, PmMsi is expenditure on raw materials, PEEsi is expenditure on energy, and Lsi is the number of employees (includ- ing both full- and part-time but not seasonal workers). To the extent that seasonal employment is more prevalent in unregistered ﬁrms than in the formal sector, we overstate the productivity of unregistered ﬁrms. We use expenditure on production inputs (such as energy) as a crude proxy for capital invested. This approach to productivity measurement has recently received con- siderable criticism, since the sales measure obviously combines physical output and prices. But in a competitive equilibrium, prices may vary inversely with efﬁciency exactly to eliminate any variation in productivity as measured by sales (or value added) per employee. The recognition of this problem in the absence of firm-specific price indices is credited to Tor Jakob Klette and Zvi Griliches;38 several more recent studies seek to address the problem.39 We follow the approach of Chang-Tai Hsieh and 319 RAFAEL LA PORTA and ANDREI SHLEIFER Peter Klenow,40 which assumes that all ﬁrms in an industry use the same Cobb-Douglas production technology and that industry output is a constant- elasticity-of-substitution (CES) aggregate of the outputs of all the ﬁrms. Productivity of Unregistered Firms They then show that, in a competitive equilibrium, physical productivity Asi (or real output per employee) can be estimated from nominal sales using the following formula: A P Y L si s si si si = ( ) − κ σ σ 1 , where κs is an unobserved constant and σ is the elasticity of substitution of output. Although we do not observe κs, relative productivities are unaffected by setting κs equal to 1 for each industry s. Intuitively, goods sold by very productive ﬁrms must command lower prices to induce buyers to demand the higher output. Raising sales to the power σ/(σ −1) yields Ysi, making it possible to infer real output from nominal revenue. Since registered ﬁrms tend to have higher sales, productivity differences between registered and unregistered ﬁrms are increasing in σ. Empirically, estimates of σ range from 3 to 10. We follow Hsieh and Klenow and conservatively set σ equal to 3.41 Before turning to the results, we note the empirical ﬁnding of Lucia Foster and coauthors that the correlation between the sales-based and the corrected measures of productivity is incredibly high, well over 0.9.42 Thus, although the theoretical objection to the traditional measures is compelling, its empirical signiﬁcance appears minor. Indeed, Foster and coauthors have data on both prices and sales. The correlation that they report between nominal and real output is based on actual data rather than on a model. 40. Hsieh and Klenow (2007). 42. Foster and others (2008). 41. Hsieh and Klenow (2007). 40. Hsieh and Klenow (2007). 41. Hsieh and Klenow (2007). 42. Foster and others (2008). 43. De Mel, McKenzie, and Woodruff (2007). 44. Harris and Todaro (1970). Measurement Error Even aside from the theoretical concerns, we need to deal with the fact that our sales numbers come from unofﬁcial ﬁrms, raising concerns about measurement error. There is good reason to worry that our productivity measures may be biased, since unregistered entrepreneurs may choose to hide output not only from the government but also from the World Bank contractors. For example, Suresh de Mel, David McKenzie, and Christo- pher Woodruff ﬁnd that microenterprises underreport proﬁts by 30 percent 320 Brookings Papers on Economic Activity, Fall 2008 to researchers, although they attribute this more to lack of recall than to intentional understatement.43 We offer two pieces of evidence that support the view that such biases are unlikely to drive our main results. First, table 11 shows the available information regarding expenditure on various production inputs (scaled by sales). If unregistered entrepreneurs lied only about sales, inputs as a fraction of sales would be higher for unregistered ﬁrms than for other ﬁrms. In fact, expenditure on raw materials by small ﬁrms in the control group is 12.7 percentage points higher than for unregistered ﬁrms in the Informal sample, and 2.7 percentage points higher than for unregistered ﬁrms in the Micro sample. Moreover, expenditure on energy by unregistered firms is comparable to that by firms in the control group. Other variables show a mixed pattern. In particular, expenditure on labor by small firms in the control group is 8.1 percentage points higher than that by unregistered ﬁrms in the Informal sample, but 1.7 percentage points lower than that by unregistered ﬁrms in the Micro sample. Similarly, expenditure on machines by small ﬁrms in the control group is 14.8 percentage points higher than that by unregistered ﬁrms in the Micro sample, but equal to that by unregistered ﬁrms in the Informal sample. Finally, there is weak evidence that unregis- tered ﬁrms in the Informal Survey spend more on rent than do small ﬁrms in the control group. In sum, there is no evidence that unregistered ﬁrms consistently spend a larger fraction of their sales on inputs than do small ﬁrms in the control group, as would be the case if unregistered entrepre- neurs lied only about their sales. We offer two pieces of evidence that support the view that such biases are unlikely to drive our main results. First, table 11 shows the available information regarding expenditure on various production inputs (scaled by sales). Measurement Error If unregistered entrepreneurs lied only about sales, inputs as a fraction of sales would be higher for unregistered ﬁrms than for other ﬁrms. Second, table 12 shows the available data on wages per employee. Under the dual hypothesis, unregistered firms should pay low wages.44 These low wages may be consistent with some on-the-job home production by workers in unregistered firms. Alternatively, workers in these firms may be less skilled than those in registered ﬁrms. Either way, the dual view pre- dicts that the measured output of unregistered ﬁrms should be low relative to that of workers in the control group. In contrast, wages in the formal and informal sectors should be comparable if observed differences in produc- tivity are due only to measurement error. The top panel of table 12 shows wages per employee in Cape Verde, the only country in the Informal sam- ple with wage data. The bottom panel shows wages per employee for the countries covered by the Micro sample. Wages in both panels are scaled by income per capita. Table 11. Expenditure on Production Inputs by Informal and Micro Survey Firms Percent of sales Informal Survey sample Enterprise Survey control group Differencea Enterprise v. Registered v. Small v. Big v. Indicator Unregistered Registered All Small Medium Big All informal unregistered unregistered small Raw materials 30.5 35.2 31.0 43.2 47.2 41.3 46.4 15.4* 4.7 12.7** −1.9 Energy 6.8 6.8 6.8 6.8 6.8 6.8 6.8 0.0 0.0 0.0 0.0 Labor 13.4 21.8 14.9 21.5 17.8 17.3 18.9 4.0 8.4 8.1 −4.2* Machines 0.1 0.1 0.1 0.1 0.1 0.0 0.1 0.0 0.0 0.0 0.0 Land 8.3 13.2 10.4 4.2 1.6 2.0 3.6 −6.8 4.9 −4.1 −2.2 Rent 7.5 9.7 7.9 3.9 2.2 1.3 2.8 −5.1* 2.3 −3.6 −2.6** Average 1.3 3.4 2.2 −1.8 difference Micro Survey sample Enterprise Survey control group Difference Enterprise v. Registered v. Small v. Big v. Indicator Unregistered Registered All Small Medium Big All micro unregistered unregistered small Raw materials 38.5 39.7 39.6 41.3 44.4 49.3 42.6 3.0 1.2 2.7 8.0 Energy 3.6 2.9 2.9 4.2 3.8 4.6 4.1 1.2* −0.7 0.6 0.4 Labor 23.3 21.0 21.5 21.6 19.7 17.7 20.9 −0.5 −2.3 −1.7 −3.9* Machines 2.9 3.3 3.1 17.8 44.1 32.9 18.6 15.5 0.4 14.8 15.1 Land . . . . . . . . . 0.7 0.7 0.7 0.7 . . . . . . . . . Measurement Error 0.0 Rent 7.4 8.3 8.0 6.7 3.5 2.3 5.8 −2.2* 0.9 −0.6 −4.4*** Average 3.4 −0.1 3.2 2.5 difference Sources: World Bank Informal, Micro, and Enterprise Surveys; authors’ calculations. a. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. Enterprise Survey Informal Survey sample control group Differenceb Enterprise v. Registered v. Small v. Big v. Country Unregistered Registered All Small Medium Big All informal unregistered unregistered small Cape Verde 0.90 1.25 0.96 2.92 4.03 2.62 3.19 2.23* 0.35 2.03* −0.30 Micro Survey sample Difference Enterprise v. Registered v. Small v. Big v. Country Unregistered Registered All Small Medium Big All micro unregistered unregistered small Angola 1.35 2.23 2.17 3.26 3.02 1.51 3.20 1.03* 0.88* 1.91* −1.74* Botswana 0.35 0.58 0.52 0.89 1.05 1.03 0.95 0.43* 0.23* 0.54* 0.14 Burundi 1.76 3.13 2.97 5.84 7.29 4.82 6.04 3.07* 1.37* 4.08* −1.02 Congo, Dem. Rep. 5.64 5.45 5.52 8.25 11.35 9.26 8.93 3.41* −0.18 2.62* 1.01* Gambia, The 0.54 1.04 0.85 1.52 2.41 1.92 1.78 0.94* 0.49* 0.98*** 0.40 Guinea 0.83 1.23 1.13 1.30 1.13 0.91 1.27 0.15* 0.40 0.47* −0.39 Guinea-Bissau 6.11 7.21 6.97 9.64 6.92 . . . 9.25 2.29** 1.10 3.53* . . . India 1.31 1.43 1.39 1.54 1.82 1.62 1.64 0.25* 0.12* 0.22* 0.09 Mauritania 2.12 2.10 2.11 3.88 3.98 4.33 3.91 1.80* −0.02 1.76* 0.44 Namibia 0.27 0.79 0.55 2.48 2.56 2.30 2.49 1.94* 0.51* 2.21* −0.19 Rwanda 1.29 1.52 1.47 4.01 5.70 3.12 4.36 2.89* 0.23 2.72* −0.89 Swaziland 0.50 1.20 1.05 1.92 2.21 1.88 1.97 0.92* 0.69* 1.42* −0.04 Tanzania 1.44 1.59 1.53 3.59 5.07 5.72 4.21 2.68* 0.16 2.15* 2.13* Uganda 3.08 3.93 3.60 4.32 4.90 3.91 4.45 0.85 0.85 1.24 −0.42 Average 1.90 2.39 2.27 3.75 4.24 3.26 3.89 1.62 0.49 1.85 −0.04 Source: World Bank Informal, Micro, and Enterprise Surveys; authors’ calculations. a. See tables 3 and 4 for the survey years for each country. b. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and *1 percent level. Table 12. Ratio of Wages per Employee to GDP per Capita in Informal and Micro Survey Firmsa Enterprise Survey control group Source: World Bank Informal, Micro, and Enterprise Surveys; authors calculations. a. See tables 3 and 4 for the survey years for each country. b. Measurement Error Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. 323 RAFAEL LA PORTA and ANDREI SHLEIFER Three facts stand out. First, there is no clear correlation between ﬁrm size and wages within the control group. Big ﬁrms pay higher wages than do small ﬁrms in Congo and Tanzania. The reverse is true in Angola. On average, wages in big and small ﬁrms are essentially indistinguishable from each other. Second, unregistered ﬁrms consistently pay lower wages than do small ﬁrms in the control group. Cape Verde illustrates this point. Wages in unregistered ﬁrms there are 10 percent lower than income per capita. In contrast, wages in the control group of small ﬁrms are 2.92 times income per capita. On average, in the Micro sample, wages are 1.90 times income per capita in unregistered ﬁrms and 3.75 times income per capita in small firms in the control group. Third, although there is considerable hetero- geneity across countries, the workers of unregistered firms are not the poorest among the poor. In India, for example, wages for the employees of unregistered ﬁrms exceed GDP per capita by 31 percent. Similarly, in the Micro sample, the average wage of unregistered workers is roughly twice GDP per capita. Taken at face value, the large wedge in wages between unregistered ﬁrms and the control group is strongly consistent with the dual view of unregistered ﬁrms. Of course, we cannot rule out the alternative interpretation that respondents shrewdly lie to the World Bank about sales, inputs, and wages. However, the ﬁndings on inputs and wages should allay some of the concerns regarding data quality. As a ﬁnal point, it seems to us that concerns about intentional understate- ment of revenues should not be exaggerated for our data. Firms participating in the surveys do so voluntarily. Virtually all of them answer questions about sales, even though they do not have to. They also give answers suggesting massive underpayment of taxes and bribe payments by “ﬁrms like theirs.” This is not the behavior one would expect of those fearful that World Bank contractors will turn them in (or that the authorities would do anything about it if they did). Measurement Error Our view is that most informal ﬁrms operate in the open, that they have done so for years, that they pay the police and other authorities to leave them alone, and that fear of reprisals for truly reporting revenues to the World Bank is very far from most of their minds. This particular concern is a rich-country fear rather than a poor-country reality. Productivity of Unregistered Firms Tables 13 and 14 present the main ﬁndings of the paper. Table 13 shows estimates of log value added (top panel), log sales per employee (middle panel), and log real output per employee (bottom panel) for the Informal sample and its Enterprise Survey control group. Table 14 shows analogous data for the Micro sample. Three key facts stand out. First, registered Log units Informal Survey sample Enterprise Survey control group Differenceb Registered v. Small v. Big v. Big v. Country Unregistered Registered All Small Medium Big All unregistered unregistered small unregistered Log of value added per employee Bangladesh 7.09 7.92 7.10 7.96 8.53 8.69 8.61 0.83 0.87* 0.73 1.60* Brazil 8.30 8.77 8.47 9.22 9.58 10.36 9.74 0.48* 0.92* 1.14* 2.06* Cambodia 7.19 8.01 7.22 . . . . . . . . . . . . 0.82 . . . . . . . . . Cape Verde 8.12 7.85 8.07 8.47 9.21 9.78 8.78 −0.27 0.35 1.30 1.65 Guatemala 7.37 8.59 7.48 8.95 9.39 9.42 9.21 1.22 1.57* 0.48* 2.05* India 7.64 8.29 7.69 9.16 9.43 9.90 9.36 0.64* 1.52* 0.74* 2.26* Indonesia 7.73 . . . 7.73 8.53 8.39 9.16 8.80 . . . 0.80 0.64 1.44* Kenya 7.76 8.04 7.83 9.58 9.99 10.30 9.94 0.28 1.82* 0.71* 2.54* Niger 9.32 7.16 8.24 11.44 10.01 9.98 10.83 −2.16 2.12 −1.46 0.66 Pakistan 7.21 6.59 7.20 9.78 9.76 9.18 9.58 −0.62 2.58* −0.60 1.98* Senegal 7.19 7.22 7.20 9.09 9.81 9.96 9.54 0.03 1.90* 0.87* 2.77* Tanzania 6.23 . . . 6.23 8.65 9.51 9.83 9.40 . . . 2.43* 1.18 3.61* Uganda 7.15 7.92 7.30 8.71 9.33 10.02 9.09 0.76 1.56* 1.31* 2.87* Average 7.56 7.85 7.52 9.13 9.41 9.72 9.41 0.18* 1.54* 0.59 2.12* Log of sales per employee Bangladesh 7.82 8.82 7.83 9.39 10.00 9.61 9.70 1.00 1.57*** 0.22* 1.79* Brazil 8.63 9.18 8.83 9.84 10.23 11.02 10.38 0.55* 1.21* 1.18* 2.40* Cambodia 7.77 9.10 7.80 8.84 8.95 8.61 8.84 1.33* 1.08* −0.24 0.84* Cape Verde 8.35 8.33 8.34 9.82 10.35 9.94 9.96 −0.02 1.48* 0.12 1.60 Guatemala 7.80 8.12 7.81 9.70 10.14 10.19 9.96 0.32 1.90* 0.49* 2.39* India 8.20 8.83 8.25 10.09 10.32 10.77 10.27 0.63* 1.89* 0.67* 2.56* Indonesia 8.38 . . . 8.38 7.66 9.07 10.04 9.58 . . . Productivity of Unregistered Firms −0.72 2.38 1.66* Kenya 8.11 8.34 8.15 10.76 11.07 10.98 10.95 0.24 2.65* 0.22 2.87* Niger 7.80 7.45 7.61 11.40 10.76 10.95 11.18 −0.35* 3.60* −0.45 3.15* Pakistan 7.73 7.30 7.73 10.73 10.83 10.17 10.62 −0.44 2.99* −0.56 2.43* Senegal 7.81 7.95 7.84 10.16 10.77 11.34 10.61 0.14 2.35* 1.19* 3.53* Tanzania 7.26 8.08 7.32 8.96 10.28 10.68 9.79 0.82* 1.70* 1.73* 3.42* Uganda 7.73 8.12 7.81 9.42 10.02 10.69 9.79 0.38 1.69* 1.27* 2.96* Average 7.95 8.30 7.98 9.75 10.21 10.38 10.12 0.38 1.80* 0.63 2.43* Log of real output per employee Bangladesh 12.49 14.79 12.51 15.35 16.90 17.29 17.05 2.30 2.86* 1.94* 4.80* Brazil 13.49 14.34 13.80 16.12 17.21 19.25 17.59 0.85* 2.63* 3.13* 5.76* Cambodia 12.56 14.66 12.62 14.23 15.26 16.00 14.57 2.10* 1.67* 1.77* 3.44* Cape Verde 13.02 13.09 13.03 15.87 17.28 17.66 16.24 0.06 2.84* 1.80 4.64 Guatemala 12.46 13.00 12.49 15.74 17.04 18.15 16.72 0.54 3.28* 2.40* 5.69* India 13.14 14.18 13.21 16.29 17.24 18.98 17.02 1.04* 3.15* 2.69* 5.84* Indonesia 13.32 . . . 13.32 12.69 15.42 18.25 16.90 . . . −0.62 5.55* 4.93* Kenya 12.82 13.29 12.91 17.36 18.51 19.31 18.36 0.47 4.54* 1.95* 6.49* Niger 12.27 12.01 12.13 18.31 18.10 19.16 18.29 −0.26 6.03* 0.85 6.88* Pakistan 12.38 11.63 12.37 17.26 18.08 18.08 18.03 −0.75 4.88* 0.82 5.70* Senegal 12.51 12.69 12.55 16.35 17.99 19.71 17.61 0.18 3.84* 3.36* 7.20* Tanzania 11.55 12.85 11.65 14.55 17.28 18.72 16.40 1.30* 2.99* 4.17* 7.17* Uganda 12.39 13.08 12.53 15.05 16.66 18.59 16.05 0.69 2.66* 3.54* 6.20* Average 12.65 13.30 12.70 15.78 17.15 18.40 16.99 0.71* 3.14* 2.61* 5.75*** Source: World Bank Informal and Enterprise Surveys; authors’ calculations. a. See tables 3 and 4 for the survey years for each country. b. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and *1 percent level. p y a. See tables 3 and 4 for the survey years for each country. b. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. Source: World Bank Informal and Enterprise Surveys; authors’ calculations. a. See tables 3 and 4 for the survey years for each country. b. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. Table 14. Productivity of Unregistered Firms Productivity of Firms in the Micro Sector Survey Log units Micro Survey sample Enterprise Survey control group Differenceb Registered v. Small v. Big v. Big v. Country Unregistered Registered All Small Medium Big All unregistered unregistered small unregistered Log of value added per employee Angola 7.48 8.35 8.30 8.97 8.86 9.34 8.97 0.87 1.50* 0.36 1.86* Botswana 9.00 8.85 8.88 9.49 10.02 9.52 9.66 −0.15 0.48 0.03 0.51 Burundi 8.52 7.81 7.91 8.19 9.23 9.11 8.47 −0.72 −0.33 0.92 0.59 Congo, Dem. Rep. 6.91 7.65 7.38 8.25 8.89 8.53 8.47 0.74 1.34* 0.28 1.62*** Gambia, The 6.86 7.39 7.24 8.23 8.76 9.35 8.44 0.52* 1.37* 1.12 2.49* Guinea 8.01 8.65 8.49 8.34 8.67 9.60 8.41 0.64* 0.33 1.26* 1.59 Guinea-Bissau 7.78 8.39 8.31 8.28 8.47 . . . 8.32 0.61 0.50 . . . . . . India 8.05 8.40 8.25 8.75 8.99 9.44 8.93 0.35* 0.70* 0.68* 1.39* Mauritania 8.43 7.50 8.16 8.69 9.23 9.34 8.92 −0.93 0.26 0.66 0.91* Namibia 6.76 7.82 7.51 9.81 10.21 10.44 10.04 1.06 3.05* 0.63 3.68* Rwanda 7.51 8.38 8.32 9.15 9.36 9.10 9.21 0.86 1.64*** −0.05 1.59* Swaziland 7.63 8.64 8.54 9.83 9.55 9.62 9.67 1.00 2.20* −0.21 1.99* Tanzania 7.88 8.21 8.09 8.92 9.74 10.37 9.32 0.33 1.05* 1.44* 2.49* Uganda 8.13 8.40 8.30 8.66 8.92 9.71 8.80 0.27 0.52* 1.05* 1.57* Average 7.78 8.17 8.12 8.83 9.21 9.50 8.97 0.39 1.04* 0.63* 1.71* Log of sales per employee Angola 8.16 8.90 8.85 9.58 9.50 9.92 9.58 0.74* 1.43* 0.34 1.77* Botswana 8.53 9.49 9.23 10.33 10.78 10.62 10.48 0.95* 1.80* 0.28 2.08* Burundi 9.09 8.69 8.73 9.25 9.86 10.15 9.37 −0.40 0.16 0.91 1.06** Congo, Dem. Rep. 7.91 8.38 8.20 8.91 9.52 9.57 9.06 0.48* 1.01* 0.66 1.67* Gambia, The 7.42 8.02 7.79 8.76 9.41 10.30 9.00 0.60* 1.34* 1.55* 2.88* Guinea 8.88 9.53 9.36 8.92 9.18 9.90 8.98 0.66 0.05 0.98** 1.03 Guinea-Bissau 8.49 9.05 8.93 9.27 9.35 . . . 9.28 0.57* 0.79* . . . . . . Productivity of Unregistered Firms India 8.66 9.12 8.93 9.79 9.93 10.14 9.85 0.46* 1.13* 0.35* 1.48* Mauritania 9.14 8.79 8.99 9.98 10.24 11.14 10.05 −0.35 0.84* 1.17* 2.00* Namibia 7.17 8.21 7.68 10.34 10.65 10.96 10.45 1.04* 3.16* 0.63* 3.79* Rwanda 7.39 8.62 8.41 9.26 9.96 10.01 9.49 1.23* 1.87* 0.74 2.61* Swaziland 7.62 8.94 8.55 9.87 10.25 10.06 9.96 1.32* 2.25* 0.19 2.44* Tanzania 8.51 8.93 8.77 9.36 10.21 11.12 9.77 0.42 0.85* 1.76* 2.61* Uganda 8.66 9.17 8.97 9.32 9.69 10.36 9.49 0.50* 0.66* 1.04* 1.70* Average 8.26 8.85 8.67 9.50 9.89 10.33 9.63 0.59* 1.24* 0.81* 2.09* Log of real output per employee Angola 12.97 14.13 14.05 15.57 15.94 17.27 15.65 1.16* 2.60* 1.71* 4.30* Botswana 13.38 14.89 14.49 16.63 18.01 18.54 17.20 1.52* 3.25* 1.92* 5.16* Burundi 14.20 13.67 13.73 14.96 16.58 17.71 15.30 −0.53 0.75 2.76* 3.51 Congo, Dem. Rep. 12.44 13.32 12.98 14.46 16.02 16.92 14.87 0.88 2.02* 2.46* 4.48* Gambia, The 11.77 12.72 12.35 14.22 15.94 18.11 14.85 0.95* 2.46* 3.89* 6.35* Guinea 14.06 15.02 14.77 14.47 15.55 17.57 14.66 0.96 0.42 3.10* 3.51* Guinea-Bissau 13.23 14.01 13.84 15.01 15.78 . . . 15.12 0.78* 1.78* . . . . . . India 13.76 14.68 14.30 15.33 16.71 18.04 15.80 0.92* 1.57* 2.71* 4.28* Mauritania 14.42 13.92 14.20 16.07 17.15 19.31 16.35 −0.49 1.65* 3.24* 4.89* Namibia 11.17 12.92 12.02 16.64 17.72 19.15 17.04 1.75* 5.47* 2.52* 7.99* Rwanda 11.62 13.44 13.13 15.00 16.75 17.89 15.65 1.82* 3.38* 2.89* 6.27* Swaziland 11.71 14.03 13.35 15.91 17.17 17.85 16.36 2.31* 4.20* 1.94* 6.14*** Tanzania 13.33 14.13 13.82 15.17 17.16 19.36 16.15 0.80* 1.84* 4.19* 6.03* Uganda 13.79 14.72 14.36 15.14 16.33 18.28 15.66 0.94* 1.35* 3.15* 4.50* Average 12.99 13.97 13.67 15.33 16.63 18.16 15.76 0.98* 2.34* 2.80* 5.19*** Sources: World Bank Micro and Enterprise Surveys; authors’ calculations. a. See tables 3 and 4 for the survey years for each country. b. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. 328 Brookings Papers on Economic Activity, Fall 2008 firms in both the Informal and the Micro Surveys are more productive than unregistered ones in the same survey. Firms in India in the 2006 Micro Survey illustrate this pattern. Productivity of Unregistered Firms Value added per employee for registered ﬁrms is 35 percent higher than for unregistered ﬁrms (8.40 versus 8.05), sales per employee are 46 percent higher (9.12 versus 8.66), and real output per employee is 92 percent higher (14.68 versus 13.76). Most countries exhibit a similar pattern, although Burundi, Mauritania, Niger, and Pakistan are exceptions. On average, value added per employee for registered firms in the Informal and Micro samples is, respectively, 18 percent and 39 per- cent higher than for their unregistered counterparts. Differences in sales per employee are even larger: 38 percent for the Informal sample and 59 per- cent for the Micro sample. Differences between unregistered and registered ﬁrms are most extreme for real output per employee: 71 percent in the Informal Survey sample and 98 percent in the Micro Survey sample. Second, these differences become much more dramatic when we com- pare Informal or Micro Survey ﬁrms with the Enterprise Survey ﬁrms. The productivity gap between unregistered ﬁrms and even the small ﬁrms in the control groups is truly enormous. Take the case of India in 2006 again. Value added per employee for small Enterprise Survey ﬁrms is 70 percent higher than for unregistered Micro Survey ﬁrms, and sales and real output per employee for small firms are 113 percent and 157 percent higher, respectively, than for unregistered ones. The example of India is represen- tative of the results for other countries, except that value added and real output per employee in Burundi and sales per employee in Indonesia do not conform to this pattern. Bearing in mind that the observations are unevenly distributed across size groups (only two small ﬁrms in Indonesia have nonmissing sales), the consistency of the results across countries is striking. On average, based on the Informal sample, the productivity of small firms in the Enterprise Survey is around 154, 180, or 314 percent higher than for unregistered ﬁrms depending on whether we look at value added, sales per employee, or real output per employee, respectively. Similarly, based on the Micro sample, the productivity wedge between small firms in the Enterprise Survey and unregistered firms is 104, 124, or 234 percent depending on whether we look at value added, sales per employee, or real output per employee, respectively. Third, big ﬁrms are signiﬁcantly more productive than small ones. Productivity of Unregistered Firms Con- tinuing with the example of India in 2006, the productivity wedge between big and small ﬁrms in the control group for the Micro sample is 68 percent for value added, 35 percent for sales per employee, and 271 percent for real output per employee. This large heterogeneity in ﬁrm productivity is RAFAEL LA PORTA and ANDREI SHLEIFER 329 consistent with work by Hsieh and Klenow showing sizable gaps in the marginal products of labor and capital across plants within narrowly deﬁned industries in China and India.45 On average, depending on the measure and the sample, productivity of big ﬁrms is between 59 and 280 percent higher than that of small ones. The cumulative effect of these productivity differences is large. Return- ing to the example of India in 2006, big ﬁrms are 139 to 428 percent more productive than unregistered ﬁrms. On average, the productivity wedge between big and unregistered ﬁrms in the Informal sample is 212 percent for value added, 243 percent for sales per employee, and 575 percent for real output per employee. The numbers for the Micro sample are of the same order of magnitude: 171 percent for value added, 209 percent for sales per employee, and 519 percent for real output per employee. To illustrate what these differences in productivity mean in practice, consider the average unregistered Micro Survey ﬁrm in India. It has sales of $2,420 per employee and value added of $1,279 per employee. In contrast, an average small ﬁrm in the control group has sales of $12,285 per employee and value added of $4,335 per employee. If the unregistered ﬁrm could achieve the value added of a small Enterprise Survey ﬁrm sim- ply by registering, would it choose to do that? By assumption, changing its legal status would generate $3,056 (= $4,335 −$1,279) in additional cash ﬂow per employee. However, the ﬁrm would have to pay registration fees and taxes as well as comply with regulations. The registration fee— including the value of the entrepreneurs’ time—would probably amount to roughly $400.46 The firm would also need to pay labor taxes (17 percent of wages), corporate taxes (35 percent of profits), and value-added taxes (12.5 percent of proﬁts).47 Recall that our value-added estimates are based on expenditure on energy and materials and do not exclude labor costs. 45. Hsieh and Klenow (2007). 46. Djankov and others (2002). 47. Djankov and others (2008b). Productivity of Unregistered Firms To keep things simple, assume that wages are 20 percent of sales ($2,457) and that there are no additional costs. Moreover, to bias the example against the firm choosing to register, assume that the firm would evade all taxes if unregistered but comply fully if registered. Under these assumptions, the ﬁrm would owe additional payments of $418 (= 0.17 × $2,457) in labor taxes, $657 in corporate taxes (= 0.35 × [$4,335 −$2,457]), and value- added tax of $235 (= 0.125 × [$4,335 −$2,457]), for a total of $1,710 per employee in taxes and fees. In this back-of-the-envelope calculation, the ﬁrm would pocket $1,346 (= $3,056 −$1,710) per employee by registering. 330 Brookings Papers on Economic Activity, Fall 2008 Of course, the gains would be even larger if the unregistered ﬁrm could, merely by registering, replicate the value added per employee of big ﬁrms in the control group. On average, such ﬁrms have value added per employee of $8,715 on sales per employee of $20,301. Calculations similar to the preceding ones suggest that the unregistered ﬁrm would gain $4,135 per employee if, simply by registering, it could replicate the value added per employee of big ﬁrms. A similar set of calculations illustrates that unregistered entrepreneurs can simply not afford to pay taxes unless sales sharply increase from merely registering. Assuming wages equal 20 percent of sales ($484), the average unregistered ﬁrm has a pre-tax proﬁt per employee of $795 (= $1,279 −$484) and owes taxes of $460 per employee.48 Unless sales dramatically increased as a result of registering, the average unregistered ﬁrm would have consid- erable difﬁculty paying $400 to register. In practice, these calculations mean that believers in the romantic view need to blame the precarious existence of unregistered ﬁrms on something beyond costly entry procedures and high tax rates. Given the very large dif- ference in productivity between unregistered ﬁrms and the control group, the cost of complying with government regulations would have to be implausi- bly high to justify operating as an unregistered ﬁrm. A more realistic sce- nario is that—consistent with the dual view—unregistered ﬁrms would not be able to achieve the performance of small ﬁrms in the control group just by registering. Perhaps, for example, unregistered ﬁrms lack the human capital necessary to match the quality of the goods produced by formal ﬁrms. 48. Such a ﬁrm would owe $82 in labor taxes (= 0.17 × $484), $278 in corporate taxes (= 0.35 × [$1,279 −$484]), and $99 in value-added taxes (= 0.125 × [$1,279 −$484]). Productivity of Unregistered Firms The image of unregistered ﬁrms that is consistent with their observed productivity is not that of predators but rather that of relics of the past. What accounts for the large difference in productivity between unregis- tered ﬁrms and the control group? We begin by running simple ordinary least squares (OLS) regressions and discuss self-selection issues later. In principle, the productivity differences that we document in tables 13 and 14 could be driven by industry effects, by differences in inputs (including human capital), or by differences in size. The goal of these regressions is to examine whether unregistered ﬁrms remain unusually unproductive after we control for these factors. In simple terms, we interpret the estimated coefficient on the unregistered dummy as a measure of our ignorance regarding the production function of unregistered firms. Omitting the unregistered dummy would not mean that unregistered ﬁrms are as pro- RAFAEL LA PORTA and ANDREI SHLEIFER 331 ductive as registered ones, but that differences in productivity are captured by differences in inputs, as in Rauch’s selection story.49 ductive as registered ones, but that differences in productivity are captured by differences in inputs, as in Rauch’s selection story.49 All speciﬁcations include dummy variables equaling 1 under the fol- lowing conditions: the ﬁrm is in the Informal Survey; the ﬁrm is registered and in the Informal Survey; the ﬁrm is in the Micro Survey; and the ﬁrm is registered and in the Micro Survey. Firms in the Enterprise Survey are the omitted category. We then add to the regression—one at a time—log income per capita, eight industry dummies, expenditure on raw materials, expenditure on energy, expenditure on machines, the index of manager education, and log sales.50 All three expenditure variables are scaled by the number of employees. Table 15 reports the results of OLS regressions in which log value added per employee is the dependent variable. Tables 16 and 17 show similar regressions for log sales and real output per employee, respectively. All three sets of results are qualitatively similar. We discuss the ﬁndings on value added in some detail and point out where the results for sales and real output per employee differ. 50. Errors are clustered at the country level. We do not include country ﬁxed effects since the frequency of unregistered ﬁrms in our sample may not reﬂect the incidence of unregistered ﬁrms in the population. 49. Rauch (1991). 49. Rauch (1991). 50 E l d h l l W d i l d ﬁ d ff Productivity of Unregistered Firms The ﬁrst regression reported in each table includes as independent variables only the dummies for whether the ﬁrm is in the Informal Survey sample or in the Micro Survey sample and the interactions between each of those two variables and whether the ﬁrm is registered. g The results conﬁrm the ﬁndings in tables 13 and 14. The estimated coef- ﬁcients in column 15-1 of table 15 are −1.78 for the Informal sample dummy and −1.29 for the Micro sample dummy. The coefficients for the interactions of the Informal and the Micro dummies with whether the ﬁrm is registered equal 0.81 and 0.35, respectively. All four dummies are highly statistically signiﬁcant. Adding GDP per capita to the regression (column 15-2) does not change the basic pattern. Similarly, the estimated coefﬁcients for the four dummies barely change as we add industry con- trols (column 15-3). The coefﬁcients do change when we add expenditure on raw materials: the estimated coefﬁcients for each of the four dummies are roughly cut in half (column 15-4). Adding expenditure on energy further lowers the estimated coefﬁcients on the four dummies, but not signiﬁcantly (column 15-5). The four coefﬁcients barely change as we add expenditure on machinery (column 15-6). The coefficients for expenditure on raw materials, energy, and machines are not only statistically signiﬁcant but also economically important. For example, increasing raw materials by Table 15. Productivity of Unregistered Firms Regressions Explaining Value Added per Employeea Regression Independent variable 15-1 15-2 15-3 15-4 15-5 15-6 15-7 15-8 Informal Survey dummy −1.7788* −1.7894* −1.8135* −0.8875* −0.7075* −0.6901* −0.5574* 0.1247 (0.1455) (0.1265) (0.1160) (0.1788) (0.1517) (0.1543) (0.1597) (0.1225) Informal Survey dummy 0.8077* 0.6241* 0.5906* 0.3032 0.1612 0.1705 0.1281 0.2948* × registered (0.2476) (0.1499) (0.1204) (0.1449) (0.1223) (0.1237) (0.1226) (0.0939) Micro Survey dummy −1.2910* −1.2810* −1.2488* −0.7925* −0.6626* −0.5711* −0.4746* 0.3720* (0.1464) (0.0963) (0.1035) (0.1127) (0.1256) (0.1218) (0.1133) (0.0660) Micro Survey dummy 0.3454* 0.3763* 0.3115* 0.1821* 0.1510* 0.1387* 0.0986 −0.0728 × registered (0.0368) (0.0436) (0.0330) (0.0350) (0.0467) (0.0418) (0.0398) (0.0527) Log of GDP per capita 0.3960* 0.4279* 0.3272 0.2985 0.2739 0.2665 0.0584 (at PPP) (0.1164) (0.1333) (0.1184) (0.1166) (0.0983) (0.0966) (0.0760) Log of expenditure on raw 0.2873* 0.2140* 0.2034* 0.1977* 0.0381 materials per employee (0.0509) (0.0491) (0.0489) (0.0483) (0.0392) Log of expenditure on 0.2059* 0.1909* 0.1834* 0.1093* energy per employee (0.0348) (0.0317) (0.0326) (0.0303) Log of expenditure on 0.0570* 0.0544* 0.0078 machines per employee (0.0102) (0.0100) (0.0112) Manager education 0.0986* −0.0231 (4 = attended college) (0.0292) (0.0160) Log of sales 0.4204* (0.0564) Industry dummies No No Yes Yes Yes Yes Yes Yes Constant 9.2615* 6.1996* 5.7435* 4.0303* 3.6547* 3.7987* 3.6445* 1.9736* (0.1389) (0.9186) (1.0810) (0.9428) (0.9646) (0.8459) (0.8393) (0.6059) Adjusted R2 (percent) 24.19 28.07 29.90 43.13 46.83 47.94 48.47 64.00 Source: Authors’ regressions. a. Results of ordinary least squares regressions on data from the 27 countries covered by the Informal and Micro Surveys. The dependent variable is the logarithm of value added per employee at purchasing power parity. The number of observations in all regressions is 8,478. Standard errors are clustered at the country level and reported in parentheses. Asterisks indicate statistical signiﬁcance at the 10 percent, 5 percent, and 1 percent level. Table 16. Productivity of Unregistered Firms Regressions Explaining Sales per Employeea Regression Independent variable 16-1 16-2 16-3 16-4 16-5 16-6 16-7 16-8 Informal Survey dummy −1.9768* −1.9861* −1.9875* −0.5355* −0.3742 −0.3625 −0.2536 0.3420 (0.1395) (0.1190) (0.1173) (0.1783) (0.1468) (0.1442) (0.1548) (0.1349) Informal Survey dummy 0.7707* 0.6036* 0.5618* 0.1077 −0.0215 −0.0155 −0.0501 0.0916 × registered (0.2493) (0.1560) (0.1211) (0.1797) (0.1615) (0.1601) (0.1620) (0.1069) Micro Survey dummy −1.4258* −1.4163* −1.3843* −0.6690* −0.5474* −0.4840* −0.4048* 0.3288* (0.1389) (0.1087) (0.1151) (0.1002) (0.1126) (0.1107) (0.1018) (0.0515) Micro Survey dummy 0.4048* 0.4335* 0.3677* 0.1634* 0.1350* 0.1265* 0.0937* −0.0554 × registered (0.0447) (0.0423) (0.0532) (0.0292) (0.0326) (0.0328) (0.0314) (0.0217) Log of GDP per capita 0.3685* 0.4094* 0.2533* 0.2271* 0.2101* 0.2040* 0.0233 (at PPP) (0.0903) (0.1081) (0.0762) (0.0736) (0.0605) (0.0590) (0.0420) Log of expenditure on raw 0.4536* 0.3835* 0.3759* 0.3713* 0.2300* materials per employee (0.0509) (0.0479) (0.0478) (0.0475) (0.0449) Log of expenditure on 0.1934* 0.1829* 0.1768* 0.1126* energy per employee (0.0331) (0.0313) (0.0322) (0.0273) Log of expenditure on 0.0400* 0.0380* −0.0031 machines per employee (0.0088) (0.0086) (0.0096) Manager education 0.0807* −0.0256 (4 = attended college) (0.0247) (0.0155) Log of sales 0.3675* (0.0485) Industry dummies No No Yes Yes Yes Yes Yes Yes Constant 10.0297* 7.1802* 6.6180* 3.8967* 3.5466* 3.6473* 3.5206* 2.0659* (0.1430) (0.7136) (0.8958) (0.6490) (0.6878) (0.6190) (0.6062) (0.3207) Adjusted R2 (percent) 28.95 32.20 34.72 68.86 70.06 70.60 70.94 82.59 Source: Authors’ regressions. a. Results of ordinary least squares regressions on data from the 27 countries covered by the Informal and Micro Surveys. The dependent variable is the logarithm of sales per employee at purchasing power parity. The number of observations in all regressions is 8,564. Standard errors are clustered at the country level and reported in parenthe- ses. Asterisks indicate statistical signiﬁcance at the 10 percent, 5 percent, and 1 percent level. Table 17. Productivity of Unregistered Firms Regressions Explaining Real Output per Employeea Regression Independent variable 17-1 17-2 17-3 17-4 17-5 17-6 17-7 17-8 Informal Survey dummy −3.9489* −3.9681* −3.9265* −1.6477* −1.3877* −1.3577* −1.0464* 0.3713 (0.2373) (0.1678) (0.1992) (0.2838) (0.2387) (0.2307) (0.2412) (0.1343) Informal Survey dummy 1.1202* 0.7732* 0.7754* 0.0627 −0.1455 −0.1301 −0.2290 0.1083 × registered (0.3540) (0.1764) (0.1682) (0.2821) (0.2535) (0.2387) (0.2425) (0.1122) Micro Survey dummy −3.1495* −3.1298* −3.1033* −1.9807* −1.7847* −1.6211* −1.3948* 0.3514* (0.2743) (0.1637) (0.1765) (0.1908) (0.2106) (0.2069) (0.1814) (0.0486) Micro Survey dummy 0.7682* 0.8279* 0.7794* 0.4588* 0.4130* 0.3911* 0.2974* −0.0575 × registered (0.0945) (0.0786) (0.0972) (0.0585) (0.0580) (0.0643) (0.0663) (0.0243) Log of GDP per capita 0.7650* 0.7877* 0.5428* 0.5006* 0.4567* 0.4393* 0.0093 (at PPP) (0.1502) (0.1856) (0.1343) (0.1311) (0.0987) (0.0945) (0.0399) Log of expenditure on raw 0.7118* 0.5989* 0.5794* 0.5662* 0.2297* materials per employee (0.0820) (0.0775) (0.0769) (0.0751) (0.0459) Log of expenditure on 0.3117* 0.2848* 0.2671* 0.1144* energy per employee (0.0538) (0.0494) (0.0515) (0.0284) Log of expenditure on 0.1032* 0.0973* −0.0005 machines per employee (0.0147) (0.0151) (0.0097) Manager education 0.2307* −0.0224 (4 = attended college) (0.0595) (0.0152) Log of sales 0.8749* (0.0505) Industry dummies No No Yes Yes Yes Yes Yes Yes Constant 16.7985* 10.8838* 10.4597* 6.1889* 5.6246* 5.8843* 5.5221* 2.0591*** (0.2667) (1.2225) (1.5417) (1.1699) (1.2260) (1.0445) (1.0103) (0.3085) Adjusted R2 (percent) 28.95 32.20 34.72 68.86 70.06 70.60 70.94 82.59 Source: Authors’ regressions. a. Results of ordinary least squares regressions on data from the 27 countries covered by the Informal and Micro Surveys. The dependent variable is the logarithm of real output per employee at purchasing power parity. The number of observations in all regressions is 8,564. Standard errors are clustered at the country level and reported in parentheses. Asterisks indicate statistical signiﬁcance at the 10 percent, 5 percent, and 1 percent level. 335 RAFAEL LA PORTA and ANDREI SHLEIFER 1 standard deviation is associated with a 43 percent increase in value added per employee.51 Similar increases in expenditure on energy and machines have somewhat smaller effects (32 and 16 percent, respectively). Coefﬁcients fall another notch when we add manager education to the regression (column 15-7). Interestingly, ignoring selection issues, the esti- mated coefﬁcient on manager education suggests that a top manager with some college education increases value added per employee by 27 percent (= 0.09 × 3) relative to a top manager with only some primary school edu- cation. 51. The standard deviations for raw materials, energy, and machines are 2.11, 1.66, and 2.83, respectively. Productivity of Unregistered Firms Finally, there is no evidence that unregistered ﬁrms are unusually unproductive once we control for log sales: the estimated coefﬁcients on both the Informal and the Micro dummies switch signs when we add log sales to the regression (column 15-8). In fact, the coefﬁcient on the Micro dummy is not only positive but also signiﬁcant. The interaction between the registration dummy and the Informal dummy is the only interaction term that remains statistically signiﬁcant. Again, the results on sales and real output per employee (tables 16 and 17, respectively) are very similar to those for value added. In the full speciﬁcation, the estimated coefﬁcients for both the Informal and the Micro dummies are positive and signiﬁcant. The interaction between the registered and the Informal dummies is insigniﬁcant, whereas that between the registered and the Micro dummies takes a small—but statistically signiﬁcant—negative value. Selection The OLS results in this section suggest that unregistered firms are not unusually unproductive once we take into account their expenditure on inputs, the human capital of their top managers, and their small size. Of course, these are all endogenous variables. In fact, a key distinguishing factor between the dual view and the other views of unregistered ﬁrms is the emphasis on the sorting process that matches able managers with good assets. High-quality managers are willing to pay taxes and bear the cost of government regulation in exchange for being able to advertise their prod- ucts, raise outside capital, and access public goods. In contrast, low-quality managers avoid taxes and regulations, since the beneﬁts of operating in the formal economy are less valuable for small ﬁrms. Table 18 examines the sorting process. Specifically, we examine the relationship between the quality of the ﬁrm’s assets and the human capital Table 18. Probit and OLS Regressions Investigating Manager Ability and Self-Selectiona Independent variables Dummy for highest level of education Pseudo-R2 attended by top manager Log of GDP No. of or R2 Dependent variable Secondary Vocational College per capita Constant observations (percent) F-testb Probit regressions Firm is registered with 0.2064* 0.2090* 0.4096* −0.0029 . . . 5,478 10.07 96.54* central government (0.0420) (0.0452) (0.0449) (0.0555) . . . Firm has ever had a −0.0250 0.0115 −0.0521 0.0415 . . . 3,763 2.75 2.81 commercial loan (0.0254) (0.0369) (0.0548) (0.0379) . . . Firm’s main customers 0.0361* 0.0349* 0.0323 0.0037 . . . 2,869 9.14 78.08* are large ﬁrms (0.0060) (0.0110) (0.0152) (0.0054) . . . Firm occupies a 0.0397 0.0954 0.0778 −0.0762* . . . 1,429 4.22 4.64 permanent structure (0.0468) (0.0434) (0.0538) (0.0265) . . . Firm is located in 0.0561* 0.0868 0.0076 −0.0258 . . . 1,439 2.31 36.87* owner’s house (0.0210) (0.0355) (0.0249) (0.0253) . . . Firm owns building 0.0847 0.0863 0.1118 0.0167 . . . 5,682 3.03 4.75 it occupies (0.0527) (0.0691) (0.0542) (0.0466) . . . Firm owns land it 0.0497 0.0352 0.0982 0.0197 . . . 11,760 5.82 6.11 occupies (0.0453) (0.0539) (0.0490) (0.0369) . . . Firm uses its own 0.0031 0.1265 0.1184* 0.0097 . . . 1,438 3.33 78.56* transportation (0.0510) (0.0574) (0.0284) (0.0216) . . . equipment Firm owns a generator 0.1280* 0.1390 0.3675* −0.1349* . . . 12,794 13.01 75.84* (0.0455) (0.0550) (0.0454) (0.0464) . . . Selection Firm uses e-mail to 0.1662* 0.2309* 0.4799* 0.1495 . . . 11,081 21.62 158.61* communicate with (0.0460) (0.0453) (0.0438) (0.0736) . . . clients Firm uses website to 0.1159* 0.1676* 0.2574* 0.1358 . . . 11,044 16.91 61.46* communicate with (0.0353) (0.0361) (0.0274) (0.0648) . . . clients Firm has electrical 0.1837* 0.1901* 0.2833* −0.0200 . . . 1,439 12.82 33.1* connection (0.0503) (0.0579) (0.0597) (0.0517) . . . OLS regressions Percent of investment 0.9852 −2.9315 −10.1707* −5.3383 111.3720* 13,006 5.10 35.06* ﬁnanced with (3.6083) (1.9982) (2.1024) (1.9468) (14.7773) internal funds Expenditure on raw 0.0378* 0.0338 0.0874* −0.0080 0.4461* 11,966 3.83 11.32* materials as percent (0.0130) (0.0147) (0.0193) (0.0154) (0.1340) of sales Expenditure on energy −0.0015 −0.0040 −0.0077 −0.0035 0.0747 12,546 1.72 1.78 as percent of sales (0.0039) (0.0041) (0.0047) (0.0040) (0.0302) Expenditure on machines −0.0047 0.0162* 0.0019 −0.0023 0.0432 12,577 3.26 7.91* as percent of sales (0.0040) (0.0052) (0.0041) (0.0046) (0.0361) Capacity utilization 0.6230 1.0456 5.4810* 1.5761 57.7676* 9,380 4.18 7.15*** (percent) (1.0430) (1.8476) (1.3208) (1.1737) (10.6062) Source: Authors’ regressions. a. Marginal effects of probit (top panel) or OLS (bottom panel) regressions on data from the 27 countries included in the Informal and Micro Surveys. All regressions include industry dummies. Robust standard errors are clustered at the country level and presented in parentheses. Asterisks indicate statistical signiﬁcance at the 10 percent, 5 percent, and 1 percent level. b. Test of the null hypothesis that true coefﬁcients on all three top-manager education dummies are zero. 338 Brookings Papers on Economic Activity, Fall 2008 of its top manager—our only proxy for managers’ ability. The depen- dent variables fall into two categories: dummy variables (top panel, which reports probit regressions) and continuous variables (bottom panel, OLS regressions). The dummy variables include indicators for whether the firm is registered; the firm has ever had a loan; the firm’s main cus- tomers are large firms; the firm occupies a permanent structure; the firm is located in the owner’s house; the firm owns the building it occupies; the firm owns the land it occupies; the firm uses its own transportation equipment; the firm owns a generator; the firm uses e-mail to communi- cate with clients; the firm uses a website to communicate with clients; and the firm has an electrical connection. Selection The five continuous variables are the percentage of investment that is financed internally; expenditure on raw materials as a fraction of sales; expenditure on energy as a fraction of sales; expenditure on machines as a fraction of sales; and capacity utilization. All regressions control for income per capita and include eight industry dummies. Many—but not all—of the correlations in table 18 are consistent with sorting on managers’ ability. Firms with more-educated managers are more likely to be registered, to sell mainly to large ﬁrms, to use their own transportation equipment, to own a generator, to communicate with clients through e-mail, to have a webpage, and to have an electrical connection. Many—but not all—of the correlations in table 18 are consistent with sorting on managers’ ability. Firms with more-educated managers are more likely to be registered, to sell mainly to large ﬁrms, to use their own transportation equipment, to own a generator, to communicate with clients through e-mail, to have a webpage, and to have an electrical connection. Along the same lines, managers who attended college are more likely to work for ﬁrms that own land and buildings. Firms with more-educated managers also use more raw materials and operate with higher capacity utilization. The economic signiﬁcance of these coefﬁcients is large. The probability of being registered increases by 41 percentage points if the top manager has some college education rather than only some primary school education. Having a top manager with some college education also has large effects on the probability of having a generator (+36.7 percentage points), the probability of using e-mail (+48.0 percentage points), the probability of having a webpage (+25.7 percentage points), and the probability of having an electrical connection (+28.3 percentage points). In contrast, the effect is moderate on the probability that the ﬁrm’s main buyers are large ﬁrms (+3.2 percentage points), the probability of owning a building (+11.2 per- centage points), the probability of owning land (+9.8 percentage points), and the probability of owning transportation equipment (+11.8 percentage points). Similarly, having a top manager with some college education increases expenditure on raw materials by a modest 8.7 percentage points (the standard deviation is 23.3 percent) and capacity utilization by 5.5 per- centage points (the standard deviation is 21.4 percent). RAFAEL LA PORTA and ANDREI SHLEIFER 339 The evidence on external ﬁnance is mixed. Selection On the one hand, ﬁrms with more-educated managers rely more on external ﬁnance (bottom panel of table 18). On the other hand, the education of managers does not signiﬁ- cantly affect the probability that the ﬁrm has ever had a loan (top panel). The evidence on investment in machines is also weak: in that regression the only signiﬁcant coefﬁcient is the one for vocational schooling. Nor is there evidence that expenditure on energy increases with managers’ education. Only one regression has statistically signiﬁcant coefﬁcients with the “wrong” sign: the likelihood that the ﬁrm operates in the house of the owner is higher when managers have attended secondary or vocational school rather than primary school only. These results suggest an explanation for the puzzlingly low productivity of unregistered ﬁrms. The productivity gap between registered ﬁrms and the control group disappears once we take into account crude proxies for physical and human capital and control for size. Of course, size is an endogenous variable. These results on manager selection are broadly con- sistent with the view that part of the reason that unregistered ﬁrms are small is that they are run by managers of low ability.52 These managers do not ﬁnd it worthwhile to pay the cost of running a formal ﬁrm. Unregistered ﬁrms are small because they are run by less able managers and, as such, face a high cost of capital, have few opportunities to advertise their prod- ucts, and are of insufﬁcient scale to own critical assets such as generators and computers. 52. Rauch (1991). Obstacles to Doing Business As a ﬁnal step, we present information on obstacles to doing business as reported by respondents in the Informal, Micro, and Enterprise Surveys. All obstacles are reported on a 0-to-4 scale for their perceived signiﬁcance, with 0 representing “no obstacle,” 1 “minor obstacle,” 2 “moderate obsta- cle,” 3 “major obstacle,” and 4 “very severe obstacle.” In table 19 we com- pare average responses about various obstacles for Informal Survey ﬁrms (top panel) and their Enterprise Survey counterparts, as well as for Micro Survey ﬁrms and their control group (bottom panel). Starting with the Informal Survey, the most striking ﬁnding is the simi- larity in many responses between the registered Informal Survey ﬁrms and the Enterprise Survey ﬁrms. Both groups consider tax rates and tax admin- istration their most signiﬁcant problems. Registered Informal Survey ﬁrms, Enterprise Survey Informal Survey sample control group Differenceb Enterprise v. Registered v. Small v. Big v. Obstaclea Unregistered Registered All Small Medium Big All informal unregistered unregistered small Access to or availability 2.05 2.38 2.07 . . . . . . . . . . . . . . . 0.33 . . . . . . Obstacles to Doing Business of markets Tax rates 1.59 2.14 1.65 2.13 2.33 2.50 2.33 0.68 0.55 0.54 0.37 Tax administration 1.40 2.05 1.46 1.79 2.14 2.37 2.05 0.59** 0.65* 0.39 0.58 Cost of ﬁnancing 2.19 2.37 2.25 1.99 2.25 2.30 2.20 −0.05 0.17 −0.20 0.31 Corruption 1.53 1.93 1.59 2.06 2.28 2.27 2.17 0.57 0.40 0.53 0.21 Macroeconomic instabilityc 1.75 1.98 1.80 1.89 2.05 2.13 1.95 0.15 0.23 0.14 0.23 Electricity supply 1.74 1.70 1.74 1.85 1.94 2.12 1.92 0.18 −0.04 0.11 0.27 Anticompetitive or unfair 1.74 2.16 1.78 1.74 1.98 2.11 1.94 0.16 0.42* 0.00 0.37 practices by other businesses Economic policy uncertainty 1.72 1.96 1.75 2.08 2.20 2.10 2.07 0.33 0.24 0.36 0.02 Customs and trade 1.00 1.51 1.06 1.24 1.61 2.09 1.53 0.46 0.51 0.25 0.85* regulations Access to ﬁnancing 2.29 2.46 2.32 1.95 1.92 1.83 1.83 −0.49 0.17 −0.33 −0.12 Legal system, conﬂict 1.04 1.33 1.07 1.10 1.47 1.78 1.24 0.17 0.29 0.06 0.67 resolution Labor regulations 0.84 1.20 0.91 0.99 1.27 1.75 1.17 0.26 0.36 0.15 0.76* Crime, theft, and disorder 1.48 1.61 1.49 1.59 1.76 1.71 1.57 0.07 0.12 0.11 0.12 Skills and education of 1.15 1.46 1.23 1.15 1.44 1.67 1.30 0.07 0.31 −0.01 0.52* available workers Transportationd 1.37 1.47 1.36 1.16 1.38 1.57 1.33 −0.04 0.11 −0.20 0.41 Procedures to register 1.26 1.64 1.49 1.20 1.21 1.42 1.12 −0.37 0.37 −0.06 0.23 ﬁrms, formalities, patents, etc. Telephone, fax, e-mail 1.00 0.84 0.99 0.85 0.94 1.32 0.99 0.00 −0.16 −0.15 0.47 Access to land 1.46 1.70 1.48 0.98 1.05 1.27 0.95 −0.53** 0.24 −0.47* 0.28 Postal services 0.07 0.00 0.06 . . . . . . . . . . . . . . . −0.07 . . . . . . Table 19. Obstacles to Doing Business in the Informal and Micro Survey Samples Index, 4 = very severe obstacle Sources: World Bank Informal, Micro, and Enterprise Surveys; authors’ calculations. a. All obstacles are reported on a 0-to-4 scale, with 0 indicating “no obstacle,” 1 “minor obstacle,” 2 “moderate obstacle,” 3 “major obstacle,” and 4 “very severe obstacle.” b. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and *1 percent level. c. High inﬂation, exchange rate instability, etc. d. Poor road quality, road blockages, difﬁculty ﬁnding ways to transport goods, etc. Enterprise Survey Micro Survey sample control group Difference Enterprise v. Registered v. Small v. Big v. Obstacles to Doing Business Obstaclea Unregistered Registered All Small Medium Big All micro unregistered unregistered small Electricity supply 1.96 1.99 1.98 2.24 2.43 2.69 2.30 0.32 0.03 0.27 0.45 Tax rates 1.35 1.69 1.59 1.75 1.90 1.84 1.78 0.19 0.34 0.40 0.09 Access to ﬁnancing 2.40 2.33 2.37 2.02 1.91 1.73 1.98 −0.39 −0.06 −0.37 −0.29 Skills and education of 0.51 0.63 0.60 0.92 1.17 1.64 1.02 0.41* 0.12 0.41* 0.72* available workers Macroeconomic instabilityc 1.38 1.67 1.63 1.47 1.51 1.53 1.50 −0.14 0.28 0.09 0.06 Tax administration 0.94 1.20 1.13 1.23 1.41 1.48 1.28 0.15 0.26 0.30* 0.25 Anticompetitive or unfair 1.54 1.43 1.47 1.40 1.46 1.48 1.41 −0.06 −0.12 −0.14 0.08 practices by other businesses Transportationd 1.34 1.30 1.31 1.23 1.25 1.40 1.25 −0.06 −0.04 −0.11 0.18 Corruption 1.09 1.06 1.07 1.20 1.46 1.37 1.27 0.20 −0.02 0.11 0.17 Crime, theft, and disorder 1.18 1.12 1.18 1.18 1.19 1.32 1.20 0.02 −0.06 0.01 0.14 Customs and trade 0.55 0.82 0.76 0.79 1.20 1.22 0.91 0.15 0.27* 0.24* 0.43* regulations Cost of ﬁnancing . . . . . . . . . 0.99 1.02 1.12 1.01 . . . . . . . . . 0.14 Procedures to register 1.22 1.11 1.10 1.00 1.08 1.00 1.01 −0.09 −0.12 −0.23 0.01 ﬁrms, formalities, patents, etc. Labor regulations 0.34 0.33 0.34 0.49 0.74 0.99 0.57 0.24 −0.01 0.15* 0.50* Legal system, conﬂict 0.38 0.44 0.43 0.55 0.68 0.98 0.60 0.17 0.07 0.17 0.43* resolution Economic policy uncertainty 0.88 0.92 0.96 1.02 1.11 0.96 1.05 0.08 0.04 0.14 −0.05 Access to land 1.44 1.14 1.22 1.06 0.90 0.89 1.02 −0.20* −0.29 −0.38** −0.17 Telephone, fax, e-mail 0.56 0.71 0.67 0.70 0.81 0.86 0.74 0.08 0.15 0.14 0.16 Sources: World Bank Informal, Micro, and Enterprise Surveys; authors’ calculations. a. All obstacles are reported on a 0-to-4 scale, with 0 indicating “no obstacle,” 1 “minor obstacle,” 2 “moderate obstacle,” 3 “major obstacle,” and 4 “very severe obstacle.” b. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. c. High inﬂation, exchange rate instability, etc. d. Poor road quality, road blockages, difﬁculty ﬁnding ways to transport goods, etc. 342 Brookings Papers on Economic Activity, Fall 2008 like Enterprise Survey ﬁrms, regard the cost of ﬁnancing and access to ﬁnancing as major obstacles as well. 53. Among big ﬁrms, concern over “anticompetitive or informal practices” ranks after concerns over tax rates, tax administration, cost of ﬁnancing, corruption, macroeconomic instability, and electricity. On the other hand, it ranks ahead of, among other things, con- cerns over economic policy uncertainty, customs and trade regulations, access to ﬁnancing, and crime, theft, and disorder. Obstacles to Doing Business Neither the Informal Survey ﬁrms nor the Enterprise Survey ﬁrms consider access to land, registration procedures, crime, low workforce skills, labor regulations (with the exception of big ﬁrms), or the legal system to be major obstacles to doing business (again with the exception of big ﬁrms). There are some signiﬁcant differences as well. Informal Survey ﬁrms consider access to or availability of markets to be a huge problem. The unregistered Informal Survey ﬁrms do not consider taxes or tax adminis- tration to be a huge problem, in obvious contrast to the registered ﬁrms. Corruption is a smaller problem for the unregistered ﬁrms than for the reg- istered ones. Indeed, both tax administration and corruption are perceived as more serious obstacles by big ﬁrms than by small ones (but only differ- ences in the perception of tax administration as an obstacle are statistically signiﬁcant). We can also use the information on obstacles to shed light on the para- site theory of the informal economy. Unfortunately, the question asked in the surveys is not ideal. Respondents assess on a 0-to-4 scale whether “anticompetitive and informal practices” are an obstacle to their business. Of course, anticompetitive practices can come not only from the informal firms, but also from formal firms with political or other connections. Nevertheless, several points emerge from these data. First, contrary to the parasite view, “anticompetitive and informal” practices are not among the key obstacles perceived by managers of ﬁrms in either the Informal Survey ﬁrms (the average score is 1.78) or their Enterprise Survey control group (1.94).53 Second, the answer is only slightly higher for the Enterprise Survey ﬁrms than for the Informal Survey ﬁrms, which is not consistent with the view that the informal ﬁrms undercut the formal ones. Third, one might have guessed that it is the small registered ﬁrms in the Enterprise Survey that would be most severely affected by the informal ﬁrms. How- ever, these ﬁrms perceive anticompetitive and informal practices to be a smaller problem, on average, than do the larger ﬁrms. None of this evidence is supportive of the parasite theory. The patterns in the Micro Survey are similar to those in the Informal Survey (except that some of the questions differ). Access to ﬁnancing and electricity emerge as by far the greatest RAFAEL LA PORTA and ANDREI SHLEIFER 343 obstacles to Micro Survey ﬁrms. y p a. Advantages are rated on a scale from 1 (“minor advantage”) to 4 (“very important”). Obstacles are rated on a 0-to-4 scale, with 0 indicating “unimportant,” 1 “minor obstacle,” 2 “moderate obstacle,” 3 “major obstacle,” and 4 “extremely important.” Sources: World Bank Informal Survey for Cape Verde; authors’ calculations. Obstacles to Doing Business These are also huge obstacles for their counterpart Enterprise Survey firms, along with tax rates. Finally, anti- competitive and informal practices are not among the top obstacles for ﬁrms in the Micro Survey. A ﬁnal piece of evidence comes from the Informal Survey, which only in Cape Verde asked respondents about the beneﬁts of and obstacles to registering. The ﬁndings are summarized in table 20. The main beneﬁts of registering are improved access to markets, to services, and to ﬁnancing— ﬁndings broadly consistent with the previous ﬁndings about the obstacles to doing business faced by informal ﬁrms. Better property rights and lower need to pay bribes are not nearly as important. On the cost side, the main obstacles to registration are taxes and the cost of registering (along with Table 20. Advantages and Obstacles to Registering in Cape Verde Table 20. Advantages and Obstacles to Registering in Cape Verde Table 20. Advantages and Obstacles to Registering in Cape Verde Percent of ﬁrms rating the advantage as very important or the obstacle as either major Advantage or obstacle or extremely importanta Advantages Better access to markets 44 Better access to services 39 Better access to ﬁnancing 39 Better access to raw materials 34 Easier to bargain with formal enterprises 25 Easier to reduce theft by employees or others 23 Better access to government subsidies 20 More solid legal basis for property rights regarding 20 real estate Less turnover of employees or better product market 18 competition Less need to pay bribes 15 Obstacles Financial burden of taxes applicable to registered ﬁrms 43 Cost of registering 38 Difﬁculties in obtaining information about how 36 to register Minimum capital legally required to register 32 Administrative burden of complying with tax laws 32 Time necessary to register 19 Labor regulations applicable to registered ﬁrms 19 Other administrative burdens 18 S W ld B k I f l S f C V d th ’ l l ti Other administrative burdens 344 Brookings Papers on Economic Activity, Fall 2008 the difﬁculty of obtaining information about how to register). Labor regu- lation and tax compliance are seen as much less important. Here as well, the picture that emerges is one in which the formal ﬁrms have better access to markets, services, and ﬁnance, and hence can be much more productive, but need to pay taxes. Obstacles to Doing Business Presumably, for the Cape Verde ﬁrms in the Infor- mal Survey, the tax price is too high to justify registration. The evidence on obstacles further supports the dual theory and seems rather inconsistent with the parasite theory. Between their extreme inefﬁ- ciency and their operation in very different markets, informal firms do not appear to pose much of a threat to formal firms, at least as perceived by the latter. Informal firms clearly recognize the many benefits of being official, including access to markets and to finance (although it is far from clear that they would gain the latter even if they registered). They do not seem to think that regulation and the cost of registration are the biggest obstacles to registration. On the other hand, they do see taxes as a huge problem. In this respect the results are consistent with the dual theory, as well as with the findings reported in the first section and by Djankov and coauthors.54 54. Djankov and others (2008b). 55. See also Lewis (2004); Banerjee and Duﬂo (2005). j 55. See also Lewis (2004); Banerjee and Duﬂo (2005). 54. Djankov and others (2008b). Conclusion Our most basic ﬁnding is that high productivity comes from formal ﬁrms, and in particular from large formal ﬁrms. Productivity is much higher in small formal ﬁrms than in informal ﬁrms, and it rises rapidly with the size of formal ﬁrms. To the extent that productivity growth is central to eco- nomic development, the formation and growth of formal ﬁrms are neces- sary for economic growth.55 Formal ﬁrms appear to be very different animals from informal ﬁrms, and this fact accounts for their sharply superior productivity. Perhaps most important, they are run by much better educated managers. As a conse- quence, besides being larger, they tend to use more capital, have different customers, and market their products and use external ﬁnance to a greater extent than do informal ﬁrms. There is no evidence that informal ﬁrms tend to become formal as they grow. Rather, virtually none of the formal ﬁrms in our sample had ever been informal. Consistent with this result, Miriam Bruhn shows that business registration reform had a large effect on new registrations in Mexico, but that the new ofﬁcial entrants were former 345 RAFAEL LA PORTA and ANDREI SHLEIFER wage earners rather than informal entrepreneurs.56 Similarly, Mondragón- Vélez and Peña-Parga find surprisingly little transition between self- employment and business ownership in Colombia.57 It does not appear from the available evidence that informal ﬁrms would sharply increase their productivity if only they registered. This interpretation raises the crucial question of what happens to infor- mal ﬁrms as the economy develops. After all, the most basic fact about the informal economy is that its role diminishes sharply as incomes grow. How does this happen? Do informal firms register or do they die? We do not have a definitive answer to this question, but the evidence we have points in the direction of death rather than registration. It is still possible, of course, that a minority of informal ﬁrms, and especially the most produc- tive ones, end up joining the formal economy, perhaps by supplying formal ﬁrms. But there is no evidence, at least in our data, that this is the typical story. The vast majority of informal ﬁrms appear to begin and end their lives as unproductive informal ﬁrms. 56. Bruhn (2008). 57. Mondragón-Vélez and Peña-Parga (2008). Conclusion Informal ﬁrms nonetheless play a crucial role in developing economies, where they represent perhaps 30 to 40 percent of all economic activity and provide a livelihood to billions of poor people. Because these ﬁrms are so inefﬁcient, taxing them or forcing them to comply with government regula- tions would likely put most of them out of business, with dire consequences for their employees and proprietors. If anything, strategies that keep these ﬁrms aﬂoat and allow them to become more productive, such as micro- ﬁnance, are probably desirable from the viewpoint of poverty alleviation. But these are not growth strategies: turning these unofﬁcial ﬁrms into ofﬁ- cial ones is unlikely to generate substantial improvements in productivity. Growth strategies, rather, need to focus on formal ﬁrms, especially the larger ones. Reducing the costs of formality, such as registration costs, is surely a good idea, but this is not the whole story. Likewise, some of the almost-standard proposals for development, such as improving land rights, the legal environment, and even the human capital of employees appear to address relatively minor factors, at least from the viewpoint of ofﬁcial entrepreneurs. The main obstacles to the operations of formal ﬁrms, according to our data, are three: taxation, uncertain supply of electricity, and lack of adequate access to ﬁnance. To us, the most striking ﬁnding is the sharply higher education of man- agers of ofﬁcial than of unofﬁcial ﬁrms, with no corresponding difference 346 Brookings Papers on Economic Activity, Fall 2008 in the human capital of the employees. This suggests that educational policies, particularly those emphasizing secondary education, might be conducive to the formation of entrepreneurial talent that can run formal ﬁrms. We do not mean to suggest that formal education is either a necessary or a sufﬁcient condition for entrepreneurial skills. But the data seem to indicate quite clearly that some aspects of management (for example, mar- keting and ﬁnance) require education. One can also think of other sources of human capital, such as immigration, as supplying the required entrepre- neurial talent. There is growing evidence that corporate income taxation deters invest- ment and formal entrepreneurship. Using a new dataset of corporate income taxes in a large number of countries, Djankov and coauthors ﬁnd strong evidence that these taxes reduce investment, foreign direct investment, and entrepreneurial activity.58 Our evidence similarly shows that ofﬁcial ﬁrms perceive taxation as the top obstacle to doing business. 59. See La Porta, Lopez-de-Silanes, and Shleifer (2008) for a survey. 58. Djankov and others (2008b). 60. Banerjee and Duﬂo (2005). Conclusion To the extent that the formation and growth of official firms are the principal engines of development, this perception must be taken seriously. Needless to say, one needs to also think about alternative sources of public ﬁnance, as well as the size of government, in developing countries to determine whether cor- porate income tax cuts are warranted. But the evidence points to a poten- tially serious problem. The evidence also suggests that ofﬁcial ﬁrms, just like unofﬁcial ones, perceive lack of access to ﬁnance to be a serious obstacle to doing business. Recent research has pointed to a broad range of legal and regulatory reforms that can underpin the development of ﬁnancial markets; in general these reforms seek to improve the legal rights of creditors and (in the case of very large ﬁrms) shareholders.59 Unlike with tax cuts, there seem to be no compelling counterarguments to improving the laws and institutions that support ﬁnancial markets. Finally, the evidence indicates that problems with electricity supply, including disruptions, afﬂict unofﬁcial as well as smaller ofﬁcial ﬁrms. This contrasts with an interesting lack of concern on the part of respondents with other limitations of infrastructure, such as transport, telephone, and mail. Most large ﬁrms have their own generators, whereas smaller ofﬁcial ﬁrms and unofﬁcial ﬁrms do not and hence are more vulnerable. The overall picture of economic development that emerges from this analysis is in many ways similar to the traditional pre-growth theory devel- RAFAEL LA PORTA and ANDREI SHLEIFER 347 opment economics, although it is related to the modern reformulations of economic growth through the lens of development economics.60 The recipe for productivity growth is the formation of ofﬁcial ﬁrms—the larger and the more productive, the better. Their formation must perhaps be promoted through tax, human capital, infrastructure, and capital markets policies, very much along the lines of traditional dual economy theories. From the perspective of economic growth, one should not expect much from the unofﬁcial economy, with its millions of entrepreneurs, except to hope that it disappears over time. This “Wal-Mart” theory of economic development receives quite a bit of support from ﬁrm-level data. ACKNOWLEDGMENTS We are grateful to Nicholas Coleman for excel- lent research assistance, to Jorge Rodriguez Mesa for help with the World Bank surveys, and to Charles Jones, Peter Klenow, James Rauch, Jeremy Stein, and William Nordhaus for helpful comments. This research was supported by the Kauffman Foundation. Conclusion of paved roads −0.28* −0.11 −0.24*** −0.22* −0.20 0.29* −0.18 −0.09 −0.33* −0.03 er km2 ruption index −0.84* −0.32* −0.67* −0.58* −0.71* 0.48* −0.57* −0.23* −0.43*** −0.26* e of law index −0.85* −0.29* −0.68* −0.58* −0.72* 0.49* −0.59* −0.26* −0.44* −0.29* ate credit as percent −0.67* −0.28* −0.49* −0.38* −0.60* 0.43* −0.43*** −0.18* −0.31*** −0.25* f GDP ket capitalization as −0.54* −0.16 −0.39* −0.37* −0.51* 0.21* −0.38* −0.09 −0.36* −0.19 ercent of GDP of GDP per capita −0.77* −0.34* −0.77* −0.66* −0.66* 0.52* −0.45* −0.27* −0.30* −0.13 Steps Index of Index of Nonwage required to Log of Private Market difﬁculty difﬁculty costs as collect on Efﬁciency of paved Rule credit capitalization of hiring a of ﬁring percent a bounced bankruptcy roads Corruption of law as percent as percent iable new worker a worker of salary check procedure per km2 index index of GDP of GDP ex of difﬁculty of 0.36* ring a worker wage costs as 0.21* 0.22* ercent of salary ps required to collect 0.47* 0.38* 0.21* n a bounced check ciency of bankruptcy −0.37* −0.15 −0.10 −0.56* rocedure of paved roads −0.12 −0.15* −0.13 −0.26* 0.29* er km2 ruption index −0.23* −0.21* 0.08 −0.37* 0.76* 0.29* e of law index −0.25* −0.25* 0.08 −0.39* 0.76* 0.31* 0.96* ate credit as percent −0.26* −0.21* −0.03 −0.39* 0.61* 0.27* 0.74* 0.75*** f GDP ket capitalization as −0.24* −0.17* −0.08 −0.51* 0.52* 0.46* 0.62* 0.59* 0.71* ercent of GDP of GDP per capita −0.25* −0.22* 0.21* −0.22 0.73* 0.27* 0.83* 0.84* 0.70* 0.52* urce: Authors’ calculations. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. Conclusion Correlations among the Main Variablesa C l ti fﬁi t Index of difﬁculty of 0.36* ﬁring a worker Nonwage costs as 0.21* 0.22* percent of salary Steps required to collect 0.47* 0.38* 0.21* on a bounced check Efﬁciency of bankruptcy −0.37* −0.15 −0.10 −0.56* procedure Log of paved roads −0.12 −0.15* −0.13 −0.26* 0.29* per km2 Corruption index −0.23* −0.21* 0.08 −0.37* 0.76* 0.29* Rule of law index −0.25* −0.25* 0.08 −0.39* 0.76* 0.31* 0.96* Private credit as percent −0.26* −0.21* −0.03 −0.39* 0.61* 0.27* 0.74* 0.75*** of GDP Market capitalization as −0.24* −0.17* −0.08 −0.51* 0.52* 0.46* 0.62* 0.59* 0.71* percent of GDP Log of GDP per capita −0.25* −0.22* 0.21* −0.22 0.73* 0.27* 0.83* 0.84* 0.70* 0.52* Source: Authors’ calculations. a. Asterisks indicate statistically signiﬁcantly different from zero at the 10 percent, 5 percent, and 1 percent level. b. Calculated as 1 minus the share of sales reported for tax purposes. Conclusion Table A1. Correlations among the Main Variables Correlation coefﬁcients Informal Self- Registered Procedures Hours per share of employed ﬁrms necessary Taxes as year needed Management GDP from Tax as share of Electricity Multiple per 1,000 to start percent to comply time dealing Variable WEF survey evasionb labor force consumption indicators population a business of proﬁts with taxes with regulations Tax evasionb 0.25 Self-employed as share 0.70* 0.25 of labor force Informal share of GDP, 0.61* 0.35 0.61* from electricity Informal share of GDP, 0.70* 0.17 0.47* 0.71* multiple indicators Registered ﬁrms per −0.43* −0.16 −0.46* −0.21 −0.38* 1,000 population Procedures necessary 0.48* 0.32* 0.37* 0.30 0.37* −0.50*** to start a business Taxes as percent of proﬁts 0.16* 0.17* −0.04 −0.13 0.16* −0.19* 0.15* Hours per year needed to 0.50* −0.02 0.34* 0.42* 0.37* −0.42* 0.38* 0.22* comply with taxes Management time dealing 0.43* 0.14 0.26** 0.24 0.20* −0.06 0.15 0.00 0.20* with regulations Index of difﬁculty of 0.32* 0.05 0.10 0.13 0.34* −0.10 0.26* 0.11 0.26* 0.24 hiring a new worker Index of difﬁculty of 0.23 0.01 0.14 0.08 0.20 −0.25 0.27* 0.20 0.32* 0.28* ﬁring a worker Nonwage costs as percent −0.10 −0.16 −0.26* −0.20 −0.08 0.05 −0.01 0.18 0.27* 0.02 of salary Steps required to collect 0.36* 0.09 0.25 0.30 0.39* −0.18 0.45* 0.27* 0.36* 0.46* on a bounced check Efﬁciency of bankruptcy −0.64* −0.32 −0.59* −0.46* −0.57* 0.35* −0.54* −0.15 −0.37* −0.35* procedure Informal share of GDP estimated from Informal Self- Registered Procedures Hours per share of employed ﬁrms necessary Taxes as year needed Management GDP from Tax as share of Electricity Multiple per 1,000 to start percent to comply time dealing able WEF survey evasionb labor force consumption indicators population a business of proﬁts with taxes with regulations Informal share of GDP estimated from A P P E N D I X Table A1. 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https://openalex.org/W4319298228	https://trialsjournal.biomedcentral.com/counter/pdf/10.1186/s13063-023-07094-6	English	null	Can multisite clinical trial results change clinical practice? Use of long-acting injectable risperidone nationally in the Veterans Health Administration	Trials	2,023	cc-by	8,191	Trials Trials Trials Rosenheck et al. Trials (2023) 24:85 https://doi.org/10.1186/s13063-023-07094-6 Open Access Can multisite clinical trial results change clinical practice? Use of long‑acting injectable risperidone nationally in the Veterans Health Administration Robert Rosenheck1,2* , Sonia T. Anand3, Stephen G. Kurtz3, Cynthia Hau3, Diane Smedberg3, James F. Pontzer4,5, Ryan E. Ferguson3,6 and Cynthia R. Davis7 Abstract Background Multisite practical clinical trials evaluate treatments in real-world practice. A multisite randomized Veterans Health Administration (VHA) cooperative study (CSP#555) published in 2011 compared the first long-acting injectable (LAI) second-generation antipsychotic (SGA), Risperidone Consta®, in veterans with a diagnosis of schizo- phrenia or schizoaffective disorder, to oral antipsychotics, with unexpected null results for effectiveness and cost- effectiveness. Whether null results of this type could change VHA practice has not been studied. Methods A longitudinal observational analysis was used to evaluate the impact of the trial findings on VHA clinical practices. National administrative data compared new starts on LAI risperidone during the 4 years before the publica- tion of CSP#555 in 2011 to new starts on LAI risperidone during the 4 years after. Results Among 119,565 Veterans with the indicated diagnoses treated with antipsychotics from 2007 to 2015, the number and proportion of new starts on LAI risperidone declined significantly following the study publication, as did the total number of annual users and drug expenditures. However, data from 2007 to 2010 showed the decline in new starts actually preceded the publication of CSP#555. This change was likely explained by the increase in new starts, total use, and expenditures on a newer medicine, LAI paliperidone, a 4-week LAI treatment, in the 2 years prior to the publication of CSP#555. Conclusions The declining use of LAI risperidone likely primarily reflects the substitution of a longer-acting LAI SGA, paliperidone, that came to market 2 years before the study publication, a substitution that may have been reinforced by null CSP#555 study results for LAI risperidone. Keywords Clinical trial impact, Long-acting antipsychotic medication, Schizophrenia, Dissemination rial impact, Long-acting antipsychotic medication, Schizophrenia, Dissemination of research 6 Boston University School of Medicine, Boston, MA, USA 7 Harvard Medical School, Boston, MA, USA 5 VA Office of Research and Development, Cooperative Studies Program, Albuquerque, NM, USA 6 Boston University School of Medicine, Boston, MA, USA 7 Harvard Medical School, Boston, MA, USA Background Since the 1940s, the Veterans Health Administration (VHA), the largest healthcare system in the USA, has committed itself to the conduct of such trials through its Cooperative Studies Program (CSP) which maintains a unique infrastructure of coordinating centers within the VHA to support comparative effectiveness research across more than 150 Veterans Affairs (VA) medical centers nationwide for the specific purpose of improving treatment in VHA [4]. The program has been responsible for landmark studies of antibiotics for tuberculosis, anti- hypertensive medications, antipsychotics for schizophre- nia, and many other major studies to “produce innovative and effective solutions to Veteran and national healthcare problems” [4]. In spite of this clear objective, there has been limited evaluation of the impact of individual VA cooperative studies on clinical practice in the VA healthcare system. There has long been concern about the often prolonged gap between published scientific findings and changes in clinical practice [5, 6]. Limited attention has been paid to the naturalistic dissemination of trial findings, such as those from the VA CSP, into clinical practice, especially in the healthcare systems that sponsored the research and in which it was conducted. The current study sought to trace the impact of CSP#555, an important null study, on VA clinical prac- tice by using national VHA administrative data to com- pare trends in the use of LAI risperidone and other types of antipsychotic medications by Veterans diagnosed with schizophrenia or schizoaffective disorder during the 4 years (2007–2010) before the publication of CSP#555 in 2011 and the 4 years after (2012–2015). As the paper was published in early March 2011, we focused on differences in the years before and after 2011 in the annual propor- tions of Veterans prescribed LAI risperidone for the first time (new starts) and in the proportions of Veterans receiving LAI risperidone at all (any use) using multivari- ate analysis to control for potentially confounding soci- odemographic and clinical characteristics. To understand contextual factors, we also examined changes in the use of other antipsychotics during these years including both LAI and oral medications, as well as changes in VA Schizophrenia, the focus of this study, is perhaps the most disabling of psychiatric conditions affecting approx- imately 1% of all adults and almost 100,000 VA patients each year at a cost of thousands of dollars per patient [7, 8]. Background in recent formulations for 4 weeks or even 6 months [11]. LAI medications are thought to reduce medication non- adherence but meta-analyses have yielded mixed results on the effectiveness of these medications compared to oral drugs [12, 13] albeit with more positive results in a recent meta-analysis, which highlighted pre-post studies that claimed to better reflect the real-world practices of prescribing LAI antipsychotics [14]. Short-term placebo-controlled randomized clinical trials (RCTs), conducted to obtain Food and Drug Administra- tion (FDA) approval for commercial marketing of new drugs, do not provide adequate information for deter- mining whether the longer-term effectiveness and safety of such medications merit widespread replacement of older, often less expensive, drugs already on the market [1]. Practical clinical trials or comparative effectiveness studies compare new drugs to widely used comparators at multiple sites, under longer-term, real-world clinical treatment conditions, with minimal exclusion criteria. These studies have become recognized as critical for the evaluation of health benefits of new medications and for fostering their widespread use [2, 3]. p g p y Beginning in the 1990s, a new type of antipsychotic medication, second-generation antipsychotics (SGAs), emerged which had fewer neurological side effects than previously approved drugs, i.e., first-generation antip- sychotics (FGAs). It was hoped they would thereby lead to greater acceptance and more consistent use of these medications, but, in fact also had mixed results [15, 16]. It was only in 2003 that the first LAI SGA, LAI risperi- done (Risperdal Consta®) became available, combining the benefits of LAI administration with the reduced neu- rological side effects of SGAs. As only 11% of diagnosti- cally appropriate VA patients [17] and 10–20% of non-VA patients [18] were prescribed LAI medications, it was of substantial importance to evaluate the potential benefits of this new treatment. In 2005, the VA CSP implemented a multisite study comparing LAI risperidone to provid- ers’ choice of oral antipsychotic medication (CSP#555) to evaluate these potential benefits before the drug entered widespread use. Results reported 6 years later found no significant reductions in hospitalization rates (the pri- mary outcome) after a year of LAI risperidone treatment and no differences in secondary outcomes including schizophrenia symptoms, major side effects, quality of life, or other health outcomes [19]. The study was thus designed to address a critical question that has not been well studied: did this costly treatment merit widespread use? This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2023. 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://creativeco mmons.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. Rosenheck et al. Trials (2023) 24:85 Rosenheck et al. Trials (2023) 24:85 Page 2 of 11 Background A major challenge in the treatment of schizophrenia is that many patients lack insight into their condition and are inconsistent in their use of prescribed medications or refuse them, leading to recidivism and costly rehospitali- zation [9, 10]. One tool to improve medication adherence in the treatment of schizophrenia is the use of long-acting injectable (LAI) antipsychotic medications—medications administered via intramuscular injection releasing active medication slowly into the bloodstream, yielding thera- peutic blood levels for 2 weeks after each injection, and Rosenheck et al. Trials (2023) 24:85 Page 3 of 11 Page 3 of 11 expenditures for LAI risperidone and other antipsychot- ics in this population. The original protocol noted that a null result could justify systemwide discouraging of LAI risperidone use in VHA as it was many times more expensive than other approved antipsychotic medica- tions [20]. Accordingly, we hypothesized that in the years following the publication of the trial use of LAI risperi- done would decline and be associated with reduced costs for LAI risperidone and for antipsychotics generally in the VHA. risperidone for each year, as well as for the other classes of antipsychotics noted above: LAI SGAs, LAI FGAs, and FGA and SGA oral medications. The proportion of aver- age costs attributable to LAI SGAs was also calculated for the entire population for each year. expenditures for LAI risperidone and other antipsychot- ics in this population. The original protocol noted that a null result could justify systemwide discouraging of LAI risperidone use in VHA as it was many times more expensive than other approved antipsychotic medica- tions [20]. Accordingly, we hypothesized that in the years following the publication of the trial use of LAI risperi- done would decline and be associated with reduced costs for LAI risperidone and for antipsychotics generally in the VHA. Sociodemographic characteristics derived from VHA EHR, included age, gender, race, marital status, and VA service-connected disability status. Comorbid psychi- atric diagnoses clustered into 12 groups included alco- hol abuse or dependence (ICD-9 303.xx or 305.00), drug abuse or dependence (292.01, 292.99, 304.xx, or 305.20–305.99), other psychoses (297.xx-299.xx), bipolar disorder (296.0x, 296.1x, or 296.40–296.89), major affec- tive disorder (296.2–296.39), dysthymia (300.4x, 296.9x, 311.xx, 301.10–301.19), posttraumatic stress disorder (309.81), anxiety disorders (300.xx excluding 300.4x), adjustment disorder (309.xx excluding 309.81), personal- ity disorder (301.0x), and any other psychiatric disorder excluding the above (290.00–312.99 excluding 305.1). Background Traumatic brain injury was identified using detailed codes published elsewhere [21]. Design and sample Data used in the present longitudinal observational analysis were derived from the national VHA electronic health records (EHRs) captured by the VA Cooperate Data Warehouse, which document sociodemographic, inpatient and outpatient services use, clinical diagnoses, and VA prescription fills for all Veterans treated by VHA clinics. The study population included all VHA patients who received an ICD-9 diagnosis of schizophrenia or schiz- oaffective disorder (295.00 to 295.95) in an inpatient discharge abstract or outpatient clinic visit in the years 2007–2015. Veterans were represented as a unique obser- vation for each year in which they received any antipsy- chotic medication regardless of whether they received a diagnosis of schizophrenia or schizoaffective disorder in that particular year. VHA service use was represented by the average num- ber of annual outpatient mental health visits for each Veteran (VA clinic stop codes 500–599), the presence of a discharge abstract for a hospitalization with a diagnostic code for schizophrenia or schizoaffective disorder, and participation in specialized VA mental health intensive case management programs, designed to treat Veterans with the most severe of mental illnesses [22]. Analysis plan D d Trials (2023) 24:85 Page 4 of 11 Rosenheck et al. Trials Page 4 of 11 Rosenheck et al. Trials (2023) 24:85 Fig. 1 Proportion of new starts on LAI risperidone, LAI paliperidone, and other LAI SGAs: 2007–2015 Fig. 1 Proportion of new starts on LAI risperidone, LAI paliperidone, and other LAI SGAs: 2007–2015 Fig. 1 Proportion of new starts on LAI risperidone, LAI paliperidone, and other LAI SGAs: 2007–2015 These analyses were repeated for any use of LAI risp- eridone, i.e., not just first starts, and other antipsychotics to assess changes and differences in the ongoing use of LAI risperidone and other antipsychotics. an asterisk on the tables). Potentially confounding soci- odemographic and diagnostic covariates (described ear- lier), representing possible confounding changes in the characteristics of the study population from year to year, were added through a stepwise procedure with forward selection. While these analyses identified significant dif- ferences in new starts on LAI risperidone for each year, as compared to 2011 (with significance marked by “^” on the tables), a more detailed evaluation of the statisti- cal significance of differences between adjacent pairs of years was determined by comparing the odds ratio (OR) for any given year with the 95% confidence interval (CI) of the OR from the year before (with significance marked by “#” on the tables). If the OR for a given year was out- side of the 95% CI of the previous year, it represented a statistically significant difference. an asterisk on the tables). Potentially confounding soci- odemographic and diagnostic covariates (described ear- lier), representing possible confounding changes in the characteristics of the study population from year to year, were added through a stepwise procedure with forward selection. While these analyses identified significant dif- ferences in new starts on LAI risperidone for each year, as compared to 2011 (with significance marked by “^” on the tables), a more detailed evaluation of the statisti- cal significance of differences between adjacent pairs of years was determined by comparing the odds ratio (OR) for any given year with the 95% confidence interval (CI) of the OR from the year before (with significance marked by “#” on the tables). If the OR for a given year was out- side of the 95% CI of the previous year, it represented a statistically significant difference. Analysis plan D d p p y Total annual VHA expenditure data were examined through random effects mixed models that compared average expenditures for LAI risperidone and other antipsychotics in each year to the reference year (2011), again adjusting for potential confounders. Expenditures for LAI risperidone were compared across the years, again with 2011 as the reference year. Costs for other LAI SGAs per Veteran were also examined along with the proportion of all antipsychotic expenditures attributable to LAI SGAs to determine the total budgetary impact of the observed changes on prescribed medications. Analysis plan D d Descriptive data on Veteran characteristics and antipsy- chotic medication use are presented for all unique Vet- erans in the study sample from 2007 to 2015. Those with data from 2007 to 2010 and those with data from 2012 to 2015 are compared with those from the reference year, 2011. Random effects mixed models were used to adjust statistical comparisons for the correlatedness of data from individuals who were included in both time periods.h The VA outpatient pharmacy records were used to iden- tify all prescriptions for antipsychotic medications from the study population. Measures were constructed to identify each year in which a Veteran had a new start on LAI risperidone and on other LAI SGAs, i.e., a year in which they had not filled a prescription for that medica- tion in the previous year. New starts were thus defined as the first year a prescription for LAI risperidone or other LAI SGAs was filled between 2007 and 2015 and were assumed to represent physician decision-making based on current knowledge, i.e., unaffected by legacy prescription decisions. Measures were also constructed representing years in which any annual LAI risperidone prescription fills were recorded, i.e., not necessarily the first, as well as prescriptions for (1) other LAI SGAs, (2) LAI FGAs, and (3) FGA and SGA oral antipsychotics. These analyses incorporate legacy prescription decisions, less likely to be influenced by recent research. The proportion of Veterans in the study sample with new starts of LAI risperidone, the primary outcome measure for this study, and new starts on other LAI SGAs are then presented graphically from 2007 to 2015 (Fig. 1).if Examination of the statistical significance of differences in the likelihood of receiving a new start on LAI risperi- done as well as other LAI SGAs over the years was con- ducted through a random effects mixed model analysis in which the dependent variable was a dichotomous variable representing a new start on LAI risperidone among all Veterans in the population seen that year, and the inde- pendent variables were the calendar years 2007–2010 and 2012–2015 with the year (2011) in which CSP#555 was published [19] as the reference year (marked with l Costs of medications to VHA (at negotiated dis- count prices) are recorded for each prescription in VA pharmacy benefit records. Measures were thus con- structed representing annual costs per Veteran for LAI Rosenheck et al. Results Sample if In addition, interrupted time series analysis was used to determine whether the slope of the curve (Fig. 1) reflect- ing a decline in new starts of LAI risperidone in the years 2012–2015, following publication, was more steeply neg- ative than in the years 2007–2010. p In the total sample of 115,172 Veterans, 74,007 (64.3%) were seen in both periods (2007–2011 and 2012–2015); 21,768 (18.9%) were seen exclusively from 2007 to 2011; and 19,397 (16.8%) exclusively from 2012 to 2015. Similar analyses were conducted for new starts on other LAI SGAs and on all LAI SGAs to provide con- text for understanding changes in prescribing for LAI risperidone. Unique Veterans in the sample averaged 56 years of age with 7.7% women, 26.3% receiving VA service-connected disability payments, 13.1% seen in the intensive case management programs, and 29.9% hospitalized for schiz- ophrenia during the study period. They had an average of 29.4 mental health outpatient visits/year and 71.9% had at least one other psychiatric comorbidity. An additional set of analyses examined the proportion of Veterans specifically prescribed LAI risperidone in each year who switched to LAI paliperidone (Invega®; a 4-week LAI SGA available after 2009) in the next year. Rosenheck et al. Trials (2023) 24:85 Rosenheck et al. Trials (2023) 24:85 Page 5 of 11 would be considered of meaningful magnitude (OR > 1.5 or Cohen’s d > 0.20) (Ferguson, 2009, Cohen 1988; Table 1). a Reported as Cohen’s d Changes in prescribed medicationsh In contrast, new starts on another LAI SGA, LAI paliperidone, once it became available to VHA in 2009, increased every year thereafter to a total of 3.4% of the entire study population by 2015 (Table 2). The intersec- tion between paliperidone and risperidone prescription thus occurs before the publication of CSP#555. New starts on other LAI SGAs increased after 2011, but by 2015 only involved 0.2% of the sample. New starts on any LAI SGA increased steadily and significantly after 2009 to 4.7% of the sample (Table 2, last column), predominantly representing growth in the use of LAI paliperidone. The proportion of Veterans diagnosed with schizo- phrenia or schizoaffective disorder who received new, first-time, prescriptions for LAI risperidone decreased steadily from 2007 to 2015 (Fig. 1), even before the pub- lication of CSP-555. In contrast, the proportion of Vet- erans receiving new starts on LAI paliperidone increased dramatically after its introduction from 2009 to 2015 (Fig. 1) while new starts on other LAI SGAs increased to a negligible degree from 2013 to 2015. Statistical analyses showed that after adjusting for other Veteran characteristics, there were a significantly greater proportion of new starts on LAI risperidone from 2007 to 2009 than in 2011 (Table 2, marked by “^”), reflect- ing a decline in new starts on LAI risperidone that pre- ceded the publication of CSP#555. The first year in which there was a statistically significant decline in new starts on LAI risperidone from the prior period was 2009–2010 (Table 2, marked by “#”). New starts on LAI risperidone continued to fall after 2011, becoming significantly differ- ent from 2011 in 2014 and 2015, several years following the publication of CSP#555. The proportion of specific switches from LAI risperi- done to LAI paliperidone doubled from 5.9% in 2009– 2010 to 13.3% in 2010 to 2011 and remained at more than 1.5 times the 2009–2010 rate of switching through 2015 (Table 3, last column). Turning to all LAI risperidone use (not just new starts), the peak year for both the total number of Veterans pre- scribed LAI risperidone and the proportion of all Veter- ans in the sample prescribed LAI risperidone occurred in 2009, 2 years before the publication of CSP#555, and declined significantly (p < 0.0001) every year after that, and by a total of 21.7% from 2007 to 2015 (Table 4). Results Sample Trials (2023) 24:85 Rosenheck et al. Trials (2023) 24:85 Page 6 of 11 Page 6 of 11 * Used as the reference year to define pre- and post-publication of a cooperative study (CSP#555) yif y # Statistically significant difference when compared to the previous year (excluding comparisons with the reference year) Statistically significant difference when compared to the previous year (excluding comparisons with the reference year) + Sample was too small to conduct a statistical model Results Sample Due to the large sample size, virtually all compari- sons between periods were statistically significant (p < 0.0001) but none of the effect size differences Table 1 Sociodemographics and psychiatric comorbidities of Veterans receiving antipsychotic medication for schizophrenia or schizoaffective disorder 2007–2015 Demographics All years: 2007–2015 N = 115,172 Pre- publication: 2007–2011 N = 95,775 Post- publication: 2012–2015 N = 93,404 OR/Cohen’s da (95% CI) p-value Race/ethnicity Black Hispanic 806 (0.7) 573 (0.6) 646 (0.7) < .0001 Black not Hispanic 39,373 (32.9) 29,182 (32.4) 31,434 (33.7) White Hispanic 7093 (5.9) 5277 (5.9) 5816 (6.2) White not Hispanic 58,693 (49.1) 44,603 (49.5) 46,456 (49.7) Other Hispanic 13,26 (1.1) 892 (1.0) 1061 (1.1) Other not Hispanic 12,274 (10.3) 9567 (10.6) 7991 (8.6) Marital status Divorced 35,956 (30.1) 27,080 (30.1) 27,881 (29.9) < .0001 Married 26,703 (22.3) 20,126 (22.3) 21,102 (22.6) Never 44,810 (37.5) 33,988 (37.7) 35,394 (37.9) Others 12,096 (10.1) 8900 (9.9) 9027 (9.7) Age Mean (SD) 56.4 (11.6) 55.3 (10.9) 57.1 (11.9) 0.164* (0.159–0.169) < .0001 Female N (%) 9348 (7.8) 6303 (7.0) 7656 (8.2) 1.19 (1.17, 1.21) < .0001 VA service-connected disability rate ≥ 50% 30,542 (25.5) 26,528 (29.4) 23,317 (25.0) 0.80 (0.79, 0.81) < .0001 < 50% 6315 (5.3) 4807 (5.3) 4480 (4.8) 0.89 (0.87, 0.92) < .0001 Participated in mental health inten- sive case management program Ever in 2007–2015 15,134 (12.7) 9615 (10.7) 10,831 (11.6) 1.10 (1.08, 1.12) < .0001 VA mental clinic visits (stop code 500–599) Annualized mean (SD) 24.6 (37.7) 28.0 (47.2) 31.6 (47.1) 0.078* (0.069, 0.087) < .0001 Deaths N (%) 17,758 (14.9) 5628 (6.3) 7269 (7.8) 1.27 (1.22, 1.31) < .0001 Inpatient diagnosis of schizophre- nia or schizoaffective 34,534 (28.9) 19,639 (21.8) 18,304 (19.6) 0.87 (0.86, 0.89) < .0001 History of mental health disorders Alcohol use disorder 47,333 (39.6) 35,753 (39.7) 38,461 (41.2) 1.06 (1.05, 1.07) < .0001 Drug use disorder 48,786 (40.8) 36,107 (40.1) 39,903 (42.7) 1.12 (1.10, 1.13) < .0001 Substance use disorder 61,346 (51.3) 45,870 (50.9) 49,828 (53.3) 1.10 (1.09, 1.11) < .0001 Other mental health diagnoses 11,475 (9.6) 8196 (9.1) 9631 (10.3) 1.15 (1.13, 1.17) < .0001 Bipolar 36,863 (30.8) 27,732 (30.8) 30,786 (33.0) 1.11 (1.10, 1.12) < .0001 Major 34,782 (29.1) 24,744 (27.5) 28,525 (30.5) 1.16 (1.15, 1.17) < .0001 Dysthymia 65,828 (55.1) 47,903 (53.2) 53,638 (57.4) 1.19 (1.18, 1.20) < .0001 PTSD 37,649 (31.5) 27,041 (30.0) 30,923 (33.1) 1.15 (1.14, 1.17) < .0001 Anxiety 49,023 (41.0) 35,486 (39.4) 40,553 (43.4) 1.18 (1.17, 1.19) < .0001 Adjustment 21,980 (18.4) 15,125 (16.8) 17,803 (19.1) 1.17 (1.15, 1.18) < .0001 Personality 22,321 (18.7) 16,729 (18.6) 18,208 (19.5) 1.06 (1.05, 1.07) < .0001 Any psychiatric comorbidity 114,924 (96.1) 87,587 (97.2) 91,401 (97.9) 1.31 (1.26, 1.35) < .0001 Alzheimer’s 2017 (1.7) 1653 (1.8) 1274 (1.4) 0.76 (0.74, 0.78) < .0001 Dementia 6562 (5.5) 5417 (6.0) 4330 (4.6) 0.74 (0.71, 0.77) < .0001 Traumatic brain injury 7966 (6.7) 5417 (6.0) 6742 (7.2) 1.22 (1.19, 1.24) < .0001 More than 1 comorbidity listed above 84,916 (71.0) 62,911 (69.8) 68,782 (73.6) 1.21 (1.19, 1.22) < .0001 d psychiatric comorbidities of Veterans receiving antipsychotic medication for schizophrenia or 15 Rosenheck et al. # Statistically significant difference when compared to the previous year (excluding comparisons with the reference year) + Sample was too small to conduct a statistical model Statistically significant difference when compared to the previous year (excluding comparisons with the reference year) + Sample was too small to conduct a statistical model ^ Statistically significant difference as compared to the reference year tatistically significant difference as compared to the reference year Changes in prescribed medicationsh As with new starts, the number and proportion of all patients prescribed LAI paliperidone increased every year after it became available in 2009 as did the pro- portion of all patients on any LAI SGA, which peaked at 8.99% in 2015. While Veterans prescribed LAI SGAs In the interrupted time series analysis, the p-value for the interruption slope term was 0.70, a non-significant value. There was thus no change in the declining slope after publication (Fig. 1). The intercept of the interrup- tion term also was insignificant. Table 2 New starts on LAI SGAs among study sample: 2007–2015 with comparison to 2011 i Year Patients with any antipsychotics New starts on LAI risperidone New starts on LAI paliperidone New starts on SGA LAIs (excluding LAI risperidone and paliperidone)+ New starts on all LAI SGAs N % N % N % N % 2007 67,023 1574 2.3^ 0 0 0 0 1574 2.3^ 2008 68,616 1542 2.2^ 0 0 0 0 1542 2.2^,# 2009 70,468 1543 2.2^ 30 0^ 0 0 1573 2.2^,# 2010 71,346 1383 1.9# 560 0.8^,# 1 0 1944 2.7^,# 2011 72,000 1244 1.7 1338 1.9 1 0 2583 3.6 2012 72,410 1102 1.5 1645 2.3^ 3 0 2750 3.8^ 2013 72,535 1100 1.5 1890 2.6^,# 23 0 3013 4.2^,# 2014 72,371 988 1.4^ 2090 2.9^,# 73 0.1 3151 4.4^,# 2015 73,008 771 1.1^,# 2503 3.4^,# 180 0.2 3454 4.7^,# All years 115,172 11,929 10.0% 13,229 11.1% 566 0.5% 25,724 21.5% Change 5985 − 803 − 1.2% 2503 3.4% 180 0.25% 1880 2.4% Percent change 8.9% − 51.0% − 52.2% –- –- –- –- 119.4% 104.3% Rosenheck et al. Changes in prescribed medicationsh Trials (2023) 24:85 Page 7 of 11 Table 3 Proportion of patients prescribed LAI risperidone who switched to LAI paliperidone in the subsequent year * Used as the reference year to define pre- and post-publication of a cooperative study (CSP#555) ^ Statistically significant difference as compared to the reference year # Statistically significant difference when compared to the previous year (excluding comparisons with the reference year) Index year Prior year Prescribed LAI risperidone in prior year Prescribed LAI paliperidone in subsequent year Switching over to paliperidone from risperidone (%) OR (95% CI) Risk ratio (subsequent year changes compared to 2009–2015 change) 2010 2009 4361 255 5.9^ 0.40 (0.34, 0.47) 2011* 2010 4344 578 13.3 2.28 2012 2011 4098 495 12.1 0.90 (0.79, 1.03) 2.06 2013 2012 3760 370 9.8^,# 0.73 (0.63, 0.83) 1.68 2014 2013 3601 384 10.7^ 0.80 (0.70, 0.92) 1.82 2015 2014 3223 403 12.5 0.97 (0.84, 1.11) 2.14 Table 4 Numbers of patients diagnosed with schizophrenia or schizoaffective disorder prescribed LAI antipsychotics from 2007 to 2016* * Used as the reference year to define pre- and post-publication of a cooperative study (CSP#555) ^ Statistically significant difference as compared to the reference year # Statistically significant difference when compared to the previous year (excluding comparisons with the reference year) Year Patients with any antipsychotics LAI risperidone Other LAI SGA LAI paliperidone Any LAI SGA Any FGA LAI Oral SGA Oral FGA N % N N % N % N N N 2007 66,985 3625 5.4^ –- –- –- 3625 5.4^ 5499 39,975 35,704 2008 68,565 3914 5.7^,# –- –- –- 3914 5.7^,# 5361 41,773 35,372 2009 70,401 4258 6.0# 29 29 0.04^ 4268 6.1^,# 5398 43,002 36,005 2010 71,250 4252 6.0^ 512 511 0.7^,# 4487 6.3^,# 5237 43,335 36,637 2011* 71,842 4024 5.6 1346 1345 1.9 5036 7.0 5061 42,986 37,032 2012 72,230 3723 5.2^ 1949 1944 2.7^ 5431 7.5^ 4855 42,617 36,976 2013 72,275 3541 4.9^,# 2585 2547 3.5^,# 5889 8.2^,# 4535 42,669 37,266 2014 71,959 3199 4.5^,# 3116 2914 4.1^,# 6075 8.4^,# 4133 43,226 35,889 2015 72,777 2838 3.9^,# 3991 3564 4.9^,# 6542 9.0^,# 4145 44,531 34,617 Change 5792 − 787 − 1.5% 3991 3564 4.9% 2917 3.6% − 1354 4556 − 1087 %Change 8.6% − 21.7% 27.9% –- –- –- 80.5% 66.1% − 24.6% 11.4% − 3.0% column 7). Expenditures on oral FGAs also declined by 92% over the same period (Table 5, column 8). Changes in prescribed medicationsh Total antipsychotic costs per Veteran declined by 39.44% 2007–2015 (Table 5, column 2), only 3.6% of which was attributable to reduced per-patient expenditures on LAI risperidone. This overall change reflected the offset- ting effect of increased expenditures on all LAI SGAs of $17.2 million (99% of which was for increased expendi- tures on LAI paliperidone) and by a reduction of $65.9 million on expenditures for oral SGA and FGA medica- tions (Table 5, sum of columns 7 and 8). As expenditures on LAI risperidone declined, expenditures on LAI pali- peridone increased substantially after its introduction in 2009, as did per Veteran expenditures on all LAI SGAs combined (Table 5, column 4). increased by 80.5% from 2007 to 2015, Veterans pre- scribed oral SGA medications increased by only 11.4%. In contrast, the proportions of Veterans on FGAs, includ- ing both LAI FGAs and oral FGAs, declined by 24.6% and 3.0% respectively (Table 4). As hypothesized, annual VHA expenditures per Vet- eran on LAI risperidone increased from 2007 to 2009 but began to decline in 2010, before CSP#555 was published, and especially in 2011, the year of publication of CSP#555 (Table 5). LAI risperidone costs per Veteran continued to decline sharply every year thereafter (2012–2015). In 2011 and 2012, two of the most widely used oral SGAs, olanzapine and quetiapine, lost their patent pro- tection and total VA expenditures on oral SGAs dropped substantially by 47.1% from 2017 to 2015 (Table 5, Rosenheck et al. Changes in prescribed medicationsh Trials (2023) 24:85 Page 8 of 11 Table 5 Annual antipsychotic medication costs among patients with schizophrenia or schizoaffective disorder * Used as the reference year to define pre- and post-publication of a cooperative study (CSP#555) ^ Statistically significant difference as compared to the reference year # Statistically significant difference when compared to the previous year (excluding comparisons with the reference year) Year All antipsychotics LAI risperidone All LAI SGAs LAI paliperidone LAI FGAs SGA oral FGA oral LAI SGA expenditures as % of total antipsychotic expenditures per Veteran 2007 $122,170,267 $12,914,274^ $12,914,274^ $0 $371,238 $76,078,149 $32,806,605 10.6^ 2008 $128,140,495 $15,085,187^ $15,085,187^,# $0 $348,750 $82,396,607 $30,309,950 11.8^ 2009 $115,066,407 $16,832,583 $16,881,109^ $48,526^ $358,936 $86,271,426 $11,554,935 14.7^,# 2010 $116,571,834 $16,878,621 18,142,921^,# $1,263,172^,# $370,713 $91,405,234 $6,652,966 15.6^,# 2011* $117,202,917 $16,062,864 $20,802,052 $4,737,495 $805,706 $90,968,967 $4,626,193 17.8 2012 $96,742,862 $14,562,180 $22,761,616^ $8,192,594^ $1,017,493 $68,812,359 $4,151,395 23.5^ 2013 $76,508,388 $12,757,391 $23,476,864^ $10,544,810^ $846,056 $48,876,894 $3,308,575 30.7^,# 2014 $70,692,177 $12,378,899 $26,402,932^,# $13,341,523^,# $919,701 $40,500,060 $2,869,484 37.4^ 2015 $73,982,381 $11,135,327^ $30,164,815^,# $17,156,228^ $869,519 $40,245,255 $2,702,792 40.8^,# Change ($48,187,886) ($1,778,947) $17,250,541 $17,156,228 $498,281 ($35,832,894) ($30,103,814) 30.2% Percent change − 39.4% − 13.8% 133.6% –- 134.2% − 47.1% − 91.8% 285.7% As a result of the large changes in per Veteran expen- ditures on oral medication, total VHA expenditures on antipsychotics for this population declined by $48 mil- lion (39%, Table 5, column 2), while the proportion of all antipsychotic expenditures on LAI SGAs, (predomi- nantly paliperidone) increased from 10 to 40% of all antipsychotic expenditures (Table 5, final column). In sharp contrast to changes in expenditures, the propor- tion of individual Veterans on LAI SGAs only increased from 5.4 to 8.99% between 2007 and 2015 (Table 3, col- umn 9). The declining use of LAI risperidone seems largely to coincide with the simultaneous and rapid increase in prescriptions for LAI paliperidone during the 2 years prior to the publication of CSP#555. However, the sharp increase in specific switches from LAI risperidone to LAI paliperidone during and after the year CSP#555 was published could possibly represent a specific clinician response to null study findings. The declining use of LAI risperidone primarily coincides with clinician choices favoring a new 4-week LAI medication, paliperidone, the active metabolite of risperidone, and manufactured by the same company. Perhaps the most striking feature of these results is that they could not have been anticipated in 2005 when CSP#555 was designed. Changes in prescribed medicationsh What was known was only that medication non-adherence was a determinant of relapse and rehospitalization in schizophrenia and that LAI medication improved compliance. There was far more limited evidence that LAI antipsychotics reduced relapse or improved symptom outcomes. Second-generation antipsychotics appeared to be better tolerated than con- ventional medications [14] and the combination of LAI delivery and SGA tolerability was promising and in need of evaluation. Discussion As hypothesized, new starts on LAI risperidone in VHA, as well as its total annual use and related expenditures declined following the publication of a multisite rand- omized clinical trial of the medicine with null results, thus failing to provide justification for use of this medi- cation. The policy option of discouraging its use had, in fact, been identified in the trial protocol [20] as a justifi- able response to null study results, although no such pol- icy has been implemented. However, further examination of data from the years prior to the publication of CSP#555 showed the decline in new starts of LAI risperidone preceded the publication of CSP#555 and thus could not be entirely attributed to the study. We also did not find evidence that the decline in new starts on LAI risperidone proceeded at an acceler- ated rate after the publication of CSP#555. Although CSP#555 offered no support for the value of LAI risperidone, or, by implication, for LAI SGAs more generally, it seems likely that the rapidly expanded use of LAI paliperidone reflected the greater face-value of a 4-week as contrasted to a 2-week LAI medication and vigorous marketing of this new but only modestly Rosenheck et al. Trials (2023) 24:85 Page 9 of 11 Page 9 of 11 different treatment whose superiority to LAI risperidone or any other treatment had not been demonstrated. subsequent national study of VHA pharmacotherapy for PTSD from 2008 to 2018 showed an annual decline of about 8.3% in risperidone use before CSP#504 publi- cation with a 7.4% annual decline (virtually unchanged) over the next 7 years. Here, again no impact of the trial was observed but rather a persistent decline in risperi- done use which also appeared to have been replaced with the new actively marketed drug, aripiprazole, albeit with- out evidence of its effectiveness in PTSD [29]. The scientific questions posed by CSP#555, in fact, remained unresolved. A RCT quite similar to CSP#555, published around the same time, also found no differ- ence in relapse or rehospitalization between LAI risp- eridone and oral antipsychotics [23] and a second RCT comparing LAI paliperidone and LAI haloperidol [24] found no clinical advantage for LAI paliperidone, while a secondary analysis of VHA data found that LAI halo- peridol was the more cost-effective treatment [25]. Limitationsh The primary limitation of this study is that temporal trends in VHA administrative data do not allow causal conclusions about the impact of CSP#555 since no infor- mation is available from a “control” healthcare system with no exposure to the results of the study. Nor are data available on the extent to which clinicians or patients and their families were aware of and understood the implica- tions of CSP#555, or on either their exposure or response to other studies of LAI antipsychotics, their medication preference, their involvement with key opinion leaders, or exposure to marketing initiatives. In addition, this study addressed only broad national trends and not small area variations in practice changes. Finally, it examines the impact of only one publication, and changes in prac- tice may only emerge in response to multiple consistent studies and meta-analyses. The discrepancy between empirical findings and sub- sequent changes in clinical practice observed in this study highlights the role of unanticipated non-research commercial factors in shaping the impact of major effec- tiveness studies. A similarly paradoxical result was docu- mented in response to the NIMH-funded CATIE study of 1432 patients diagnosed with schizophrenia that com- pared several oral SGAs to each other [15]. Following its publication, use of the drug that did best on the primary effectiveness outcome of the study (olanzapine) actually declined in clinical practice, most likely due to increasing concern about its metabolic side effects, and due to the approval of a new actively marketed drug with fewer met- abolic side effects (aripiprazole), not evaluated in CATIE, that showed substantially increased use, also without evidence of its superiority [27]. VA cooperative study #504 evaluating oral risperidone for PTSD (CSP#504) also reported unexpected null results in 2011 [28]. A Discussion In contrast, an observational study examined VHA admin- istrative data and reported cost-savings with paliperi- done as compared to oral medications, albeit without random assignment [26]. f In contrast to CSP#555, some trials quickly lead to major changes in practice. As soon as recent trials dem- onstrated that COVID-19 vaccines were highly effec- tive, they received regulatory approval and major efforts were initiated to foster their widespread use [30]. Simi- larly, once a major trial clearly revealed the risk of cardiac mortality with rofecoxib [31], the drug was withdrawn from the market altogether. These examples point to a major factor that is likely to affect the impact of indi- vidual RCTs—the magnitude of effects and seriousness of the health benefits or harms they demonstrate. CSP#555 (as well as CSP#504) found virtually no positive or nega- tive effect and this may account for its limited impact. Another influential factor is whether a study leads to explicit policy changes or directives by governmental agencies as in the case of the FDA and CDC promoting COVID-19 vaccines. The most recent meta-analysis of 137 studies, pub- lished 10 years after CSP#555 [14], found significant but small effects in both randomized trials (RR = 0.88 for relapse in 29 studies of 7833, p = 0.03) and cohort studies (RR = 0.92 in 44 studies of 106,136 patients, p = 0.004). More substantial effects were found only in studies with weak pre-post designs (RR = 0.44 in 28 studies of 17,876 patients, p < 0.0001). Perhaps the strongest evidence favoring LAIs was pub- lished in 2020 from a 489-patient cluster randomized trial showing less risk of hospitalization or relapse among patients with early episode schizophrenia at sites ran- domized to encourage the use of LAI aripiprazole as compared to sites that provided usual care. This more supportive evidence, published 9 years after CSP#555, is of unknown generalizability to adults with long-term ill- ness such as those treated by VHA. Thus, VHA prescrib- ers increasingly used LAI SGAs in the years after 2009, in spite of the null findings from CSP#555 or positive evi- dence from other studies.hi Conclusion Th d l The declining use of LAI risperidone after the publica- tion of null findings from CSP#555 likely reflected the expanded use of LAI paliperidone more than study results, although the declining use of LAI risperidone may have been reinforced by null study findings. Unan- ticipated changes in the therapeutic environment can complicate the implementation of clinical trial results and deserve further attention. 3. Califf RM, Sugarman J. Exploring the ethical and regulatory issues in pragmatic clinical trials. Clin Trials. 2015;12(5):436–41. 4. Office of Research & Development - History & timeline. U.S. Department of Veterans Affairs. Available from: https://www.research.va.gov/progr ams/csp/history.cfm. Accessed 27 Apr 2021. y 5. Rogers EM. Diffusion of innovations. 5th ed. New York: Free Press a Divi- sion of Simon and Schuster; 2003. 6. Kilbourne AM, Evans E, Atkins D. Learning health systems: driving real- world impact in mental health and substance use disorder research. FASEB Bioadv. 2021;3(8):626–38. 7. Sernyak MJ, Rosenheck R. Economic grand rounds: systemwide costs associated with second-generation antipsychotics in the treatment of schizophrenia. Psychiatr Serv. 2004;55(12):1361–2. Abbreviations CSP Cooperative Studies Program CI Confidence interval EHR Electronic health record FDA Food and Drug Administration FGA First-generation antipsychotic LAI Long-acting injectable OR Odds ratio RCT Randomized clinical trial SGA Second-generation antipsychotic VA Veterans Affairs VHA Veterans Health Administration y 8. Barnett PG, Scott JY, Rosenheck RA, Group CSPS. How do clinical trial participants compare to other patients with schizophrenia? Schizophr Res. 2011;130(1–3):34–9. 9. Thieda P, Beard S, Richter A, Kane J. An economic review of compliance with medication therapy in the treatment of schizophrenia. Psychiatr Serv. 2003;54(4):508–16. 9. Thieda P, Beard S, Richter A, Kane J. An economic review of compliance with medication therapy in the treatment of schizophrenia. Psychiatr Serv. 2003;54(4):508–16. 10. Weiden PJ, Olfson M. Cost of relapse in schizophrenia. Schizophr Bull. 1995;21(3):419–29. 10. Weiden PJ, Olfson M. Cost of relapse in schizophrenia. Schizophr Bull. 1995;21(3):419–29. 11. Citrome L. Long-acting injectable antipsychotics: what, when, and how. CNS Spectr. 2021;26(2):184. 11. Citrome L. Long-acting injectable antipsychotics: what, when, and how. CNS Spectr. 2021;26(2):184.fi 12. Fusar-Poli P, Kempton MJ, Rosenheck RA. Efficacy and safety of second- generation long-acting injections in schizophrenia: a meta-analysis of randomized-controlled trials. Int Clin Psychopharmacol. 2013;28(2):57–66. 12. Fusar-Poli P, Kempton MJ, Rosenheck RA. Efficacy and safety of second- generation long-acting injections in schizophrenia: a meta-analysis of randomized-controlled trials. Int Clin Psychopharmacol. 2013;28(2):57–66. 13. Kane JM, Kishimoto T, Correll CU. Page 10 of 11 Page 10 of 11 Page 10 of 11 Rosenheck et al. Trials (2023) 24:85 Received: 30 June 2022 Accepted: 12 January 2023 Received: 30 June 2022 Accepted: 12 January 2023 for readily adapting their design/methods should new and relevant developments emerge during the course of the trial. Planned dissemination and policy implemen- tation efforts should also be part of the study design, as is increasingly the case in the VA Cooperative Studies Program. Availability of data and materials 17. Rosenheck R, Leslie D, Sernyak M. From clinical trials to real-world prac- tice: use of atypical antipsychotic medication nationally in the Depart- ment of Veterans Affairs. Med Care. 2001;39(3):302–8. Veterans Health Administration data used for these analyses are not available to the public. 18. Glazer WM, Kane JM. Depot neuroleptic therapy: an underutilized treat- ment option. J Clin Psychiatry. 1992;53(12):426–33. 18. Glazer WM, Kane JM. Depot neuroleptic therapy: an underutilized treat- ment option. J Clin Psychiatry. 1992;53(12):426–33. References 1. Tunis SR, Stryer DB, Clancy CM. Practical clinical trials: increasing the value of clinical research for decision making in clinical and health policy. JAMA. 2003;290(12):1624–32. 1. Tunis SR, Stryer DB, Clancy CM. Practical clinical trials: increasing the value of clinical research for decision making in clinical and health policy. JAMA. 2003;290(12):1624–32. 2. Glasgow RE, Magid DJ, Beck A, Ritzwoller D, Estabrooks PA. Practical clini- cal trials for translating research to practice: design and measurement recommendations. Med Care. 2005;43(6):551–7. Funding The study was funded by the VA Cooperative Studies Program which also funded the original study which also received medication and modest sup- port from Janssen Pharmaceuticals. The funding organization had no role in the writing or editing of the manuscript. 15. Lieberman JA, Stroup TS, McEvoy JP, Swartz MS, Rosenheck RA, Perkins DO, et al. Effectiveness of antipsychotic drugs in patients with chronic schizophrenia. N Engl J Med. 2005;353(12):1209–23. 16. Rosenheck RA, Sernyak MJ. Developing a policy for second-generation antipsychotic drugs. Health Aff (Millwood). 2009;28(5):w782–93. 16. Rosenheck RA, Sernyak MJ. Developing a policy for second-generation antipsychotic drugs. Health Aff (Millwood). 2009;28(5):w782–93. Conclusion Th d l Assessing the comparative effective- ness of long-acting injectable vs. oral antipsychotic medications in the prevention of relapse provides a case study in comparative effectiveness research in psychiatry. J Clin Epidemiol. 2013;66(8 Suppl):S37-41. Authors’ contributions research in psychiatry. J Clin Epidemiol. 2013;66(8 Suppl):S37-41. RR and CD wrote the initial draft, based on analyses by SK. The text was edited with added contributions by SH, SA, and RF and substantive input from JP and DS. All of the authors approved the final version of this article. 14. Kishimoto T, Hagi K, Kurokawa S, Kane JM, Correll CU. Long-acting injectable versus oral antipsychotics for the maintenance treatment of schizophrenia: a systematic review and comparative meta-anal- ysis of randomised, cohort, and pre-post studies. Lancet Psychiatry. 2021;8(5):387–404. Declarations 19. Rosenheck RA, Krystal JH, Lew R, Barnett PG, Fiore L, Valley D, et al. Long- acting risperidone and oral antipsychotics in unstable schizophrenia. N Engl J Med. 2011;364(9):842–51. 19. Rosenheck RA, Krystal JH, Lew R, Barnett PG, Fiore L, Valley D, et al. Long- acting risperidone and oral antipsychotics in unstable schizophrenia. N Engl J Med. 2011;364(9):842–51. Ethics approval and consent to participate Ethics approval and consent to participate The study was approved with a waiver of informed consent by the Boston VA IRB. 20. The CSP #555 Research Group. VA cooperative study #555 protocol: long- acting injectable risperidone in the treatment of schizophrenia. epart- ment of Veteran Affairs Cooperative Studies Program; 2008. p. 7. Available from: https://www.nejm.org/doi/full/10.1056/NEJMoa1005987. Consent for publication All of the authors approved the final version of this article for publication. 21. Kulas JF, Rosenheck RA. A comparison of veterans with post-traumatic stress disorder, with mild traumatic brain injury and with both disorders: understanding multimorbidity. Mil Med. 2018;183(3–4):e114–22. Policy implicationsh This discussion highlights the importance of designing practical clinical trials, of critically considering their potential effect size and health impact, and of possi- ble future of prescribing, research, and marketing con- texts. Protocols may be enhanced by including a plan Rosenheck et al. Trials (2023) 24:85 Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. 22. Mohamed S. Rates and correlates of suicidality in VA intensive case man- agement programs. Community Ment Health J. 2022;58(2):356–65. 22. Mohamed S. Rates and correlates of suicidality in VA intensive case man- agement programs. Community Ment Health J. 2022;58(2):356–65. Page 11 of 11 Rosenheck et al. Trials (2023) 24:85 Rosenheck et al. Trials (2023) 24:85 23. Buckley PF, Schooler NR, Goff DC, Hsiao J, Kopelowicz A, Lauriello J, et al. Comparison of SGA oral medications and a long-acting injectable SGA: the PROACTIVE study. Schizophr Bull. 2015;41(2):449–59. 24. McEvoy JP, Byerly M, Hamer RM, Dominik R, Swartz MS, Rosenheck RA, et al. Effectiveness of paliperidone palmitate vs haloperidol decanoate for maintenance treatment of schizophrenia: a randomized clinical trial. JAMA. 2014;311(19):1978–87. 25. Rosenheck RA, Leslie DL, Sint KJ, Lin H, Li Y, McEvoy JP, et al. Cost-effec- tiveness of long-acting injectable paliperidone palmitate versus halop- eridol decanoate in maintenance treatment of schizophrenia. Psychiatr Serv. 2016;67(10):1124–30. 26. Young-Xu Y, Duh MS, Muser E, DerSarkissian M, Faust E, Kageleiry A, et al. Impact of paliperidone palmitate versus oral atypical antipsychotics on health care resource use and costs in veterans with schizophrenia. J Clin Psychiatry. 2016;77(10):e1332–41. 27. Berkowitz RL, Patel U, Ni Q, Parks JJ, Docherty JP. The impact of the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) on prescrib- ing practices: an analysis of data from a large midwestern state. J Clin Psychiatry. 2012;73(4):498–503. 28. Krystal JH, Rosenheck RA, Cramer JA, Vessicchio JC, Jones KM, Vertrees JE, et al. Adjunctive risperidone treatment for antidepressant-resistant symptoms of chronic military service-related PTSD: a randomized trial. JAMA. 2011;306(5):493–502. 29. Holder N, Woods A, Neylan TC, Maguen S, Seal KH, Bernardy N, et al. Trends in medication prescribing in patients with PTSD from 2009 to 2018: a National Veterans Administration study. J Clin Psychiatry. 2021;82(3):20m13522. 30. Safety of COVID-19 vaccines: Center for Diseases Control and Prevention; 2019 [updated 05/03/2022. Available from: https://www.cdc.gov/coron avirus/2019-ncov/vaccines/safety/safety-of-vaccines.html. Accessed 30 Dec 2021. 31. Bresalier RS, Sandler RS, Quan H, Bolognese JA, Oxenius B, Horgan K, et al. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N Engl J Med. 2005;352(11):1092–102. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. 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https://openalex.org/W4361842791	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Table_2_from_Intratumoral_Cytokines_and_Tumor_Cell_Biology_Determine_Spontaneous_Breast_Cancer_Specific_Immune_Responses_and_Their_Correlation_to_Prognosis/22382805/1/files/39828216.pdf	English	null	Supplementary Table 2 from Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer–Specific Immune Responses and Their Correlation to Prognosis	null	2,023	cc-by	206	Suppl.Tab: 2. List of factors that were quantified in breast tumour tissue lysates by multiplex analysis. Name abbreviation eotaxin fibroblast growth factor basic FGFb granulocyte colony-stimulating factor G-CSF granulocyte-macrophage colony- stimulating factor GM-CSF interferon- alpha IFN-α interleukin-1 beta IL-1ß interleukin 1 receptor-alpha IL-1Rα interleukin-2 IL-2 interleukin-4 IL-4 interleukin-5 IL-5 interleukin-6 IL-6 interleukin-7 IL-7 interleukin-8 IL-8 interleukin-9 IL-9 interleukin-10 IL-10 interleukin-12 IL-12 interleukin-13 IL-13 interleukin-15 IL-15 interleukin-17 IL-17 IFN-gamma-inducible protein-10 IP-10 monocyte chemoattractant protein 1 MCP-1 macrophage inflammatory protein 1alpha MIP-1α macrophage inflammatory protein 1beta MIP-1ß chemokine ligand 5 RANTES transforming growth factor-beta 1 TGF-ß1 tumor necrosis factor-alpha TNF-α vascular endothelial growth factor VEGF Name abbreviation eotaxin fibroblast growth factor basic FGFb granulocyte colony-stimulating factor G-CSF granulocyte-macrophage colony- stimulating factor GM-CSF interferon- alpha IFN-α interleukin-1 beta IL-1ß interleukin 1 receptor-alpha IL-1Rα interleukin-2 IL-2 interleukin-4 IL-4 interleukin-5 IL-5 interleukin-6 IL-6 interleukin-7 IL-7 interleukin-8 IL-8 interleukin-9 IL-9 interleukin-10 IL-10 interleukin-12 IL-12 interleukin-13 IL-13 interleukin-15 IL-15 interleukin-17 IL-17 IFN-gamma-inducible protein-10 IP-10 monocyte chemoattractant protein 1 MCP-1 macrophage inflammatory protein 1alpha MIP-1α macrophage inflammatory protein 1beta MIP-1ß chemokine ligand 5 RANTES transforming growth factor-beta 1 TGF-ß1 tumor necrosis factor-alpha TNF-α vascular endothelial growth factor VEGF Suppl.Tab: 2. List of factors that were quantified in breast tumour tissue lysates by multiplex analysis.
https://openalex.org/W2342466633	https://europepmc.org/articles/pmc4849675?pdf=render	English	null	Orthostatic Hypotension and Elevated Resting Heart Rate Predict Low-Energy Fractures in the Population: The Malmö Preventive Project	PloS one	2,016	cc-by	6,401	Aims To test the association between orthostatic hypotension, resting heart rate and incidence of low-energy-fractures in the general population. Copyright: © 2016 Hamrefors 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 Citation: Hamrefors V, Härstedt M, Holmberg A, Rogmark C, Sutton R, Melander O, et al. (2016) Orthostatic Hypotension and Elevated Resting Heart Rate Predict Low-Energy Fractures in the Population: The Malmö Preventive Project. PLoS ONE 11(4): e0154249. doi:10.1371/journal.pone.0154249 Background Autonomic disorders of the cardiovascular system, such as orthostatic hypotension and ele- vated resting heart rate, predict mortality and cardiovascular events in the population. Low- energy-fractures constitute a substantial clinical problem that may represent an additional risk related to such autonomic dysfunction. Editor: Tatsuo Shimosawa, The University of Tokyo, JAPAN Received: December 20, 2015 Accepted: April 10, 2016 Published: April 28, 2016 Copyright: © 2016 Hamrefors et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: December 20, 2015 Accepted: April 10, 2016 Published: April 28, 2016 Copyright: © 2016 Hamrefors 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. Orthostatic Hypotension and Elevated Resting Heart Rate Predict Low-Energy Fractures in the Population: The Malmö Preventive Project Viktor Hamrefors1,2, Maria Härstedt1, Anna Holmberg3, Cecilia Rogmark3, Richard Sutton4, Olle Melander1,5, Artur Fedorowski1,6 a1111 1 Department of Clinical Sciences, Faculty of Medicine, Lund University, Malmö, Sweden, 2 Department of Medical Imaging and Physiology, Skåne University Hospital, Malmö, Sweden, 3 Department of Orthopaedics, Skåne University Hospital, Malmö, Sweden, 4 National Heart and Lung Institute, Imperial College, St Mary’s Hospital Campus, London, UK, 5 Department of Internal Medicine, Skåne University Hospital, Malmö, Sweden, 6 Department of Cardiology, Skåne University Hospital, Malmö, Sweden Viktor.Hamrefors@med.lu.se RESEARCH ARTICLE * Viktor.Hamrefors@med.lu.se Conclusion Orthostatic blood pressure decline and elevated resting heart rate independently predict low-energy fractures in a middle-aged population. These two measures of subclinical car- diovascular dysautonomia may herald increased risks many years in advance, even if symptoms may not be detectable. Although the effect sizes are moderate, the easily acces- sible clinical parameters of orthostatic blood pressure response and resting heart rate deserve consideration as new risk predictors to yield more accurate decisions on primary prevention of low-energy fractures. Competing Interests: The authors have declared that no competing interests exist. Orthostatic Hypotension, Heart Rate and Fractures minute. When combining the orthostatic response and resting heart rate, there was a 30% risk increase (1.08–1.57) of low-energy-fractures between the extremes, i.e. between sub- jects in the fourth compared with the first quartiles of both resting heart rate and systolic blood pressure-decrease. minute. When combining the orthostatic response and resting heart rate, there was a 30% risk increase (1.08–1.57) of low-energy-fractures between the extremes, i.e. between sub- jects in the fourth compared with the first quartiles of both resting heart rate and systolic blood pressure-decrease. Ernhold Lundströms Research Foundation, the Region Skåne, the Hulda and Conrad Mossfelt Foundation, the Wallenberg Foundation, the Lennart Hanssons Memorial Foundation, and the Anna Lisa and Sven-Erik Lundgrens Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Changes in autonomic control of the cardiovascular system may adversely affect various aspects of health [1]. Orthostatic hypotension (OH) is a common manifestation of autonomic dysfunction [2, 3] that occasionally leads to cerebral hypoperfusion and syncope, either directly by the decrease in blood pressure (BP) or indirectly by triggering the vasovagal reflex, but is often asymptomatic [4]. OH conveys independent prognostic information concerning mortal- ity [5] with an underlying increased risk of death due to cardiovascular disease (CVD), injuries, neurodegenerative, and respiratory diseases [6, 7]. In parallel, an elevated resting heart rate (RHR), which correlates with OH [8], has been linked with increased general and cardiovascu- lar mortality [9, 10] independently of traditional risk factors. Hemodynamic impairment during orthostasis is a well-known risk factor for traumatic falls [6, 11, 12]. Low-energy fractures, resulting from low-energy traumas such as a fall from stand- ing position, constitute a substantial clinical problem [13, 14]. Whereas several risk factors for low-energy fractures have been identified in the population [15, 16], the specific underlying hemodynamic factors have not been thoroughly studied. Importantly, in pharmacological pri- mary prevention of low-energy fractures with e.g. bisphosphonates, there is a clinical need to sharpen risk prediction to target subjects at highest risk and thus reduce the number needed to treat [17]. Indeed, non-pharmacological primary prevention has a similar demand to focus interventions on the appropriate individuals. Also, in general terms, low-energy fractures can plausibly be regarded as a surrogate marker of falls, thus being a suitable endpoint for the assessment of risks related to autonomic dysfunction. In this study, we investigated longitudinal association of orthostatic BP fall, RHR and their combination at baseline with incident low-energy-fractures in a middle-aged cohort of 33 000 individuals. Methods and Results Using multivariable-adjusted Cox regression models we investigated the association between orthostatic blood pressure response, resting heart rate and first incident low- energy-fracture in a population-based, middle-aged cohort of 33 000 individuals over 25 years follow-up. Data Availability Statement: All relevant data are within the paper and available upon request due to the presence of identifying participant information. Please contact the corresponding author to request data access. The median follow-up time from baseline to first incident fracture among the subjects that experienced a low energy fracture was 15.0 years. A 10 mmHg orthostatic decrease in sys- tolic blood pressure at baseline was associated with 5% increased risk of low-energy-frac- tures (95% confidence interval 1.01–1.10) during follow-up, whereas the resting heart rate predicted low-energy-fractures with an effect size of 8% increased risk per 10 beats-per- minute (1.05–1.12), independently of the orthostatic response. Subjects with a resting heart rate exceeding 68 beats-per-minute had 18% (1.10–1.26) increased risk of low-energy-frac- tures during follow-up compared with subjects with a resting heart rate below 68 beats-per- Funding: Funding was provided by the European Research Council (StG 282225), Swedish Medical Research Council, the Swedish Heart and Lung Foundation, the Medical Faculty of Lund University, the governmental funding of clinical research within the National Health Services, the Albert Påhlsson Research Foundation, the Crafoord Foundation, the 1 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Definition of the clinical variables at baseline used in the current study Decrease in SBP and DBP, the components of orthostatic hemodynamic response, were assessed using a continuous/semi quantitative scale. Orthostatic SBP and DBP response (ΔSBP and ΔDBP) were defined as standing SBP/DBP minus supine SBP/DBP. The RHR was the mean of two measurements in supine position. BMI was calculated as weight in kilograms divided by the square of the height in meters. Antihypertensive treatment (AHT) was defined as a positive answer to the following question: Do you take medication for high blood pressure? Those who confirmed regular or occasional current smoking were classified as smokers. Diabe- tes was defined as fasting plasma glucose of 7.0 mmol/l, current pharmacological treatment of diabetes or self-reported history of diabetes. Previous myocardial infarction (MI) at baseline was recorded from the national Swedish patient register (which contains data from 1964- with complete coverage from 1987-). Study population The Malmö Preventive Project (MPP) is a population-based prospective cohort study in the city of Malmö, in southern Sweden [18]. The primary aim was to screen for CVD among large strata of the adult population. Subjects from birth cohorts in Malmö were invited by mail and a 2 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Orthostatic Hypotension, Heart Rate and Fractures total of 33346 (22444 men and 10902 women, mean age 45 years; range 26–61 years) inhabi- tants of Malmö, born between 1921 and 1948 attended the screening program and were exam- ined between 1974 and 1992. The overall participation among invited subjects was 71% [19]. The current analysis is a retrospective review of the prospective study of MPP. The present study included subjects from MPP with complete data on age, sex, body-mass index (BMI) and follow-up data of low-energy fractures (see below), rendering a total of 33139 subjects eligible for analysis. total of 33346 (22444 men and 10902 women, mean age 45 years; range 26–61 years) inhabi- tants of Malmö, born between 1921 and 1948 attended the screening program and were exam- ined between 1974 and 1992. The overall participation among invited subjects was 71% [19]. The current analysis is a retrospective review of the prospective study of MPP. The present study included subjects from MPP with complete data on age, sex, body-mass index (BMI) and follow-up data of low-energy fractures (see below), rendering a total of 33139 subjects eligible for analysis. Baseline examination Subjects were asked to abstain from food, alcohol and tobacco for 12 h prior to the baseline examination, which was performed by trained nurses in the morning. BP was measured using the auscultatory method with a mercury sphygmomanometer on the right arm at heart level. BP was measured twice in the supine position and twice after one minute of standing. The mean value of two readings was recorded for each position and rounded to nearest 5 mmHg. The heart rate was measured twice by palpation over one minute in supine position. After the examination, the participants were asked to fill a questionnaire focused on personal and family history of CVD, hypertension, diabetes, cancer, smoking habits, and lifestyle pattern. A detailed description of recruitment and screening procedures of MPP may be found else- where [18, 20]. The MPP was approved by the Health Department of Malmö city (1972) and the retrospective analysis of the cohort was approved by IRB in Lund. All participants gave written informed consent. The data were anonymized before the analyses. PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Population characteristics Baseline characteristics of the study population are shown in Table 1. There were 4.9 first inci- dent low-energy fractures per 1000 person-years during a median follow-up time of 25.1 years in the whole population. The median follow-up time from baseline to first incident fracture among the subjects that experienced a low energy fracture was 15.0 years. The mean age at first low-energy fracture among these subjects was 63.5 years (10.7 years) Statistics The hemodynamic parameters recorded at baseline (supine SBP/DBP, ΔSBP/DBP, and RHR) were related to first incident low-energy fracture using multivariable-adjusted Cox-regression models. We tested models entering age, sex and BMI as covariates (Model 1) as well as more comprehensive models including age, sex, BMI, AHT, diabetes, smoking, previous MI, and all measured hemodynamic parameters (supine SBP/DBP, ΔSBP/DBP and RHR) from the base- line examination (Model 2). In order to evaluate the combined effect of RHR and OH on the risk of incident low energy fractures we constructed a combined RHR-OH-score for each individual subject. The score was constructed as follows: the study population was split into quartiles according to baseline RHR and ΔSBP, respectively. Thus, each individual was given a quartile number (1 for lowest, 4 for highest) for RHR and ΔSBP. The RHR-OH-score for each individual (range 2–8) was then constructed by summing the individual quartile number for RHR and ΔSBP, respectively. Additionally, in order to further investigate the specific contribution of RHR and ΔSBP and of their combined effect to the risk of incident low-energy-fractures, the 16 specific quartile com- binations of RHR and ΔSBP that could be combined for the study subjects (i.e. subjects in [Q1 for RHR–Q2 ΔSBP], [Q1 for RHR—Q3 ΔSBP]. . .up to [Q4 for RHR- Q4 - ΔSBP] was tested in Cox-regression models in relation to the reference [Q1 for RHR–Q1 for ΔSBP]. The proportional-hazards assumption was confirmed by visual inspection of survival curves (Figs 1 and 2). Follow-up and retrieval of end-points Fracture data were obtained by linking the unique Swedish ten-digit personal identification number of included subjects with the register at the Department of Radiology at Malmö Uni- versity Hospital as previously described [16]. In the city of Malmö, all emergency radiographic examinations are performed at the Department of Radiology of Skåne University Hospital and reports and films of fractures are stored. Fractures identified were confirmed through manual search of medical and radiological files. Previous studies have shown that at least 97% of all fractures experienced by citizens of Malmö can be identified this way [21]. Follow-up extended to December 31 2006. Fractures were classified as high- or low-energy trauma depending on the cause of fracture, based on information in the radiographic reports. Fractures caused by falling from standing position or less energy were classified as low-energy fractures. Of all fractures, 97.7% had descriptions with adequate information about the degree of trauma. The fractures with 3 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Orthostatic Hypotension, Heart Rate and Fractures insufficient information were classified as low-energy fractures based on the experience that it is highly unlikely that information about high-energy accidents is not reported. Fractures caused by high-energy trauma were excluded from all analyses, as were also pathological frac- tures caused by cancer or other bone diseases. Relation between orthostatic blood pressure change and low energy fractures The mean orthostatic change in SBP predicted incident low-energy fractures, in both models. The effect size was approximately 5% increased risk per 10-mmHg decrease in SBP in the fully adjusted model (Table 2). The relative risk increase could be observed for SBP fall in the range of 0–5 mmHg (3rd quartile in the population, +12%), and it was even higher for those who demonstrated SBP fall >5 mmHg (4th quartile, + 17%) (S1 Table). In contrast, the orthostatic DBP-response did not predict low energy fractures. When analyzing the type of fractures predicted by a decrease in SBP we found (in the fully adjusted models) an association with vertebral fragility fractures (221 fractures; 24 percent risk increase per—10 mmHg; P = 0.008) whereas there were no significant associations with radius, skull and hip fractures (S2 Table). 4 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Orthostatic Hypotension, Heart Rate and Fractures Fig 1. The cumulative incidence of low-energy fractures stratified by orthostatic decrease in systolic blood pressure over 20 mmHg in the Malmö Preventive Project population (n = 33 000). doi:10 1371/journal pone 0154249 g001 Fig 1. The cumulative incidence of low-energy fractures stratified by orthostatic decrease in systolic blood pressure over 20 mmHg in the Malmö Preventive Project population (n = 33 000). Fig 1. The cumulative incidence of low-energy fractures stratified by orthostatic decrease in systolic blood pressure over 20 mmHg in the Malmö Preventive Project population (n = 33 000). doi:10.1371/journal.pone.0154249.g001 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 The combined value of OH and RHR on predicting low-energy fractures When combining orthostatic SBP-decrease and RHR there was a 30% risk increase of first inci- dent low energy fracture (95% CI 1.08–1.57; P <0.001) between the extremes of the RHR-OH- score (i.e. subjects in the fourth compared with the first quartiles of both RHR and ΔSBP). When stratifying subjects by quartiles of both RHR and ΔSBP in relation to first incident low energy fracture the effect of orthostatic SBP-decrease on risk of low energy fractures was evi- dent predominantly in the upper quartiles of RHR (Fig 3, S3 Table). Relation between RHR and low- energy fractures RHR predicted incident low-energy fractures in both models, the latter model also including supine SBP and orthostatic change in SBP as covariates. The effect size was approximately 8% increased risk per 10 beats per minute (bpm) in the fully adjusted model (Table 2). Subjects with a RHR exceeding 68 bpm (= median RHR) had 18% (95% CI 1.10–1.26; P < 0.001) increased risk of low-energy fractures during follow-up compared with subjects having a RHR below 68 bpm. There was a linear association between RHR and number of low energy fractures in individ- ual subjects (P = 0.011) and the odds ratio (OR) of suffering more than one fracture (compared with one or zero) was 1.10 (95% CI 1.03–1.16; P = 0.002) per RHR-quartile in a logistic regres- sion model, adjusted as in model 2. On analysis of specific fractures, we found (in the fully adjusted models) an association with distal radius fractures (1020 fractures; 14% risk increase per 10 bpm; P <0.001) and a border- line-significant association with vertebral fragility fractures (219 fractures; 14% risk increase per 10 bpm; P = 0.050), whereas there were no associations with hip fractures and skull frac- tures (S2 Table). 5 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Orthostatic Hypotension, Heart Rate and Fractures Fig 2. The cumulative incidence of low-energy fractures stratified by resting heart rate over 68 bpm in the Malmö Preventive Project population (n = 33 000). mulative incidence of low-energy fractures stratified by resting heart rate over 68 bpm in the Malmö j t l ti ( 33 000) Fig 2. The cumulative incidence of low-energy fractures stratified by resting heart rate over 68 bpm in the Malmö Preventive Project population (n = 33 000). Fig 2. The cumulative incidence of low-energy fractures stratified by resting heart rate over 68 bpm in the Malmö Preventive Project population (n = 33 000). Fig 2. The cumulative incidence of low-energy fractures stratified by resting heart rate over 68 bpm in the Malmö Preventive Project population (n = 33 000). Fig 2. The cumulative incidence of low-energy fractures stratified by resting heart rate over 68 bpm in the Malmö Preventive Project population (n = 33 000). Fig 2. The cumulative incidence of low-energy fractures stratified by resting heart rate over 68 bpm in Preventive Project population (n = 33 000). doi:10.1371/journal.pone.0154249.g002 Discussion Apart from this finding, we also deter- mined a strong association between elevated RHR and low-energy fractures, independently of supine SBP and orthostatic SBP-decrease. Finally, there was a strong association with risk of first incident low-energy fracture when combining RHR and orthostatic BP-fall in a model, observing a 30% risk increase between subjects at the extremes of the combination of both variables. the S1 Table, the relative risk increase could be observed for SBP fall in the range of 0–5 mmHg and it was higher for those who demonstrated SBP fall >5 mmHg. Thus, although the current guidelines recommend SBP decline > 20mmHg on standing as the diagnostic criteria for OH, any drop in BP during orthostatic challenge indicate an increased risk for low-energy fracture. It is noteworthy that OH is often asymptomatic, and the first recognized symptom may be a complete loss of consciousness resulting in trauma [22]. Apart from this finding, we also deter- mined a strong association between elevated RHR and low-energy fractures, independently of supine SBP and orthostatic SBP-decrease. Finally, there was a strong association with risk of first incident low-energy fracture when combining RHR and orthostatic BP-fall in a model, observing a 30% risk increase between subjects at the extremes of the combination of both variables. In a recent meta-analysis, Ricci and colleagues reported that OH is associated with increased risk of death and CVD [5] regardless of studied population. Further, higher RHR has been reported to predict mortality and cardiovascular events [9, 23] in the general population. Our study is, to the best of our knowledge, the first to show a prospective association between OH, RHR and incident low-energy-fractures among middle-aged adults over a long follow-up. It is likely that many low-energy-fractures are caused by falls and that these falls may in turn be provoked by a temporary cerebral hypoperfusion resulting from impaired BP response to orthostasis. As such, low-energy fractures may serve as a surrogate variable for falls in the population. Supporting this hypothesis, we found a stronger effect of both OH and RHR on fracture types traditionally associated with falls, such as distal radius and vertebral fragility fractures (even though the latter may also occasionally be non-traumatic). The main potential clinical implication of our results is the possibility that OH and RHR may be used to sharpen clinical risk prediction of low-energy fractures. Discussion In this study, we evaluated two hemodynamic parameters, orthostatic blood pressure-fall and resting heart rate, in relation to first incident low-energy fracture during a median follow-up of 25 years in a population-based middle-aged cohort of 33 000 subjects. The orthostatic systolic blood pressure-decrease at baseline predicted low-energy fractures, also after adjusting for covariates known to be associated with fragility fractures such as smoking and antihypertensive therapy [15, 16]. This is in line with our previous observation of a strong association between impaired orthostatic BP regulation and fatal injuries in the same population [6]. As can be seen 6 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Orthostatic Hypotension, Heart Rate and Fractures Table 1. Baseline characteristics. Baseline characteristics of the study population (N = 33139) Age, years 45.6 (7.4) Sex, % male 67.6 BMI, kg/m2 24.6 (3.6) Current smoker, % 44.6 Supine SBP, mmHg 129.2 (15.6) Supine DBP, mmHg 85.1 (9.5) Postural SBP response, mmHg -1.8 (7.4) Postural DBP response, mmHg 2.3 (4.5) Postural SBP decrease 20 mmHg, % 2.2 Orthostatic hypotension (postural decrease 20/10 mmHg), % 2.8 Resting heart rate, BPM 69 (10) Antihypertensive treatment, % 5.4 Diabetes, % 4.7 Previous myocardial infarction, % 0.4 Values displayed as mean (SD) if not otherwise indicated. BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; BPM = beats per minute. doi:10 1371/journal pone 0154249 t001 Table 1. Baseline characteristics. Values displayed as mean (SD) if not otherwise indicated. BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; BPM = beats per minute. Values displayed as mean (SD) if not otherwise indicated. BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; BPM = beats per minute. the S1 Table, the relative risk increase could be observed for SBP fall in the range of 0–5 mmHg and it was higher for those who demonstrated SBP fall >5 mmHg. Thus, although the current guidelines recommend SBP decline > 20mmHg on standing as the diagnostic criteria for OH, any drop in BP during orthostatic challenge indicate an increased risk for low-energy fracture. It is noteworthy that OH is often asymptomatic, and the first recognized symptom may be a complete loss of consciousness resulting in trauma [22]. PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Orthostatic Hypotension, Heart Rate and Fractures Table 2. Relation between hemodynamic parameters and first incident low energy fracture. Number of subjects (events) HR 95% CI P value Supine SBP Model 1 32672 (3597) 1.002 per mmHg 1.000–1.004 0.128 Model 2* 32672 (3597) 1.002 per mmHg 1.000–1.004 0.066 Supine DBP Model 1 32667 (3596) 1.001 per mmHg 0.997–1.004 0.691 Model 2* 32667 (3596) 1.002 per mmHg 0.998–1.005 0.390 ΔSBP Model 1 32610 (3584) 1.006 per - ΔmmHg 1.002–1.011 0.005 Model 2 32610 (3584) 1.005 per - ΔmmHg 1.001–1.010 0.022 ΔDBP Model 1 32594 (3582) 1.007 per - ΔmmHg 1.000–1.015 0.052 Model 2* 32594 (3582) 1.006 per - ΔmmHg 0.999–1.014 0.109 RHR Model 1 32530 (3573) 1.009 per BPM 1.005–1.012 <0.001 Model 2**** 32461 (3559) 1.008 per BPM 1.005–1.012 <0.001 Model 1: Includes covariates age, sex, BMI. Model 2 * Includes covariates age, sex, BMI, AHT, smoking, diabetes, previous MI Includes covariates age, sex, BMI, AHT, smoking, diabetes, previous MI, SBP supine ’ Includes covariates age, sex, BMI, AHT, smoking, diabetes, previous MI, DBP supine **’ Includes covariates age, sex, BMI, AHT, diabetes, smoking, previous MI, SBP supine, ΔSBP in standing. HR = hazard ratio; 95% CI = 95% Conﬁdence interval; SBP = systolic blood pressure; DBP = diastolic blood pressure; RHR = resting heart rate. Table 2. Relation between hemodynamic parameters and first incident low energy fracture. P value Model 1: Includes covariates age, sex, BMI. Model 2 Includes covariates age, sex, BMI, AHT, smoking, diabetes, previous MI doi:10.1371/journal.pone.0154249.t002 doi:10.1371/journal.pone.0154249.t002 improve risk prediction in order to better target subjects at highest risk and thus reduce the number needed to treat [17]. Non-pharmacological primary prevention has a similar demand to focus interventions on the right individuals. We were unable to adjust for measures of bone density; however, we find it unlikely that bone density would be a real confounder as it is likely to be related only to the outcome and not to the exposures (OH and RHR). Accordingly, even though the effect sizes are moderate, we suggest that OH and RHR may be used as potential additional predictors in clinical decisions on fracture preventive therapy. As for mechanistic aspects of the current results, we propose some explanations why OH and particularly RHR are independently associated with low-energy injuries. Discussion Low energy fractures constitute a huge clinical problem [13, 14], often being a complicating factor in patients with multiple comorbidities treated in the internal medicine ward. A number of risk factors for low- energy fractures have been identified [15, 16], however, in the primary pharmacological pre- vention of low-energy fractures with e.g. bisphosphonates, there is still a clinical need to 7 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 doi:10.1371/journal.pone.0154249.t002 The first and most obvious is that these variables are merely markers of poor physical fitness or an impaired health status, which in turn would be correlated with traditional risk factors for fragility frac- tures such as diabetes or smoking [15, 16]. However, the association between RHR and mortal- ity has been found to hold true irrespective of physical fitness [9] meaning that RHR is likely to convey an independent prognostic value. As expected, RHR correlated strongly with a number of variables in our study population (S5 Table). However, the effect size of RHR on predicting low-energy fractures was not reduced when orthostatic SBP-response or other factors suppos- edly associated with impaired health were included in the model. Furthermore, the strong pre- dictive value of RHR on incident low-energy fractures persisted when analyses were done separately in untreated hypertensives as well as in normotensives (S4 Table). This supports the hypothesis that RHR is not only a mere marker of impaired health status. Accordingly, we pro- pose that a higher RHR may be a marker of subtle autonomic dysfunction associated with sym- pathetic hyper-activation, an explanation in parallel with that between elevated RHR and CVD [9, 23]. Advancing age, diabetes, hypertension—other CVD risk factors that are all associated 8 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Orthostatic Hypotension, Heart Rate and Fractures Fig 3. Quartile specific hazard ratios concerning resting heart rate and orthostatic change in systolic blood pressure (SBP) in relation to first incident low-energy fracture. Hazard ratio = 1.00 (reference) for quartile 1/1 of resting heart rate/ΔSBP. Model includes covariates age, sex, body-mass-index, antihypertensive treatment, smoking, diabetes, previous myocardial infarction, SBP in supine. Results are detailed in S3 Table. d i 10 1371/j l 0154249 003 Fig 3. Quartile specific hazard ratios concerning resting heart rate and orthostatic change in systolic blood pressure (SBP) in relation to first incident low-energy fracture. Hazard ratio = 1.00 (reference) for quartile 1/1 of resting heart rate/ΔSBP. Model includes covariates age, sex, body-mass-index, antihypertensive treatment, smoking, diabetes, previous myocardial infarction, SBP in supine. Results are detailed in S3 Table. doi:10.1371/journal.pone.0154249.g003 doi:10.1371/journal.pone.0154249.g003 with OH—share the potential to impair or override autonomic mechanisms [3] resulting in a higher RHR. Thus, subclinical forms of the above conditions may independently and additively influence RHR. PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 Orthostatic Hypotension, Heart Rate and Fractures changes may partly be determined by genetic factors associated with cardiac and neuroendo- crine conditions [26, 27]. Regarding the long time span between baseline screening and the index event, a similar tem- poral delay was observed in MPP in relation to all-cause mortality, and cardiovascular events such as myocardial infarction [8]. Thus, the signs of subclinical cardiovascular dysautonomia may herald increased risks many years in advance, even if the symptoms may not be detectable. This might be due to compensatory mechanisms such as cerebral autoregulation, protecting the brain tissue against systemic hemodynamic fluctuations [28]. Consequently, effects of car- diovascular dysautonomia may not be directly perceived by an affected individual but ageing may eventually provoke failure of compensatory mechanisms and unexpected fall leading to trauma. Although asymptomatic to a large extent, OH is a clinically well recognized problem, espe- cially in the elderly in whom the consequences of OH (such as a fracture) may frequently be the first manifestation [4]. Various drugs have been tested as symptomatic relief of OH, how- ever, without convincing data and international consensus on their efficacy [5]. Concerning heart rate regulation, beta-blockers have a role in the primary and secondary prevention and treatment of most types of CVD [29] and are sometimes used as symptomatic treatment in inappropriate sinus tachycardia (IST) and POTS, as is the heart rate modulating drug Ivabra- dine [25]. Whether or not heart rate modulating drugs may have a role in treating subtle auto- nomic dysfunction remains to be tested. Regardless of these considerations, OH and RHR may, as previously discussed, possibly enhance decision making of fracture-preventive therapy in order to reduce a consequence of such subtle autonomic dysfunction. The main strengths of this study are the large number of subjects, long follow-up and access to reliable case data. The major limitation of this study is the lack of data on heart rate and 3-min BP on standing which were not recorded in MPP. Naturally, the current guidelines on OH [2] were not available at the time of the baseline examination of MPP. Another limitation is that we assumed that most cases of low-energy-fractures were indeed caused by a fall pro- voked by reduced cerebral circulation during a maladaptive BP response. However, some low- energy-fractures are likely to occur in other settings. Finally, for the whole MPP cohort ortho- static response was examined only at baseline, indicating that we have been unable to evaluate how prospective changes in hemodynamic parameters were related to outcome. In conclusion, we have shown that orthostatic blood pressure decline, elevated resting heart rate and their combination are strong independent predictors of low-energy fractures in a mid- dle-aged population and as such, potential signs of subtle autonomic dysfunction in these sub- jects. Orthostatic blood pressure response and resting heart rate deserve consideration as new tools for risk prediction in order to obtain more accurate clinical decisions on non-pharmaco- logical and pharmacological primary prevention of low-energy fractures. Also, elevated heart rate may be an expression of impaired baroreflex function, involved in the pathogenesis of OH [24], and characterized by enhanced sympathetic activity and withdrawal of parasympathetic control. Regardless of whether subclinical CVD or a mal- functioning baroreflex is the cause, a higher RHR is likely to indicate the presence of autonomic dysfunction. We hypothesize that when this initially subtle autonomic dysfunction evolves to a manifest condition, it may eventually lead to overt OH or possibly arrhythmias causing falls and fall-related injuries. Alternative mechanisms that might link elevated RHR with fall-related injuries include the possibility of an undiagnosed state that increases the risk of pre-syncope and syncope such as postural orthostatic tachycardia syndrome (POTS) [25]. Also, a higher heart rate implies that the cardiovascular system is already challenged at rest. Thus, subjects with a higher heart rate may be prone to cerebral hypoperfusion during orthostatic challenge, due to reduced circula- tory reserve. The elevated heart rate at rest and limited capacity for tolerating orthostatic PLOS ONE \| DOI:10.1371/journal.pone.0154249 April 28, 2016 9 / 12 References 1. Goldstein DS, Robertson D, Esler M, Straus SE, Eisenhofer G. Dysautonomias: clinical disorders of the autonomic nervous system. Annals of internal medicine. 2002; 137(9):753–63. PMID: 12416949 2. Freeman R, Wieling W, Axelrod FB, Benditt DG, Benarroch E, Biaggioni I, et al. 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https://openalex.org/W2278836975	https://europepmc.org/articles/pmc4747557?pdf=render	English	null	High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling	PloS one	2,016	cc-by	9,433	High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Xin Fu1, Fang-Nan Xie1, Ping Dong1, Qiu-Chen Li1, Guang-Yan Yu2, Ran Xiao1* 1 Research Center of Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 33 Ba Da Chu Road, Beijing, 100144, PR China, 2 Department of Oral and Maxillofacial Surgery, Peking University School of Stomatology, 22 Zhong Guan Cun South St., Beijing, 100081, PR China * xiaoran@psh.pumc.edu.cn * xiaoran@psh.pumc.edu.cn Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by grants from the National Natural Science Foundation of China (http://www.nsfc.gov.cn/) with grants numbers as 31201102 (XF), 30871433 (RX) and 31071305 (RX); and from International S&T Cooperation Program of China (http://www.istcp.org.cn) with grant number as 2011DFA32190 (GYY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However, insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs), which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore, we investigated the effects of sodium fluoride (NaF) on the proliferation, differentiation and viability of H9 hESCs. For the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology, mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and acti- vation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway, coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a p- JNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs. OPEN ACCESS Citation: Fu X, Xie F-N, Dong P, Li Q-C, Yu G-Y, Xiao R (2016) High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling. PLoS ONE 11(2): e0148819. doi:10.1371/ journal.pone.0148819 Editor: Yingmei Feng, Katholieke Universiteit Leuven, BELGIUM Received: October 30, 2015 Accepted: January 22, 2016 Published: February 9, 2016 Copyright: © 2016 Fu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Fu X, Xie F-N, Dong P, Li Q-C, Yu G-Y, Xiao R (2016) High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling. PLoS ONE 11(2): e0148819. OPEN ACCESS Citation: Fu X, Xie F-N, Dong P, Li Q-C, Yu G-Y, Xiao R (2016) High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling. PLoS ONE 11(2): e0148819. doi:10.1371/ journal.pone.0148819 Copyright: © 2016 Fu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. RESEARCH ARTICLE Abstract doi:10.1371/ journal.pone.0148819 Editor: Yingmei Feng, Katholieke Universiteit Leuven, BELGIUM Received: October 30, 2015 Accepted: January 22, 2016 Published: February 9, 2016 Copyright: © 2016 Fu 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. Effects of Fluoride on Human Embryonic Stem Cells systemic exposure to fluorides can lead to the disturbance of bone homeostasis (skeletal fluoro- sis) and enamel development (dental/enamel fluorosis) [4]. Similarly, acute and high-dose exposure to fluorides can result in renal toxicity [5], liver damage [6], neurological defects [7], reproductive toxicity [8], infertility [9] and mental retardation [10]. Moreover, high-dose fluo- ride can readily cross the placental barrier to directly damage the developing mammalian fetus, thus resulting in embryonic and fetal developmental abnormalities in a number of species, including frogs [11] rats [12] and mice [13] Positive correlations between the fluoride content systemic exposure to fluorides can lead to the disturbance of bone homeostasis (skeletal fluoro- sis) and enamel development (dental/enamel fluorosis) [4]. Similarly, acute and high-dose exposure to fluorides can result in renal toxicity [5], liver damage [6], neurological defects [7], reproductive toxicity [8], infertility [9] and mental retardation [10]. Moreover, high-dose fluo- ride can readily cross the placental barrier to directly damage the developing mammalian fetus, thus resulting in embryonic and fetal developmental abnormalities in a number of species, including frogs [11], rats [12] and mice [13]. Positive correlations between the fluoride content and pathological changes in the femur of aborted human fetuses have also been reported [14]. Numerous epidemiological and clinical studies have also demonstrated that high-dose fluo- rides could lead to changes in teeth and bone structure and adversely affect neurodevelopment by lowering the intelligence quotient (IQ) in children [10]. All of these findings suggested that high-dose fluoride could influence the development of the human embryo [11]. However, very little is known about the potential developmental toxicity and the underlying mechanism of high-dose fluorides on the early development of human embryos due to the lack of appropriate humanized models. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: hESCs, human embryonic stem cells; MSC, mesenchymal stem cell; EB, embryoid body; NaF, sodium fluoride; RT-PCR, real-time polymerase chain reaction; ROS, reactive oxygen species; MMP, mitochondrial membrane potential. The successful in vitro culture of pluripotent human embryonic stem cells (hESCs) isolated from human blastocyst [15] created a new avenue to analyze the cytotoxicity and embryotoxi- city of chemical compounds and substances in humans [16], as the in vitro differentiation of hESCs can partially recapitulate cellular developmental processes and gene expression patterns of early human embryogenesis [17]. Introduction Fluorides are inorganic and organic fluorine compounds that are widely used in numerous dental products for the prevention and remineralization of dental caries [1–2]. Low-dose fluo- rides are beneficial to bone health and have been used in the treatment of age-related osteopo- rosis for the last 40 years [3]. However, the biphasic actions of fluoride suggest that excessive 1 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 For instance, formation of embryoid bodies (EBs), which are cell aggregates produced during the course of hESCs differentiation in suspension [18], indicating the onset of differentiation of hESCs during early embryogenesis [17]. Accumulating evidence also indicates that EB is spatially and temporally patterned [18] and the expressions of developmental marker genes can be used to define the EB morphogenesis [19–20]. There- fore, hESCs-based systems are currently being explored as alternatives for assessing the embry- otoxic potential of compounds [21–23]. The effects of fluoride on cellular metabolism and physiology are diverse and are dependent upon the cell type, duration of exposure and concentration [24]. For instance, low-dose fluo- ride can exhibit a specific mitogenic effect on osteoblasts [25], enhance the osteoblastic differ- entiation of mesenchymal stem cells (MSC) [26], and induce the early differentiation of murine bone marrow cells along the granulocytic pathway [27]. In contrast, growth arrest and apoptosis induction are among the most common toxic effects of high fluoride levels on many types of cells, including ameloblasts [28], osteoblasts [29], epithelial cells [30] and mouse embryonic stem cells [31]. Elucidating the effects of high-dose fluorides on hESCs is thus important for understanding the impairment of early human embryogenesis. In this study, we examined the differentiation, proliferation, viability and apoptosis of H9 hESCs under treatment with different concentrations of sodium fluoride (NaF). Furthermore, the mechanism of high-dose NaF on apoptosis in hESCs was investigated by assessing the mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). Our findings sug- gested that high-dose NaF suppressed proliferation and induced apoptosis in hESCs and dis- turbed the gene expression patterns of hESCs during EB differentiation. High-dose NaF- induced apoptosis in hESCs is facilitated by MAPK-mediated and caspase-dependent pathways and is independent of ROS production. To our knowledge, this is the first report to study the effects of high-dose fluorides on the biological characteristics of hESCs. Our findings may pro- vide new insights into the mechanism of high-dose NaF-induced toxicities during early human embryogenesis. In this study, we examined the differentiation, proliferation, viability and apoptosis of H9 hESCs under treatment with different concentrations of sodium fluoride (NaF). Furthermore, the mechanism of high-dose NaF on apoptosis in hESCs was investigated by assessing the mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Our findings sug- gested that high-dose NaF suppressed proliferation and induced apoptosis in hESCs and dis- turbed the gene expression patterns of hESCs during EB differentiation. High-dose NaF- induced apoptosis in hESCs is facilitated by MAPK-mediated and caspase-dependent pathways d i i d d f ROS d i T k l d hi i h fi d h 2 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Effects of Fluoride on Human Embryonic Stem Cells Real-Time Polymerase Chain Reaction (PCR) For cDNA synthesis, 500 ng of RNA was reverse transcribed using M-MLV reverse transcrip- tase (Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. Real- time PCR was performed using the Fast SYBR Green Master Kit and LightCycler 480 system (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The expression level of each gene was normalized to the expression of GAPDH. Primer sequences were summarized in Table 1. Chemicals Fluorescent probe JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-imidacarbocyanine iodide) and inhibitors for pan-caspase (z-VAD-fmk), JNK (SP600125) and ERK (PD98059) were pur- chased from Beyotime (Jiangsu, China) and dissolved in dimethyl sulfoxide (DMSO). Unless otherwise specified, the chemicals used in this study were purchased from Sigma (St. Louis, MO, USA). Cell Culture The H9 hESCs (WA09) was obtained from the WiCell Research Institute (Madison, WI, USA) under a Materials Transfer Agreement (No. 11-W0039). Cells were cultured on plates pre- coated with Matrigel (BD, Franklin Lakes, NJ, USA) and maintained in mTeSR-1 medium (StemCell Technologies, Vancouver, Canada) with daily medium change. When the cells reached 90% confluence, cells were scraped from the plate mechanically after dispase digestion (1 mg/ml, 2–3 min) and seeded on a new Matrigel-coated plate at a splitting ratio of 1:6. EB Formation Upon reaching 90% confluence, hESCs were digested with 1 mg/ml dispase and scraped from the plate, followed by suspension culture in EB differentiation medium containing DMEM/F- 12 (Invitrogen, Carlsbad, CA, USA), 20% knockout serum replacement (Invitrogen), 1 mM L-glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen) and 0.1 mM β-mercap- toethanol. After 24 h, EB medium was supplemented with 0.5 or 1 mM of NaF, and the media were changed every other day. EB samples were collected on the 5th day (5D), 7th day (7D) and 14th day (14D) for further analysis. The circularities of 5D EB were measured from 50 ran- domly selected EBs that formed in each culture condition using Adobe Photoshop CS3, San Jose, CA, USA). Cell Proliferation and Viability Assay The hESCs (5×103 cells/well) were plated into 96-well plates containing 100 μl of mTeSR-1 medium and exposed to increasing concentrations of NaF (1~6 mM) at 37°C for 24 to 120 h in the presence or absence of pharmacological inhibitors. The number of viable cells was quanti- fied by the CellTiter 96 AQueous One Solution Cell Proliferation kit (Promega, Wisconsin, USA) following the manufacturer’s instructions. In brief, 20μl of (3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added in each well and incubated for 4 h at 37°C. The absorbance was measured at 490 nm on a PerkinElmer EnSpireTM Multimode Plate Reader. All experiments were repeated three times with triplicates in each experiment. 3 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Effects of Fluoride on Human Embryonic Stem Cells Table 1. List of primers used in the real-time PCR. Genes Primer sequences 4-Oct F: 5’-CCCCTGGTGCCGTGAA-3’ R: 5’-GCAAATTGCTCGAGTTCTTTCTG-3’ Nanog F: 5’-ATGCCTCACACGGAGACTGT-3’ R: 5’-AAGTGGGTTGTTTGCCTTTG-3’ SOX2 F: 5'-TTGCTGCCTCTTTAAGACTAGGA-3’ R: 5'-CTGGGGCTCAAACTTCTCTC-3’ NeuroD1 F: 5’-CGCTGGAGCCCTTCTTTG-3’ R: 5’-GCGGACGGTTCGTGTTTG-3’ Brachyury F: 5’-CCTCYCCCYCCCCYCCACGC-3’ R: 5’-GGTGGGCTGGCATTGTGGCT-3’ AFP F: 5’-TGCAGCCAAAGTGAAGAGGGAAGA-3’ R: 5’-CATAGCGAGCAGCCCAAAGAAGAA-3’ NOS F: 5’-CACTCAGCTGTGCATCGAC-3’ R: 5’-CAGTTCCCGAAACCACTCGT-3’ SOD2 F: 5’-AACAACCTGAACGTCACCGA-3’ R: 5’-AGCAACTCCCCTTTGGGTTC-3’ CYBA F: 5’-GAGCGGCATCTACCTACTGG-3’ R: 5’-TGATGGTGCCTCCGATCT-3’ PRDX5 F: 5’-GTTCGGCTCCTGGCTGATCC-3’ R: 5’-TGCCATCCTGTACCACCAT-3’ RUNX2 F: 5’-ACTGGCGCTGCAACAAGAC-3’ R: 5’-CCCGCCATGACAGTAACCA-3’ OPN F: 5’-CAGCAACCGAAGTTTTCACTCCAG-3’ R: 5’-CACCATTCAACTCCTCGCTTTCC-3’ COL1A F: 5’-GTGAACCTGGTGCTCCTG-3’ R: 5’-GTGAACCTGGTGCTCCTG-3’ PPAR-γ F: 5’-TGGAATTAGATGACAGCGACTTGG-3’ R: 5’-CTGGAGCAGCTTGGCAAACA-3’ CEBP/α F: 5’-AGGAACACGAAGCACGATCA-3’ R: 5’-ACAGAGGCCAGATACAAGTG-3’ Adiponectin F: 5’-CTTCCGTCACCTCTAAATCC-3’ R: 5’-GTCATCCCTAACTTCAGTGG-3’ GAPDH F: 5’-GCACCGTCAAGGCTGAGAAC-3’ R: 5’-TGGTGAAGACGCCAGTGGA-3 The oligonucleotide sequences of primers used in the real-time PCR analysis Table 1. List of primers used in the real-time PCR. Genes Primer sequences 4-Oct F: 5’-CCCCTGGTGCCGTGAA-3’ R: 5’-GCAAATTGCTCGAGTTCTTTCTG-3’ Nanog F: 5’-ATGCCTCACACGGAGACTGT-3’ R: 5’-AAGTGGGTTGTTTGCCTTTG-3’ SOX2 F: 5'-TTGCTGCCTCTTTAAGACTAGGA-3’ R: 5'-CTGGGGCTCAAACTTCTCTC-3’ NeuroD1 F: 5’-CGCTGGAGCCCTTCTTTG-3’ R: 5’-GCGGACGGTTCGTGTTTG-3’ Brachyury F: 5’-CCTCYCCCYCCCCYCCACGC-3’ R: 5’-GGTGGGCTGGCATTGTGGCT-3’ AFP F: 5’-TGCAGCCAAAGTGAAGAGGGAAGA-3’ R: 5’-CATAGCGAGCAGCCCAAAGAAGAA-3’ NOS F: 5’-CACTCAGCTGTGCATCGAC-3’ R: 5’-CAGTTCCCGAAACCACTCGT-3’ SOD2 F: 5’-AACAACCTGAACGTCACCGA-3’ R: 5’-AGCAACTCCCCTTTGGGTTC-3’ CYBA F: 5’-GAGCGGCATCTACCTACTGG-3’ R: 5’-TGATGGTGCCTCCGATCT-3’ PRDX5 F: 5’-GTTCGGCTCCTGGCTGATCC-3’ R: 5’-TGCCATCCTGTACCACCAT-3’ RUNX2 F: 5’-ACTGGCGCTGCAACAAGAC-3’ R: 5’-CCCGCCATGACAGTAACCA-3’ OPN F: 5’-CAGCAACCGAAGTTTTCACTCCAG-3’ R: 5’-CACCATTCAACTCCTCGCTTTCC-3’ COL1A F: 5’-GTGAACCTGGTGCTCCTG-3’ R: 5’-GTGAACCTGGTGCTCCTG-3’ PPAR-γ F: 5’-TGGAATTAGATGACAGCGACTTGG-3’ R: 5’-CTGGAGCAGCTTGGCAAACA-3’ CEBP/α F: 5’-AGGAACACGAAGCACGATCA-3’ R: 5’-ACAGAGGCCAGATACAAGTG-3’ Adiponectin F: 5’-CTTCCGTCACCTCTAAATCC-3’ R: 5’-GTCATCCCTAACTTCAGTGG-3’ GAPDH F: 5’-GCACCGTCAAGGCTGAGAAC-3’ R: 5’-TGGTGAAGACGCCAGTGGA-3 The oligonucleotide sequences of primers used in the real-time PCR analysis The oligonucleotide sequences of primers used in the real-time PCR analysis Cell Apoptosis Analysis by Annexin V/propidium iodide (PI) Staining The hESCs exposed to NaF were incubated with the Muse Annexin V Dead Cell Kit (EMD Millipore, Billerica, MA, USA) following the manufacturer’s instructions. The percentages of apoptotic cells were analyzed with a program for Annexin V and dead cells on the Muse Cell Analyzer (EMD Millipore). Measurement of Intracellular ROS A stock solution of 2,7-dichlorodihydrofluorescein-diacetate (DCFH-DA, 25 mM) was pre- pared in DMSO and stored at -20°C in the dark. The hESCs exposed to increasing concentra- tions of NaF (1~4 mM) were incubated with DCFH-DA (5 μM) for 10 min. The single cell suspension was prepared after accutase treatment and 1×104 events/cell sample were recorded by FACSAria™II (BD). The percentage of green-fluorescence positive cells and the mean value of green fluorescence intensities were calculated using the Flowjo v7.6.1 software (Tree Star, OR, USA). Western Blot Analysis After NaF treatment, total protein from hESCs was isolated using the mirVana™miRNA Isola- tion kit (Life technologies). Protein lysates (50 μg/sample) were analyzed by western blot using primary antibodies, including mouse anti-human JNK (SC-7345, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human p-JNK (SC-293136, Santa Cruz Biotechnology), rabbit anti-human ERK (GTX59618, Genetex, Irvine, CA), rabbit anti-human p-ERK (2219–1, Epi- tomics, Burlingame, CA), rabbit anti-human BCL-2 (GTX100064, Genetex), rabbit anti- human BAX (GTX109683, Genetex) and mouse anti-human GAPDH (TA08, ZSGB-BIO, Beijing, China). The horseradish peroxidase (HRP)–conjugated donkey anti-mouse IgG (ZB5305, ZSGB-BIO) or donkey anti-rabbit IgG (ZB5301, ZSGB-BIO) were used as secondary antibodies. SuperSignal West Pico Trial Kit (Thermo Scientific, Rockford, IL) was applied for protein detection. The intensity of individual bands was quantified using the ImageJ densitom- etry software. Transmission Electron Microscopy (TEM) Alternations in the ultramicrostructure of the hESCs after NaF exposure were observed by TEM. In brief, the cells were dissociated into single cells by accutase (Stemcell Technologies) PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 4 / 15 Effects of Fluoride on Human Embryonic Stem Cells treatment and centrifuged to remove supernatant. The cell pellets were then fixed with 2.5% glutaraldehyde overnight at 4°C, followed by fixation, staining, dehydration, embedding and sectioning as described previously [32]. The ultrathin sections were stained with uranyl acetate and lead citrate and then observed by TEM (H-7650, Hitachi, Japan) at 80 kV. Measurement of MMP and Caspase Activity The hESCs (1×104 cells/well) were seeded into 96-well plates and cultured in mTeSR-1 medium for 4 days. On day 5, hESCs were exposed to increasing concentrations of NaF (0.5, 1, 2 and 4 mM) for 24 h. To run the JC-1 assay, cells were rinsed with PBS once and then incu- bated with JC-1 working solution (mTeSR-1 medium supplemented with 10 μg/ml JC-1) at 37°C for 1 h, followed by two washes with PBS. The mTeSR-1 medium was then added back to the plate, and the fluorescence intensities of the cells were monitored at Ex/Em = 490/525 nm (green) and 490/590 (red) nm on a Multimode Plate Reader. The ratio of red-to-green fluores- cence was calculated, and the loss of MMP was indicated by the decrease in the ratio. The cas- pase activities of the cells were assessed by the Caspase-Glo1 3/7 Assay System (Promega, Wisconsin, USA) in the absence or presence of 20 μM z-VAD-fmk. NaF Affected the Gene Expression Patterns of hESCs during EB Differentiation This study was the first to examine the effects of NaF on the biological characteristics of H9 hESCs, including the morphology, expression of pluripotent markers (OCT4, NANOG and SOX2) and differentiation potential by inducing the differentiation of hESCs into EB. Observa- tions at 40× magnification showed that H9 hESCs grew as compact colonies with distinct bor- ders in both the untreated (0 mM) and NaF-treated (1 mM, 2 mM) groups; in contrast, at higher magnification (200×), the untreated hESCs exhibited typical hESC morphology (small and tightly packed with prominent nuclei), but the NaF-treated hESCs became larger and flat- tened (Fig 1A). The expression of pluripotent markers (OCT4, NANOG and SOX2) in hESCs was not significantly affected after NaF treatment (Fig 1B). After 5 days of hESC differentia- tion, the EBs exhibited a circular shape in both untreated and 0.5 mM NaF-treated culture but appeared irregular in the 1 mM NaF-treated culture, with a significant decrease in circularity (, p < 0.001, Fig 1C). Moreover, 1 mM NaF significantly up-regulated the expressions of the ectoderm marker NeuroD1 in 14D EB and the mesoderm marker Brachyury in 7D and 14D EB but markedly decreased the expression of the endoderm marker AFP in 14D EB (Fig 1Di). Fig 1. Sodium Fluorides (NaF) affected the differentiation of H9 hESCs. (A) The morphology of hESCs was characterized under an inverted microscope. NaF-treated hESCs were larger and flattened than untreated cells were. (B) The expressions of pluripotent markers (OCT4, NANOG and SOX2) in untreated and NaF-treated hESCs were comparable, as quantified by real-time polymerase chain reaction (PCR). (C) The morphology of embryoid bodies (EBs) derived from hESCs after 5 days (5D) of differentiation in the presence or absence of NaF. (i) 5D EBs in untreated and 0.5 mM NaF-treated groups exhibited a circular in shape while EBs in the 1 mM NaF-treated group were irregular in shape. (ii) The circularities of 5D EBs in the 1 mM NaF group were significantly lower than in the untreated and 0.5 mM NaF-treated groups. (D) The gene expression patterns of EBs were disturbed by high-dose NaF treatment. (i) The expression of three germ layer markers (Ectoderm: NeuroD1, Mesoderm: Brachyury, Endoderm: AFP) in EBs. (ii) Expression of osteogenesis markers (RUNX2, OPN and COL1A) in EBs. (iii) Expression of adipogenesis markers (PPAR-γ, CEBP/α, Adiponectin) in EBs. 40×: 40× magnification. 200×: 200× magnification. , p < 0.05. , p < 0.01. Statistical Analysis Statistical analysis was performed using the SPSS statistical software package. Differences were evaluated by an independent Student’s t-test and paired t-test. A p-value < 0.05 was considered statistically significant. 5 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Effects of Fluoride on Human Embryonic Stem Cells doi:10.1371/journal.pone.0148819.g001 NaF Affected the Gene Expression Patterns of hESCs during EB Differentiation (C) Cell viability of hESCs was dramatically decreased in the 2, 4 and 6 mM NaF-treated groups, with a time-dependent reduction in the 2 mM NaF-treated group. , p < 0.05. , p < 0.01. , p < 0.001. Fig 2. High-dose NaF impaired the proliferation and viability of hESCs. (A) Proliferation of hESCs in the 2 mM NaF-treated group was significantly lower than in the untreated and 1 mM NaF-treated groups. (B) Cell cycle arrest in G0/G1 phase was induced in H9 hESCs after exposure to 2 and 4 mM NaF. (C) Cell viability of hESCs was dramatically decreased in the 2, 4 and 6 mM NaF-treated groups, with a time-dependent reduction in the 2 mM NaF-treated group. , p < 0.05. , p < 0.01. , p < 0.001. doi:10.1371/journal.pone.0148819.g002 Notably, 1 mM NaF could also disturb the osteogenesis of the EBs by significantly up-regulat- ing the osteogenesis markers RUNX2, OPN and COL1A at different time points (, p < 0.05, Fig 1Dii). However, no significant impact of NaF on the expression of adipogenesis markers (PPAR-γ, CEBP/α, Adiponectin) in EBs was detected (Fig 1Diii). Notably, 1 mM NaF could also disturb the osteogenesis of the EBs by significantly up-regulat- ing the osteogenesis markers RUNX2, OPN and COL1A at different time points (, p < 0.05, Fig 1Dii). However, no significant impact of NaF on the expression of adipogenesis markers (PPAR-γ, CEBP/α, Adiponectin) in EBs was detected (Fig 1Diii). Notably, 1 mM NaF could also disturb the osteogenesis of the EBs by significantly up-regulat- ing the osteogenesis markers RUNX2, OPN and COL1A at different time points (, p < 0.05, Fig 1Dii). However, no significant impact of NaF on the expression of adipogenesis markers (PPAR-γ, CEBP/α, Adiponectin) in EBs was detected (Fig 1Diii). High-Dose NaF Affected the Proliferation and Viability of hESCs To assess the influence of high-dose NaF on hESC proliferation, the number of viable cells was determined by measuring the absorbance at 490 nm using an MTS assay during the experimen- tal periods. A time-dependent increase in the absorbance was observed in both the untreated and 1 mM NaF-treated groups, but in the 2 mM NaF-treated group, the proliferation of hESCs was markedly suppressed after 96 h of culture (Fig 2A, , p < 0.05). Flow cytometry analysis after PI staining showed that 2 and 4 mM NaF could induce cell cycle arrest in hESCs, thus leading to a significant increase in the number of cells in G0/G1 phase with a concomitant decrease of cells in S and G2/M phase (Fig 2B, , p < 0.05, , p < 0.01, , p < 0.001). Consis- tently, the viability of hESCs was dramatically decreased in the 2, 4 and 6 mM NaF-treated groups, with a time-dependent reduction in the 2 mM NaF-treated group (Fig 2C, , p < 0.05, , p < 0.01, , p < 0.001). NaF Affected the Gene Expression Patterns of hESCs during EB Differentiation *, p < 0.001. Fig 1. Sodium Fluorides (NaF) affected the differentiation of H9 hESCs. (A) The morphology of hESCs was characterized under an inverted microscope. NaF-treated hESCs were larger and flattened than untreated cells were. (B) The expressions of pluripotent markers (OCT4, NANOG and SOX2) in untreated and NaF-treated hESCs were comparable, as quantified by real-time polymerase chain reaction (PCR). (C) The morphology of embryoid bodies (EBs) derived from hESCs after 5 days (5D) of differentiation in the presence or absence of NaF. (i) 5D EBs in untreated and 0.5 mM NaF-treated groups exhibited a circular in shape while EBs in the 1 mM NaF-treated group were irregular in shape. (ii) The circularities of 5D EBs in the 1 mM NaF group were significantly lower than in the untreated and 0.5 mM NaF-treated groups. (D) The gene expression patterns of EBs were disturbed by high-dose NaF treatment. (i) The expression of three germ layer markers (Ectoderm: NeuroD1, Mesoderm: Brachyury, Endoderm: AFP) in EBs. (ii) Expression of osteogenesis markers (RUNX2, OPN and COL1A) in EBs. (iii) Expression of adipogenesis markers (PPAR-γ, CEBP/α, Adiponectin) in EBs. 40×: 40× magnification. 200×: 200× magnification. , p < 0.05. , p < 0.01. , p < 0.001. doi:10.1371/journal.pone.0148819.g001 doi:10.1371/journal.pone.0148819.g001 6 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Effects of Fluoride on Human Embryonic Stem Cells Fig 2. High-dose NaF impaired the proliferation and viability of hESCs. (A) Proliferation of hESCs in the 2 mM NaF-treated group was significantly lower than in the untreated and 1 mM NaF-treated groups. (B) Cell cycle arrest in G0/G1 phase was induced in H9 hESCs after exposure to 2 and 4 mM NaF. (C) Cell viability of hESCs was dramatically decreased in the 2, 4 and 6 mM NaF-treated groups, with a time-dependent reduction in the 2 mM NaF-treated group. , p < 0.05. , p < 0.01. , p < 0.001. Fig 2. High-dose NaF impaired the proliferation and viability of hESCs. (A) Prolifer Fig 2. High-dose NaF impaired the proliferation and viability of hESCs. (A) Proliferation of hESCs in the 2 mM NaF-treated group was significantly lower than in the untreated and 1 mM NaF-treated groups. (B) Cell cycle arrest in G0/G1 phase was induced in H9 hESCs after exposure to 2 and 4 mM NaF. High-Dose NaF Induced Apoptosis in hESCs doi:10.1371/journal.pone.0148819.g003 doi:10.1371/journal.pone.0148819.g003 characteristics of the early phases of apoptosis, including nuclear fragmentation (black arrow) and chromatin condensation (white arrow), were observed in 0.5 and 1 mM NaF-treated hESCs (Fig 3B). Additionally, 2 mM NaF-treated hESCs showed characteristics of late stage of apoptosis, including shrinkage and blebbing of the plasma membrane (red arrowhead), cyto- plasmic vacuoles (yellow arrowhead) and the formation of apoptotic bodies (black arrowhead). h h h d d d f h f d b h f characteristics of the early phases of apoptosis, including nuclear fragmentation (black arrow) and chromatin condensation (white arrow), were observed in 0.5 and 1 mM NaF-treated hESCs (Fig 3B). Additionally, 2 mM NaF-treated hESCs showed characteristics of late stage of apoptosis, including shrinkage and blebbing of the plasma membrane (red arrowhead), cyto- plasmic vacuoles (yellow arrowhead) and the formation of apoptotic bodies (black arrowhead). The high-dose NaF-induced apoptosis was further verified by measuring the activities of caspase3/7, the cellular levels of MMP and the expression of apoptosis regulators (BCL-2 and Bax) in hESCs. The caspase3/7 activity was significantly increased in 2 and 4 mM NaF-treated hESCs, which could be inhibited by the pan-caspase inhibitor z-VAD-fmk (Fig 3C). In these two NaF-treated groups, mitochondrial function analysis also revealed a significant decrease in both the cellular MMP levels and the Bcl-2/BAX ratio in hESCs (Fig 3D). y The high-dose NaF-induced apoptosis was further verified by measuring the activities of caspase3/7, the cellular levels of MMP and the expression of apoptosis regulators (BCL-2 and Bax) in hESCs. The caspase3/7 activity was significantly increased in 2 and 4 mM NaF-treated hESCs, which could be inhibited by the pan-caspase inhibitor z-VAD-fmk (Fig 3C). In these two NaF-treated groups, mitochondrial function analysis also revealed a significant decrease in both the cellular MMP levels and the Bcl-2/BAX ratio in hESCs (Fig 3D). High-Dose NaF Induced Apoptosis in hESCs Because a reduction in cell viability indicates the occurrence of cell death, NaF-treated hESCs were analyzed by flow cytometry after Annexin V/PI staining. The percentage of cells gated in G4 (early apoptotic cells) and G3 (late apoptotic cells) increased after exposure to NaF (Fig 3A). The TEM results confirmed that the untreated hESCs exhibited normal morphologies with an intact plasma membrane and clear nucleus. In contrast, the morphological 7 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Effects of Fluoride on Human Embryonic Stem Cells Fig 3. High-dose NaF induced the occurrence of apoptosis in H9 hESCs. (A) The percentage of early apoptotic cells (G3) and late apoptotic cells (G4) was significantly increased after exposure to 2 mM and 4 mM NaF, as demonstrated by the Annexin V/ Propidium Iodide staining assay. (B) The sub-cellular morphology of hESCs was characterized by transmission electron microscopy, and the data showed that NaF-treated hESCs exhibited morphological and nuclear features typical of apoptosis, including nuclear fragmentation (black arrow), chromatin condensation (white arrow), shrinkage and blebbing of the plasma membrane (red arrowhead), cytoplasmic vacuoles (yellow arrowhead) and the formation of apoptotic bodies (black arrowhead). (C) Caspase activities were significantly increased, whereas the (D) mitochondrial membrane potential (MMP) and the (E) BCL-2/BAX ratio were significantly decreased in hESCs after exposure to 2 and 4 mM NaF for 24 h. , p < 0.05. *, p < 0.01. doi:10 1371/journal pone 0148819 g003 Fig 3. High-dose NaF induced the occurrence of apoptosis in H9 hESCs. (A) The percentage of early apoptotic cells (G3) and late apoptotic cells (G4) was significantly increased after exposure to 2 mM and 4 mM NaF, as demonstrated by the Annexin V/ Propidium Iodide staining assay. (B) The sub-cellular morphology of hESCs was characterized by transmission electron microscopy, and the data showed that NaF-treated hESCs exhibited morphological and nuclear features typical of apoptosis, including nuclear fragmentation (black arrow), chromatin condensation (white arrow), shrinkage and blebbing of the plasma membrane (red arrowhead), cytoplasmic vacuoles (yellow arrowhead) and the formation of apoptotic bodies (black arrowhead). (C) Caspase activities were significantly increased, whereas the (D) mitochondrial membrane potential (MMP) and the (E) BCL-2/BAX ratio were significantly decreased in hESCs after exposure to 2 and 4 mM NaF for 24 h. , p < 0.05. *, p < 0.01. Effects of Fluoride on Human Embryonic Stem Cells Fig 4. Production of intracellular reactive oxygen species (ROS) in hESCs was decreased after NaF treatment. (A) Assessment of ROS levels by 2,7-dichlorodihydrofluorescein-diacetate (DCFH-DA) staining revealed that the percentage of fluorescent dichlorofluorescein (DCF)-positive hESCs was significantly decreased in the 2 and 4 mM NaF-treated groups, and the mean fluorescence intensities of hESCs were significantly lower in the 1, 2 and 4 mM NaF-treated groups. (B) The expression of the cellular antioxidant defense enzyme SOD and the oxidative stress genes PRDX5 and CYBA did not show significant changes after NaF treatment, except for NOS, whose expression was significantly decreased in the 2 and 4 mM NaF-treated hESCs. , p < 0.05. , p < 0.01. , p < 0.001. Fig 4. Production of intracellular reactive oxygen species (ROS) in hESCs was decreased after NaF treatment. (A) Assessment of ROS levels by 2,7-dichlorodihydrofluorescein-diacetate (DCFH-DA) staining revealed that the percentage of fluorescent dichlorofluorescein (DCF)-positive hESCs was significantly decreased in the 2 and 4 mM NaF-treated groups, and the mean fluorescence intensities of hESCs were significantly lower in the 1, 2 and 4 mM NaF-treated groups. (B) The expression of the cellular antioxidant defense enzyme SOD and the oxidative stress genes PRDX5 and CYBA did not show significant changes after NaF treatment, except for NOS, whose expression was significantly decreased in the 2 and 4 mM NaF-treated hESCs. , p < 0.05. , p < 0.01. , p < 0.001. doi:10.1371/journal.pone.0148819.g004 the mean fluorescence intensities of hESCs were significantly lower in the 1, 2 and 4 mM NaF groups than in the untreated group. The expression of the cellular antioxidant defense enzyme SOD and the oxidative stress genes PRDX5, NOS and CYBA were further characterized by real- time PCR, and the results showed that NaF treatment did not alter the expression levels of SOD, PRDX5 and CYBA in hESCs, except for NOS, whose expression was significantly decreased in the 2 and 4 mM NaF-treated hESCs (Fig 4B). High-Dose NaF-Induced Apoptosis in hESCs Was ROS-Independent Because the involvement of increased ROS production has been proven in NaF-induced apo- ptosis in some cell types, the intracellular ROS levels in NaF-treated hESCs were investigated by DCFH-DA staining followed by flow cytometry analysis. The representative flow cytometry histograms showed decreased ROS levels in NaF-treated hESCs in a dose-dependent manner (Fig 4A). Three independent experiments revealed that the percentages of dichlorofluorescein (DCF)-positive cells were significantly decreased in 2 and 4 mM NaF-treated hESCs and that 8 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 JNK-Mediated Signaling Was Involved in High-Dose NaF-Induced Apoptosis in hESCs To investigate the roles of MAPKs in high-dose NaF-induced apoptosis, the activation of MAPK family members, extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) were examined in NaF-treated hESCs by western blot analysis. The results showed that the levels of phosphorylated-JNK1/2 (P-JNK1/2) were notably increased in NaF- treated cells in a time- (Fig 5A) and dose-dependent manner (Fig 5B). P-JNK1/2 levels in 2 mM NaF-treated hESCs reached a peak after 60 min of treatment (Fig 5Aii, , p < 0.05). The levels of phosphorylated-ERK (P-ERK) in hESCs were only mildly increased after exposure to PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 9 / 15 Effects of Fluoride on Human Embryonic Stem Cells Fig 5. High-dose NaF induced apoptosis in hESCs via the mitogen-activated protein kinase (MAPK)- dependent pathway. (A) Time-dependent effects of 2 mM NaF on the activation of MAPKs in hESCs. (i) Western blot analysis showed a time-dependent increase in the levels of phosphorylated-c-Jun N-terminal kinases 1/2 (P-JNK1/2) and a transient increase in the levels of phosphorylated-extracellular signal-regulated kinases (P-ERKs) in 2 mM NaF-treated hESCs. (ii) Three independent experiments demonstrated that the levels of P-JNK1/2, but not P-ERK, were significantly increased after 60 min of NaF treatment. (B) Dose- dependent effects of NaF on the activation of MAPKs in hESCs. (i) Western blot analysis showed a dose- dependent increase in the levels of P-JNK1/2 and a transient increase in the levels of P-ERK in hESCs treated with increasing doses of NaF for 180 min. (ii) Three independent experiments demonstrated no significant difference in the levels of P-JNK1/2 and P-ERK between untreated and NaF-treated hESCs. (C) Verification of the effects of NaF-induced phosphorylation of JNK and ERK on the apoptosis of hESCs. (i) The viabilities of the 2 and 4 mM NaF-treated hESCs were significantly increased by pre-treating hESCs with the P-JNK1/2 specific inhibitor SP600125 but not with the P-ERK specific inhibitor PD98059. (ii) SP600125 but not PD98059 could significantly suppress the decreased cell viability of hESCs exposed to 2 mM NaF for 24 h and 48 h. , p < 0.05. *, p < 0.01. Fig 5. High-dose NaF induced apoptosis in hESCs via the mitogen-activated protein kinase (MAPK)- dependent pathway. (A) Time-dependent effects of 2 mM NaF on the activation of MAPKs in hESCs. JNK-Mediated Signaling Was Involved in High-Dose NaF-Induced Apoptosis in hESCs (i) Western blot analysis showed a time-dependent increase in the levels of phosphorylated-c-Jun N-terminal kinases 1/2 (P-JNK1/2) and a transient increase in the levels of phosphorylated-extracellular signal-regulated kinases (P-ERKs) in 2 mM NaF-treated hESCs. (ii) Three independent experiments demonstrated that the levels of P-JNK1/2, but not P-ERK, were significantly increased after 60 min of NaF treatment. (B) Dose- dependent effects of NaF on the activation of MAPKs in hESCs. (i) Western blot analysis showed a dose- dependent increase in the levels of P-JNK1/2 and a transient increase in the levels of P-ERK in hESCs treated with increasing doses of NaF for 180 min. (ii) Three independent experiments demonstrated no significant difference in the levels of P-JNK1/2 and P-ERK between untreated and NaF-treated hESCs. (C) Verification of the effects of NaF-induced phosphorylation of JNK and ERK on the apoptosis of hESCs. (i) The viabilities of the 2 and 4 mM NaF-treated hESCs were significantly increased by pre-treating hESCs with the P-JNK1/2 specific inhibitor SP600125 but not with the P-ERK specific inhibitor PD98059. (ii) SP600125 but not PD98059 could significantly suppress the decreased cell viability of hESCs exposed to 2 mM NaF for 24 h and 48 h. , p < 0.05. **, p < 0.01. doi:10.1371/journal.pone.0148819.g005 2 mM NaF for 10 min to 30 min or exposure to 1 mM NaF for 180 min, but no significant dif- ference was observed between untreated and NaF-treated hESCs (Fig 5Aii and 5Bii). 2 mM NaF for 10 min to 30 min or exposure to 1 mM NaF for 180 min, but no significant dif- ference was observed between untreated and NaF-treated hESCs (Fig 5Aii and 5Bii). The effects of NaF-induced phosphorylation of JNK1/2 and ERK on apoptotic cell death were further verified by pre-treating H9 hESCs with the P-JNK1/2 inhibitor SP600125 (10 μM) or the P-ERK inhibitor PD98059 (20 μM) for 1 h before NaF treatment. The viabilities of the 2 and 4 mM NaF-treated hESCs were significantly increased by pre-treating hESCs with SP600125 (Fig 5C, , p < 0.05), but not with PD98059. Neither SP600125 nor PD98059 could effectively prevent the cell death of hESCs induced by 6 mM NaF. Moreover, SP600125 could suppress the decreased cell viability of hESCs exposed to 2 mM NaF for 24 h and 48 h, but not for 96 h and 120 h (Fig 5Cii, , p < 0.05). JNK-Mediated Signaling Was Involved in High-Dose NaF-Induced Apoptosis in hESCs In contrast, PD98059 did not attenuate the high-dose NaF-induced cell viability decrease in hESCs by a significant level (Fig 5Cii). Discussion It has been reported that hESCs can recapitulate both cellular developmental processes and gene expression patterns of early embryogenesis during in vitro differentiation [33]. Therefore, hESC is considered as an in vitro model to investigate the molecular mechanisms of embryonic cell differentiation [34–36] and embryotoxicity of developmental toxicants [37]. Previous 10 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Effects of Fluoride on Human Embryonic Stem Cells studies have shown that ESCs require the expression of various transcription factors such as OCT4, NANOG and SOX2 to specify the stem cell state [38–39]. In addition, EB formation recapitulates features of pregastulation and early gastrulation [40–41] and the embryonic dif- ferentiation event is tightly regulated by the actions of specific gene products [20]. Therefore, expression of pluripotent and differentiation-related marker genes have been detected by tran- scriptome analysis to assessed the chemical impact on cell differentiation more sensitively, quantitatively, objectively, and speedily [20–21,23,42–43]. Fluoride is a ubiquitous natural substance that is present in the environment and is often used as an effective prophylactic for dental caries. However, the excess intake of fluorides impairs metabolism and cellular functions in different tissues and organs and ultimately leads to cell death [24]. Although harmful effects of high-dose fluorides on embryonic development have been reported in animal models [11–13] and in the developing brain of human fetuses [14], little infor- mation is available about the effects of fluorides on early human embryogenesis. In our study, for the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb gene expression patterns during EB differentiation by suppressing the expression of endoderm markers while enhancing the expression of ectoderm, mesoderm and osteogenesis markers. Higher doses of NaF (2 mM and above) markedly decreased the viability and prolifera- tion potential of hESCs and led to apoptosis via a ROS-independent and JNK-mediated pathway. p p p p p y Our findings suggest that high-dose NaF might interfere with early human embryogenesis by disturbing both the regulation of the specification of the three germ layers and osteogenic lineage commitment. We observed a marked increase in NEUROD1 expression in 14D EB after 1 mM NaF treatment. PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Discussion Our data demonstrated the reduction of MMP and the BCL-2/BAX ratio coupled with caspase 3/7 activation in high-dose NaF-treated hESCs and indicated that NaF induced apoptosis in hESCs occurs via a mitochondria-mediated and cas- pase-dependent pathway. The mechanisms of fluoride-mediated apoptosis are under extensive study but are still not fully understood due to the complexity and diversity of the molecular events underlying cell– fluoride interactions [54]. Accumulating evidence has suggested that the increased production of cellular ROS is closely involved in high-dose fluoride-induced apoptosis in many cell types [24, 31, 54]. ROS are free radicals and chemically reactive molecules that contain oxygen, are normally generated during mitochondrial oxidative metabolism and induce oxidative stress in cells [55–56]. However, a lack of ROS production during high-dose fluoride-induced apoptosis has also been reported in several cell types [57–58]. Moreover, oxidative stress was absent in patients with skeletal fluorosis [59]. Therefore, the roles of ROS in high-dose fluoride-induced apoptosis might be cell-type dependent. It has been reported that stem cells possess low levels of intracellular ROS because they engage scavenger antioxidant enzyme systems [60]. Our find- ing of a reduced rather than increased production of ROS in NaF-treated hESCs might be par- tially explained by the robust expression of the antioxidant enzyme SOD and the decreased expression of the stress response gene NOS in hESCs. MAPKs are a group of protein serine/threonine kinases that play important roles in com- plex cellular programs such as proliferation, differentiation and apoptosis [61]. Three subfami- lies of MAPKs, including ERKs, JNKs and p38-MAPKs, have been identified, and ERKs have been shown to be important for cell survival, whereas JNKs and p38-MAPKs were deemed to be stress responsive and thus involved in apoptosis [62]. Indeed, a few scientific studies have demonstrated that activation of MAPKs was associated with fluoride-induced apoptosis [30– 31]. In parallel with these reports, our results demonstrated a direct correlation between high- dose NaF-induced apoptotic cell death and JNK-activation in hESCs, thus confirming the involvement of JNK signaling. However, pretreatment with SP600125 could not effectively recover the decreased cell viability of hESCs exposed to 6 mM NaF for 24 h or 2 mM NaF for longer than 48 h. Discussion NEUROD1 is a basic helix-loop-helix transcription factor that has been identified as a differentiation factor for neurogenesis [44] and was found to strikingly coincide with terminal neural differentiation; further, its over-expression could lead to the premature and ectopic neural differentiation of neural stem cells both in vitro and in vivo [45]. Adverse effects of fluorides on neurodevelopment, such as DNA damage in cultured neural cells [7], behavioral deficits in rats [46] and lowered IQ in children [10], have been reported by numer- ous research groups. NaF-induced over-expression of NEUROD1 in hESCs-differentiated EB suggested that the toxicity of high-dose fluorides on human neurodevelopment might be par- tially attributed to the ectopic and premature differentiation of neuronal precursors during early embryogenesis. In addition, NaF is a potent stimulator of ongoing osteogenesis from already differentiated osteoblasts, and it can substitute for a normal bone inducer and permit the initiation of osteoblastic differentiation of MSCs [26] as well as osteogenesis from the embryonic mesenchyme [47–48]. Consistently, our findings showed that 1 mM NaF increased the expression of RUNX2 followed by the up-regulation of OCN and COL1A during EB differ- entiation, thus indicating that NaF could accelerate the osteogenic differentiation of hESCs. Apoptosis, which is also known as programmed cell death, is a complex and highly regu- lated phenomenon that is characterized by a series of cellular processes, including chromatin condensation, DNA fragmentation, mitochondria disintegration, cell shrinkage, membrane blebbing and apoptotic body formation [24]. In our study, TEM analysis showed that high- dose NaF-treated hESCs exhibited morphological and nuclear features typical of apoptosis. Mitochondria are active participants in apoptosis and play central roles in both caspase-depen- dent and caspase-independent death pathways [49–50]. An important mitochondrial event during apoptosis is the reduction of MMP, which is accompanied by the alteration of Bcl-2 family proteins [31, 51]. The Bcl-2 family proteins, whose members may be anti-apoptotic or pro-apoptotic, regulate apoptosis by controlling mitochondrial permeability [52]. The down- regulation of anti-apoptotic proteins such as BCL-2 and the up-regulation of pro-apoptotic proteins such as BAX could cause MMP loss, thus allowing the release of cytochrome c out of the mitochondria and ultimately lead to the activation of caspase3/7, which act as executioners PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 11 / 15 Effects of Fluoride on Human Embryonic Stem Cells to initiate apoptotic cell death [53]. Conclusions In summary, our findings demonstrated that treatment with 1 mM NaF could disturb the gene expression patterns of hESCs during EB differentiation but that high-dose NaF (greater than 2 mM) caused suppressed proliferation and apoptosis in hESCs. Our data further revealed that JNK signaling was involved in NaF-induced hESC apoptosis in a concentration- and time- dependent manner. These findings suggested that chronic exposure to NaF over a threshold concentration might interfere with early human embryogenesis by disturbing the regulation of the specification of the three germ layers, impairing osteogenic lineage commitment, and caus- ing apoptotic cell death in stem cells. Nevertheless, upstream factors that connect NaF treat- ment with MAPK activation were not discussed in our study, and further investigations are needed to expand on our findings. Discussion These findings suggested that other signaling pathways might be associated with the NaF-mediated cell death of hESCs undergoing acute exposure to NaF at concentra- tions higher than 6 mM or chronic exposure to 2 mM NaF for more than 2 days. References 1. Griffin SO, Regnier E, Griffin PM, Huntley V. Effectiveness of fluoride in preventing caries in adults. J Dent Res. 2007; 86: 410–415. PMID: 17452559 2. Lee YE, Baek HJ, Choi YH, Jeong SH, Park YD, Song KB. Comparison of remineralization effect of three topical fluoride regimens on enamel initial carious lesions. J Dent. 2010; 38:166–171. doi: 10. 1016/j.jdent.2009.10.002 PMID: 19819290 3. Fordyce. FM. Fluoride: human health risks. Encyclopedia of environmental health. 2011; 5:776–785. 4. Everett ET. Fluoride's effects on the formation of teeth and bones, and the influence of genetics. J Dent Res. 2011; 90:552–560. doi: 10.1177/0022034510384626 PMID: 20929720 5. Lantz O, Jouvin MH, De Vernejoul MC, Druet P. 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Acknowledgments We thank Dr. Li Yan, Dr. Xia Liu, Dr. Ning Kang and Dr. Qian Wang for support in data inter- pretation and analysis. We thank Miss Xun Yang for technical support in flow cytometry analysis. 12 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0148819 February 9, 2016 Effects of Fluoride on Human Embryonic Stem Cells Author Contributions Conceived and designed the experiments: XF RX. Performed the experiments: XF FNX PD QCL. Analyzed the data: XF RX. Contributed reagents/materials/analysis tools: XF FNX PD QCL GYY RX W h XF RX P id d f di XF RX GYY Conceived and designed the experiments: XF RX. Performed the experiments: XF FNX PD QCL. Analyzed the data: XF RX. Contributed reagents/materials/analysis tools: XF FNX PD QCL GYY RX. Wrote the paper: XF RX. Provided funding support: XF RX GYY. 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https://openalex.org/W248737065	http://www.jafs.com.pl/pdf-66747-6084?filename=Influence of dietary CLA.pdf	English	null	Influence of dietary CLA isomers and selenium compounds on the fatty acid and amino acid profiles in blood plasma of rats	Journal of Animal and Feed Sciences	2,007	cc-by	8,233	The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences 05-110 Jabłonna, Poland (Received 5 April 2007; accepted 2 May 2007) * Supported in part by the Ministry for Science and Information, Grant No. 2 P06Z 016 29 1 Corresponding author: e-mail: m.czauderna@ifzz.pan.pl Journal of Animal and Feed Sciences, 16, 2007, 257–273 Journal of Animal and Feed Sciences, 16, 2007, 257–273 KEY WORDS: CLA isomers, selenate, high-selenized yeast, fatty acids, amino acids, blood plasma, rat Inﬂuence of dietary CLA isomers and selenium compounds on the fatty acid and amino acid proﬁles in blood plasma of rats* .M. Niedźwiedzka, K. Korniluk, J. Kowalczyk and M. Czauderna1 K.M. Niedźwiedzka, K. Korniluk, J. Kowalczyk and M. Czauderna1 The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences 05-110 Jabłonna, Poland The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences 05-110 Jabłonna, Poland FA AND AA PROFILES IN BLOOD PLASMA OF RATS FA AND AA PROFILES IN BLOOD PLASMA OF RATS 258 INTRODUCTION ABSTRACT The inﬂuence of diets enriched in CLA isomers and/or selenium on the concentration of fatty acids and amino acids in blood plasma was studied on 20 groups of 7-8 rats aged 8 weeks and with a body weight (BW) of about 201 g. Rats were fed a basal diet for 29 days or a diet enriched with a combination of 1 or 2% CLA isomer(s) and/or 2 ppm as selenate, 1.2% as selenized yeast. Diets enriched in 1% of t10c12CLA and Se compounds caused the most efﬁcient body weight gain (BWG) and feed conversion. Diets enriched in Se compounds only showed a tendency to decrease BWG and feed conversion efﬁciency compared with control rats. Diets with CLA isomers increased isomer concentrations in plasma with preferential accumulation c9t11CLA in comparison with t10c12CLA. Supplemental Se and CLA isomer mixture usually increased β-oxidation of t10c12CLA compared with supplementing the CLA isomer mixture only. Supplementing 1% of the CLA isomer mixture showed tendency to decrease the concentration of C16:0 and SFA in plasma but supplementing 1 or 2% of the isomer mixture with Se compounds tended to or signiﬁcantly decreased C16:0 and SFA in plasma. The t10c12CLA isomer reduced, whereas c9t11CLA, stimulated the yield of Δ9-desaturation. An increase of linoleic and linolenic acid concentrations was found in the plasma of rats fed the diet with CLA isomer(s) and Se sources compared with the control diet. Supplementing Se sources or CLA isomer(s) stimulated MUFA and PUFA accumulation in plasma due to stimulation of Δ9-, Δ6- and Δ4-desaturation and elongation of fatty acids. Supplementing CLA isomers increased the concentration of amino acids in plasma, while the interaction between supplemented c9t11CLA and t10c12CLA in rats resulted in reduction of the concentration of amino acids in plasma. KEY WORDS: CLA isomers, selenate, high-selenized yeast, fatty acids, amino acids, blood plasma, rat Animals and experimental design The experimental protocol was approved by the Local Animal Care and Use Committee (The Agricultural University, Warsaw, Poland). Ten groups of 7-8 female rats (Wistar, Ifz: BOA), at 8 weeks of age, each weighing 201±1 g at the beginning of the experiment, were housed individually in plastic cages at a temperature of 22±1oC with a 12 h light-dark cycle and relative humidity of 50-60%. During a one-week preliminary period the animals were fed a standard Labofeed H diet produced by the Feeds and Concentrates Production Plant in Kcynia, (Poland) (Pastuszewska et al., 2000) given at a submaintenance level (9 g of diet daily per rat) to reduce body fat content. During that time the rats decreased their body weight by about 10% of their initial weight. Then for 29 days the rats were fed the experimental diets (Table 1) enriched with 1 or 2% of the mixture of CLA isomers (CLAmix), 1% of individual isomers (i.e. c9t11 and t10c12), 2 ppm Se as SeVI (Experiment I) or 1.2 ppm Se as Se-yeast (Experiment II). The composition of dietary CLA isomer(s) is presented in Table 1. The rations were adjusted each day to ensure an ad libitum feeding level. After day 29 rats were killed by CO2 and the liver and femoral muscles removed and freeze-dried. Blood samples were collected into heparinized tubes kept in an ice bath and centrifuged at 1500-1700 g for 15 min (at 2-4oC). Muscle, liver and blood plasma samples were stored at -28oC until analysed for the concentrations of fatty acids and amino acids. NIEDŹWIEDZKA K.M. ET AL. MATERIAL AND METHODS INTRODUCTION Recent investigations have documented that conjugated linolenic acid (CLA) isomers could exert many beneﬁcial effects on human health thanks to their antiproliferative, antitumour, antiinﬂammatory, antiatherogenic, antidiabetogenic, and antiadipogenic properties (Wahle et al., 2004; De La Tore et al., 2006). A beneﬁcial regulatory inﬂuence of CLA isomers on cytokine and immunoglobulin production, and on immune function was found. CLA isomers affected the metabolisms of lipids and eicosanoids, as well as of other fatty acids (Naumann et al., 2006). Our recent studies also clearly evidence that dietary selenium (Se) and CLA isomers inﬂuenced the concentration of mono- (MUFA) and polyunsaturated (PUFA) fatty acids, minerals and amino acids in laboratory animals (Czauderna et al., 2004; Korniluk et al., 2006; Niedźwiedzka et al., 2006a,b). The nutritional requirements of animals and humans should met by provision of ultra-trace nutrients like Se or essential fatty acids. In humans and animals, numerous disorders related to Se have been recognized, including liver necrosis, white muscle dystrophy, exudative diathesis, and cardiomyopathy (Arthur, 2003; Lyons and Jacques, 2004). The discovery that Se was an essential component of radical- or/and peroxide-metabolizing seleno-proteins (e.g., glutathione peroxidase) led to the hypothesis that physiologically advantageous supplementation with Se results in elevation of unsaturated fatty acid concentrations in animals. Recent investigations revealed that dietary selenate and high-selenized yeast increased the accumulation of CLA isomers and other PUFA in the bodies of rats (Czauderna et al., 2004a,b). Therefore, a protective effect of dietary Se (seleno-cysteine is an essential component of more than 25 seleno-proteins) on the deposition of CLA isomers and other unsaturated fatty acids in the body of mammals may also be plausible. Considering the above evidence, we decided to carry out experiments to test the hypothesis that dietary CLA isomer(s), inorganic and organic Se (as selenate and high-selenized yeast) improve the fatty acid proﬁle in blood plasma of rats. The inﬂuence of diets enriched in CLA isomer(s) and Se on the concentrations of free essential (E-AA) and non-essential (NE-AA) amino acids in plasma and protein E-AA and NE-AA amino acids in the liver and femoral muscles was also examined. On the basis of these investigations we intended to compare the efﬁcacy of dietary selenate (SeVI) and organic Se (Se-yeast) on the capacity for accumulation of CLA isomers, other unsaturated fatty acids and, in particular, E-AA in plasma, liver, and muscles. NIEDŹWIEDZKA K.M. ET AL. MATERIAL AND METHODS 259 Reagents and chemicals Sodium selenate (Na2SeO4) and amino acid (AA) standards were provided by Sigma (USA), whereas methanol, 99.9% acetonitrile and 95% heptane were HPLC grade and purchased from Lab-Scan (Ireland). The CLA isomer mixture (CLAmix), c9t11CLA and t10c12CLA isomers were supplied by Larodan Fine Chemicals AB (Sweden). Composition details (Table 1) and the purity of CLA isomer mixtures and individual isomers were examined using our Ag+ -HPLC and GLC method (Czauderna et al., 2003, 2005). All fatty acid (FA) standards, 50% BF3 in methanol, o-phthaldialdehyde (OPA) and butylated hydroxytoluene were provided by SIGMA (USA) and Suppelco. Ethanethiol, tetrahydrofuran and sodium hypochlorite water solution (4% available Cl) were from Fluka. High-selenized yeast (Se-yeast) was donated by Sel-Plex (Alltech Inc., USA). Eighty-three per cent of the total Se content of Se-yeast represents Se in the form of Se-methionine incorporated into the proteins of Saccharomyces cerevisiae (Rayman, 2004). Other reagents, FA AND AA PROFILES IN BLOOD PLASMA OF RATS 260 Table 1. Reagents and chemicals Dietary effects1 of 1%, 2% CLA isomer mixture (CLAmix), cis9trans11CLA (c9t11), trans10cis12CLA (t10c12), selenate (SeVI) and high- selenized yeast (Se-yeast) on body weight of rats after 7 days of adaptation2, the body weight gain (BWG)3 and the feed conversion efﬁciency (FCE)4 of rats after 29 days feeding with experimental diets Group Experiment I Rats fed diets enriched in CLA isomer(s) and selenate(SeVI) Experiment II Rats fed diets enriched in CLA isomer(s) and Se-yeast Additives Content8 Body weight, g BWG g FCE g/g Additives Content8 Body weight, g BWG g FCE G initial after 7 days initial after 7 days Control5 - - 200.1 184.9 (8)9 59.4a 0.1365 Control5 - 199.5 177.0 (8)9 61.1a 0.1221a SeVI or Se-yeast selenate 2 ppm 200.1 185.3 (8) 52.8b 0.1220 Se-yeast 1.2 ppm 200.4 179.0 (8) 60.9 0.1215 1%CLA CLAmix6 1% 200.5 184.4 (7) 54.8 0.1285 CLAmix7 1% 201.1 183.0 (7) 62.9 0.1258 c9t11 c9t11 1% 200.8 185.6 (7) 59.7 0.1359 c9t11 1% 200.6 177.5 (7) 67.5b 0.1337b t10c12 t10c12 1% 200.9 184.4 (7) 54.1 0.1287 t10c12 1% 201.1 180.0 (7) 61.6c 0.1282 2%CLA CLAmix6 2 ppm 200.4 183.3 (7) 56.8 01376 CLAmix7 2% 200.1 178.0 (7) 56.4 0.1227 1%CLA + SeVI or Se-yeast CLAmix6 +selenate 1% 2 ppm 200.2 181.9 (7) 56.1 0.1319 CLAmix7 +Se-yeast 1% 1.2 ppm 202.6 180.0 (7) 63.9 0.1302 c9t11+ SeVI or Se-yeast c9t11 +selenate 1% 2 ppm 199.6 184.2 (7) 55.5a 0.1289 c9t11 +Se-yeast 1% 1.2 ppm 201.2 181.0 (7) 56.8b 0.1185b t10c12+ SeVI or Se-yeast t10c12 +selenate 1% 2 ppm 200.3 183.8 (7) 62.2b 0.1427 t10c12 +Se-yeast 1% 1.2 ppm 200.3 178.0 (7) 68.8ac 0.1366a 2%CLA + SeVI or Se-yeast CLAmix6 +selenate 2% 2 ppm 200.2 182.8 (7) 58.4 0.1409 CLAmix7 +Se-yeast 1% 1.2 ppm 204.1 182.0 (8) 60.4 0.1293 1 means in columns with the same letter are signiﬁcantly different: A,B -P<0.01; a,b - P<0.05. Analyses were performed by one Mann-Whitney U and two factorial ANOVA tests. Statistical analyses of simultaneously the CLA isomer(s) and SeVI or Se-yeast treatments were performed applying two- factorial ANOVA analysis 2 body weight of individually adapted rats after 7 days of submaintenance feeding (daily: 9 g/the standard diet per rat). Chromatographic equipment An alliance separation module (model 2690, Waters) with a Waters 996 photodiode array detector (DAD) and Waters 474 ﬂuorescence detector (FD) was used for determination of the concentration of free amino acids (AAs) and fatty acids in assayed blood plasma samples. The underivatized CLA isomers and other fatty acids containing conjugated double bonds (CFA) in blood plasma of rats fed all experimental diets were determined according to Czauderna et al. (2003). The derivatized non-CLA fatty acids (FAs) in plasma of rats fed the diets enriched in CLA isomer(s) and SeVI (selenate) were determined using an HPLC system according to Czauderna and Kowalczyk (2001). Methylated non-CLA FAs in plasma of rats fed the diets enriched in CLA isomer(s) and high-selenized yeast (Se-yeast) were determined using long-capillary gas-liquid chromatography (GLC) with a ﬂame-ionization detector (FID) (Czauderna et al., 2005). The method of free amino acid (AAs) conversion to o-phthaldialdehyde (OPA)-derivatives (OPA-AAs) followed by reversed-phase HPLC separations and quantiﬁcations of OPA-AAs in plasma of rats fed the all experimental diets were as previously described (Czauderna et al., 2002; Niedźwiedzka et al., 2006c). Reagents and chemicals Initial body weight of rats and after 7 days of adaptation did not statistically differ among group at the P<0.1 level 3 after feeding for 29 days with experimental diet enriched in CLA isomer(s) and/or SeVI or Se-yeast (2 ppm or 1.2 ppm, respectively) 4 FCE: g body weight gain/g feed intake 5 the concentrations of Se (as Na2SeO2), Zn, Fe, Mg and Ca in the standard Labofeed H diet found: 0.63, 137, 698, 1653 and 10683 µg/g, respectively 6 the dietary CLA isomer mixture contains, %: t11t13 - 2.9; t10t12 - 5.1; t9t11 - 4.3; t8t10 - 2.9; c11t13 - 13.4; c10t12 - 28.0; c9t11 - 28.6; c8t10 - 9.6; c11c13 - 1.6; c10c12 - 1.5; c9c11 - 1.4; c8c10 - 0.7. The ratio of the concentration of the isomers (c9t11/ t10c12) in the dietary CLA isomer mixture: 1.0242. The composition of individual isomers: c9t11 and t10c12 - 95% of c9t11 and t10c12 isomer, respectively; ttCLA isomers - ca. 2%; no other fatty acids were detected 7 the CLA isomer mixture contained: 1.94% t,tCLA isomers, 95.22% c9t11CLA and t10c12CLA, and 1.48% c,cCLA isomers and 1% linoleic acid (LA); the ratio of the c9t11CLA to t10c12; CLA contents in the CLA isomer mixture was 0.981 (i.e., 47.3 and 48.2% respectively). The composition of individual isomers: c9t11CLA and t10c12CLA - 98% of c9t11CLA and t10c12CLA, respectively; t,tCLA isomers - 0.2%; LA - 1% (Czauderna et al., 2003a, 2005) 8 the concentration of CLA isomer(s) and Se in the rats’ diets 9 i h i b i 261 NIEDŹWIEDZKA K.M. ET AL. includingdichloromethane (DCM), KOH, NaOH, Na2SO4 and concentrated HCl, were analytical grade and were purchased from POCh (Gliwice, Poland). includingdichloromethane (DCM), KOH, NaOH, Na2SO4 and concentrated HCl, were analytical grade and were purchased from POCh (Gliwice, Poland). Statistical analysis The results in Tables 1-6 are presented as means of 7-8 individually analysed rat body parameters, and liver, femoral muscle, and plasma blood samples. Statistical analyses of the effects of the CLA isomer(s) and/or Se (as SeVI or Se-yeast) in the diets were conducted using the nonparametric Mann-Whitney U test for comparing pairs in an independent experimental group (single-factor analysis), while statistical analyses of the interaction between the CLA isomer(s) and Se (as SeVI or Se-yeast) were performed using two-factorial ANOVA (CLA isomer(s)×Se). The statistical analyses were performed using the Statistica ver. 6 package (Statistica, 2002). Differences were considered signiﬁcant at the P<0.05 or P<0.01 level, while tendencies, at the P<0.1 level. Statistical analyses of the interaction between the CLA isomer and Se were performed using two-factorial ANOVA analysis (the CLA isomer(s)×Se); -P<0.05 and -P<0.01 were considered statistically signiﬁcant. FA AND AA PROFILES IN BLOOD PLASMA OF RATS 262 Table 2. Statistical analysis The concentration of selected fatty acids and the sum of amino acids (ΣAA), essential (ΣE-AA), non-essential (ΣNE-AA) amino acids in blood plasma of rats fed the diets enriched in cis9trans11CLA (c9t11), trans10cis12CLA (t10c12) or/and SeVI (selenate) Group Control SeVI c9t11 t10c12 1%CLA4 c9t11 +SeVI t10c12 +SeVI 4 1%CLA +SeVI Signiﬁcance of effect1 one factorial analysis interaction Item3 µg/ml blood plasma2 Se c9t11 t10c12 CLA c9t11 × Se t10c12 × Se CLA × Se C16:0 82.0 137.0 53.1 82.4 56.9 83.1 80.9 131.9 NS NS NS NS NS NS NS SFA18+16 133 223ab 108a 140 181 143 139b 213 NS NS NS NS NS NS c9C18:1 19.5 37.4 22.0 17.9 20.2 24.0 36.7 19.6 NS NS NS NS NS * NS Δ9index 0.128 0.143 0.169 0.114 0.100 0.147 0.110 0.121 NS * NS NS NS NS * LA 24.7 42.0 28.0 32.6 30.9a 42.1 54.1 37.4a NS NS NS NS NS NS NS α-LNA 10.8 19.8 13.9a 18.5 14.2 23.9a 25.2 18.9 NS NS NS NS NS * * c9t11 NQ NQ 28.7 0.53 12.1 48.4 1.8 16.3 - - - - - - - t10c12 NQ NQ 0.94 31.2 10.0 1.7 33.0 12.7 - - - - - - - ct/tcCLA NQ NQ 29.6 32.0 23.5 50.3 37.1 30.9 - - - - - - - ttCLA NQ NQ 9.4 6.6 15.2 13.5 15.2 9.5 - - - - - - - ccCLA NQ NQ 1.1 1.3 2.7 1.3 2.1 2.3 - - - - - - - ΣCLA NQ NQ 42.3 43.6 45.6 69.5 62.1 48.2 - - - - - - - SFA 136 235 116 146 101 145 323 150 NS NS NS NS NS NS NS UFA 86.6 166 208 149a 158 203 285a 182 * * NS NS NS * NS ΣFA 223 401 324 295 259a 348 608 332a NS NS NS NS NS NS NS ΣE-AA 337 377Aa 415b 392B 324 327b 307AB 335a NS NS NS ΣNE-AA 460 476A 550B 532 424 419B 380A 445 NS * NS NS ΣAA 797 853A 965a 924B 748 746a 687AB 780 NS ** NS * NS 1 signiﬁcance of effects: - P<0.01; * - P<0.05; NS - P≥0.05; interactions were analysed by two-factorial ANOVA test followed by one-factorial Mann-Whitney U analysis 2 means in rows with the same letters are signiﬁcantly different: A,B - P<0.01 and a,b - P<0.05; analyses were performed using one-factorial Mann- Whitney U analysis 3 NQ - below the quantiﬁcation limit. Feed conversion efﬁciency, body weight gain of rats Feed conversion efﬁciency, body weight gain of rats Feed conversion efﬁciency, body weight gain of rats It is well established that Se as inorganic Se (particularly selenate) is less effectively accumulated in the body of laboratory rats in comparison with dietary organic Se (e.g., Se-cysteine, methionine or Se-yeast) (Lyons and Jacques, 2004; Rayman, 2004). Therefore, in the current study, the concentration of Se as selenate in the rats’ diets was higher than the dietary concentration of organic Se as high- selenized yeast (i.e. 2 ppm vs 1.2 ppm, respectively). No macroscopic lesions or toxic symptoms of CLA isomers, selenate and Se- yeast were observed in the animals fed experimental diets. The diet containing 2 ppm of Se would not be toxic for rodents like rats or mice because only dietary inorganic Se compounds, selenite in particular, chronically consumed at a rate of more than 5 ppm can be hepatotoxic and teratogenic in humans and animals (Tapiero et al., 2003; Tinggi, 2003). The value of LD50 is about 5 mg Se/kg of body mass for rats, thus, this correspond to a diet containing ~50 ppm Se (i.e. 20 g of the Labofeed H diet enriched in 50 ppm Se per rat and day). In contrast to selenate and particularly selenite, Se-Met (the main Se-compound in Se-yeast) is less reactive because tRNAMet does not discriminate between Se-Met and methionine (Met). Therefore, the Se-Met residue in general proteins is a stable and safe-storage mode for Se in the body of rats fed the diet enriched in 1.2 ppm Se as Se-yeast. In the current study, the inﬂuence of dietary CLA isomer(s) and/or Se (as SeVI and Se-yeast) on the body weight gain (BWG) of rats and feed conversion efﬁciency (FCE) was conﬁrmed as being the highest in animals fed the diet enriched simultaneously in t10c12 and SeVI or Se-yeast (Table 1). Interestingly, the addition of organic Se (as Se-yeast) to the diet with t10c12 resulted in a more efﬁcient increase of BWG (11.2%) of animals and FCE (11.9%) compared with BWG (4.7%) of rats and FCE (4.5%) fed inorganic Se (as selenate) in the diet containing t10c12 (i.e. 68.8 g vs 62.2 g). NIEDŹWIEDZKA K.M. ET AL. 263 Statistical analysis SFA18+16 - the sum of C18:0 and C16:0. ΣFA - the sum of SFA and UFA; UFA - the sum of MUFA and PUFA without CLA isomer Δ9index - (c9C16:1+c9C18:1)/(c9C16:1+c9C18:1+C16:0+C18:0); SFA - the sum of C8:0, C10:0, C12:0, C14:0, C16:0, C18:0, C20:0 and C22:0 4 value ratios of c9t11/t10c12: 1.209 and 1.277 for the groups 1%CLA and 1%CLA+Se, respectively FA AND AA PROFILES IN BLOOD PLASMA OF RATS FA AND AA PROFILES IN BLOOD PLASMA OF RATS 2004a,b; Niedźwiedzka et al., 2006a; Korniluk et al., 2006, 2007). Consistently, other investigation (Terpstra et al., 2002) reported an elevation in the concentration of insulin and a decrease in the level of leptin in blood plasma of mice fed a diet containing CLA isomers. Feed conversion efﬁciency, body weight gain of rats In contrast, the addition of Se (as SeVI and Se-yeast) to the diet enriched in c9t11 tended to or statistically signiﬁcantly decreased the BWG and FCE of rats, although the diet containing c9t11 and Se-yeast resulted in the strongest reduction of BWG (-7.0%) and FCE (-2.95%). Consistently with these results, the diet containing selenate most efﬁciently reduced BWG (-11.1%) and FCE (-10.6%), whereas the diet containing only Se-yeast resulted in a minute decrease in BWG (-0.33%) and FCE (-0.49%). We also found a tendency towards decreased BWG of rats fed the diet enriched in 2% CLAmix. Similar results were also reported by Terpstra et al. (2002) and Wahle et al. (2004) in mice and rats. These studies and our recent investigations conﬁrm that CLA isomers decrease the BWG of laboratory animals by increasing energy expenditure and also by increasing energy loss in the excreta (Czauderna et al., 264 2 means in rows with the same letters are signiﬁcantly different: A,B - P<0.01; a,b - P<0.05 4 value ratios of c9t11/t10c12: 1.426 and 1.234 for the groups 2%CLA and 2%CLA+Se, respectively means in rows with the same letters are signiﬁcantly different: P 0.01; P 0.05 3 abbreviations for FA and other items see Table 2 reviations for FA and other items see Table 2 NIEDŹWIEDZ Table 4. The concentration of fatty acids in blood plasma of rats fed the diets enriched in cis9trans11CLA (c9t11), trans10cis12CLA (t10c12) or/and Se-yeast (high-selenized yeast) Group Control Se-yeast c9t11 t10c12 1%CLA3 c9t11 +Se-yeast t10c12 +Se-yeast 1%CLA3 +Se-yeast Signiﬁcance of effect1 one factorial analysis interaction Item µg/ml blood plasma2 Se c9t11 t10c12 CLA c9t11 x Se t10c12 x Se CLA x Se Table 4. CLA isomer concentrations in plasma of rats We found that feeding the diets with CLA isomer(s) increased the concentration of CLA isomer(s) in plasma, regardless of the geometrical conﬁguration of CLA isomers (Tables 2, 3 and 5). However, detailed analysis of e concentration of fatty acids in blood plasma of rats fed for 4 weeks 2%CLA isomer hout or with SeVI (selenate) mixture without or with SeVI (selenate) Group Control Se(VI) 2%CLA4 4 2%CLA +SeVI Signiﬁcance of effect1 2%CLA interaction Item3 µg/g blood plasma2 2%CLA × Se C16:0 82.0 137 61.7 92.4 NS NS SFA18+16 133 223 108 147 NS NS c9C18:1 19.5 37.4a 14.2 25.4a NS NS Δ9-index 0.128 0.143 0.116 0.147 NS NS LA 24.7 42.0 33.3 42.2 NS NS α-LNA 10.8 19.8 18.7 22.8 NS NS c9t11 NQ NQ 24.3 25.5 - - t10c12 NQ NQ 17.0 20.6 - - ct/tcCLA NQ NQ 44.1 49.0 - - ttCLA NQ NQ 13.3 16.4 - - ccCLA NQ NQ 3.0 4.0 - - ΣCLA NQ NQ 67.3 79.8 - - SFA 136 235 114 156 NS NS UFA 86.6 166 177 214 * * ΣFA 223 401 291 370 NS NS ΣE-AA 337 377a 351 314a NS * ΣNE-AA 460 476 480A 405A NS NS ΣAA 797 853 831 719 NS NS 1 signiﬁcance of effects: *- P<0.01, - P<0.05; NS-P≥0.05; interactions were analysed by two- factorial ANOVA test 2 means in rows with the same letters are signiﬁcantly different: A,B - P<0.01; a,b - P<0.05 3 abbreviations for FA and other items see Table 2 4 value ratios of c9t11/t10c12: 1.426 and 1.234 for the groups 2%CLA and 2%CLA+Se, respectively 265 NIEDŹWIEDZKA K.M. ET AL. NIEDŹWIEDZ Table 4. The concentration of fatty acids in blood plasma of rats fed the diets enriched in cis9trans11CLA (c9t11), trans10cis12CLA (t10c12) or/and Se-yeast (high-selenized yeast) Group Control Se-yeast c9t11 t10c12 1%CLA3 c9t11 +Se-yeast t10c12 +Se-yeast 1%CLA3 +Se-yeast Signiﬁcance of effect1 one factorial analysis interaction Item µg/ml blood plasma2 Se c9t11 t10c12 CLA c9t11 x Se t10c12 x Se CLA x Se The concentration of fatty acids in blood plasma of rats fed the diets enriched in cis9trans11CLA (c9t11), trans10cis12CLA (t10c12) or/and Se-yeast (high-selenized yeast) Group Control Se-yeast c9t11 t10c12 1%CLA3 c9t11 +Se-yeast t10c12 +Se-yeast 1%CLA3 +Se-yeast Signiﬁcance of effect1 one factorial analysis interaction Item µg/ml blood plasma2 Se c9t11 t10c12 CLA c9t11 x Se t10c12 x Se CLA x Se C16:0 114 95.2AB 95.7 81.0 97.3 154A 150 332B NS NS NS NS NS SFA18+16 253 226ABC 221 205 235 381A 370B 640C NS NS NS NS ** * c9C18:1 54.7 40.0ABa 68.7 30.8a 40.1 100A 102 351B NS NS NS NS NS NS Δ9-index 0.184 0.162A 0.224 0.140 0.151B 0.204 0.180 0.348AB NS NS ** NS NS * LA 167 139AB 172 147 156 229A 191 644B NS NS NS NS NS NS αLNA 31.9 25.1ABC 26.8 27.2 30.1 40.2A 48.3B 139C NS NS NS NS * NS c9t11 NQ NQ 29.2A 1.3 14.5B 127.6A 4.8 76.5B - - - - - - t10c12 NQ NQ NQ 14.8B 13.3C 4.3 59.2B 57.9C - - - - - - ct/tcCLA NQ NQ 30.0A 17.4 27.9B 139A 71.6 134B - - - - - - ttCLA NQ NQ 1.68A 4.1a 0.71B 4.2A 1.36a 6.8B - - - - - - ccCLA NQ NQ NQ NQ NQ NQ NQ NQ - - - - - - ΣCLA NQ NQ 31.7A 21.5 28.6B 144A 73.0 141B - - - - - - SFA 257 230ABCDE 591A 323B 240 452C 474D 1334E NS NS NS NS NS NS MUFA 70.6 51.5ABab 93.5 42.6a 50.8 140A 124b 577B NS NS NS NS NS NS ** PUFA 408 347AB 530 349 405 601A 466 1689B NS NS NS NS * NS ** UFA 479 399 624 392 456 741 590 2266 NS * NS NS * NS ΣFA 735 628ABC 1114 715 696 1193A 1064B 3600C NS NS NS NS NS 1 signiﬁcance of effects: - P<0.01; - P<0.05; NS - P≥0.05; interactions were analysed by two-factorial ANOVA test followed by one-factorial Mann-Whitney U analysis 2 means in rows with the same letters are signiﬁcantly different: A,B - P<0.01 and a,b - P<0.05; analyses were performed using one-factorial Mann-Whitney U analysis 3 value ratios of c9t11/t10c12: 1.0903 and 1.3212 for the groups 1%CLA and 1%CLA+Se, respectively abbreviations for FA and other items see Table 2 FA AND AA PROFILES IN BLOOD PLASMA OF RATS 266 Table 5. NIEDŹWIEDZ Table 4. The concentration of fatty acids in blood plasma of rats fed the diets enriched in cis9trans11CLA (c9t11), trans10cis12CLA (t10c12) or/and Se-yeast (high-selenized yeast) Group Control Se-yeast c9t11 t10c12 1%CLA3 c9t11 +Se-yeast t10c12 +Se-yeast 1%CLA3 +Se-yeast Signiﬁcance of effect1 one factorial analysis interaction Item µg/ml blood plasma2 Se c9t11 t10c12 CLA c9t11 x Se t10c12 x Se CLA x Se The concentration of fatty acids in blood plasma of rats fed for 4 weeks 2% CLA isomer mixture without or with Se-yeast Group Control Se-yeast 2%CLA4 2%CLA +Se-yeast4 Signiﬁcance of effect1 2%CLA interaction Item3 µg/g blood plasma2 2%CLA × Se C16:0 114 95.2A 119 142A NS * C18:0 139 131A 164 218A NS * SFA18+16 253 226 283 360 NS ** C9C18:1 54.7 40.0a 59 55a NS NS Δ9-index 0.184 0.162 0.156 0.144 NS NS LA 167 139A 227 199A NS NS αLNA 31.9 25.1A 43.4 48.3A NS * c9t11 NQ NQ 17.8a 43.1a - - t10c12 NQ NQ 14.8A 30.4A - - ct/tcCLA NQ NQ 32.6a 73.5a - - ttCLA NQ NQ 1.48 4.7 - - ccCLA NQ NQ NQ 1.04 - - ΣCLA NQ NQ 34.1a 79.3a - - SFA 257 230A 340a 509Aa NS * MUFA 70.6 51.5A 75.8 144A NS NS PUFA 408 347A 647 499A NS NS UFA 479 399 723 643 NS NS ΣFA 735 628A 1063 1152A NS NS 1 signiﬁcance of effects: *- P <0.01, - P<0.05; NS-P≥0.05; interactions were analysed by two- factorial ANOVA test 2 means in rows with the same letters are signiﬁcantly different: A,B - P<0.01; a,b - P<0.05 3 abbreviations for FA and other items see Table 2 4 value ratios of c9t11/t10c12: 1.2027 and 1.4178 for the groups 2%CLA and 2%CLA+Se, respectively Table 5. The concentration of fatty acids in blood plasma of rats fed for 4 weeks 2% CLA isomer mixture without or with Se-yeast our results revealed a slight preference towards accumulation of c9t11 in comparison with t10c12 in the plasma of rats fed the diets enriched in 1 or 2% CLAmix, irrespective of the presence of Se (as SeVI and Se-yeast). Indeed, the concentration of t10c12 as well as t10t12 isomers tended to be lower than c9t11 and t9t11 isomers in tissues of rats due to more efﬁcient β-oxidation of t10c12 and t10t12 isomers than their 9,11 homologues (Alasnier et al., 2002; Czauderna et al., 2004a,b). Therefore, the value of the concentration ratios of c9t11/t10c12 in the plasma of rats fed the diets enriched in 1 or 2% CLAmix, irrespective of the presence of Se (as SeVI and Se-yeast), was higher compared with the value of the concentration ratios of these isomers in the dietary CLAmix (see data in Tables 2-5 vs Table 1). 2 means in rows with the same letters are signiﬁcantly different: A,B - P<0.01; a,b - P<0.05 NIEDŹWIEDZKA K.M. ET AL. β-oxidation of t10c12 in comparison with the β-oxidation of this isomer in the blood of rats fed the diet enriched in only 2% CLAmix (Tables 2 and 3). The different chemical forms of Se may have different metabolic roles (Tapiero et al., 2003; Suzuki, 2005), as observed in our previous studies (Czauderna et al., 2004a,b; Korniluk et al., 2006). Indeed, in our current investigation we found that dietary Se-yeast (a rich-source of seleno-methionine) signiﬁcantly stimulated β-oxi-dation of t10c12 regardless of the amount of CLAmix added to the diets (Tables 4 and 5). Moreover, as can be seen from the results summarized in Tables 2-5, the addition of organic Se as Se-yeast to the diets enriched in CLA isomer(s) resulted in signiﬁcantly stronger stimulation of the accumulation of CLA isomer(s) in plasma in comparison with SeVI supplied to the diets containing CLA isomer(s). Different chemical forms of dietary Se (as SeVI or Se-yeast) could be attributed to the different inﬂuence of Se additives to the diets on the proﬁle and yield of the accumulation of CLA isomers in plasma of rats. Indeed, selenate (SeVI), unlike selenite (SeIV), is metabolized in the liver to give selenide, seleno-diglutathione, seleno-cysteine (Se-Cys), etc. (Combs, 2004; Suzuki, 2005). Therefore, Se-Cys- proteins are the predominant Se-compound in blood plasma of rats fed diets enriched in SeVI. On the other hand, dietary Se-yeast stimulated accumulation of Se-Cys-proteins and, particularly, seleno-methionine-proteins in the body of rats (Suzuki, 2005). Therefore, these changes in the proﬁle and concentration of CLA isomer(s) could be attributed to the higher concentration of seleno-methionine- proteins in the plasma of rats. NIEDŹWIEDZ Table 4. The concentration of fatty acids in blood plasma of rats fed the diets enriched in cis9trans11CLA (c9t11), trans10cis12CLA (t10c12) or/and Se-yeast (high-selenized yeast) Group Control Se-yeast c9t11 t10c12 1%CLA3 c9t11 +Se-yeast t10c12 +Se-yeast 1%CLA3 +Se-yeast Signiﬁcance of effect1 one factorial analysis interaction Item µg/ml blood plasma2 Se c9t11 t10c12 CLA c9t11 x Se t10c12 x Se CLA x Se Addition of SeVI to the diet enriched in 1% CLAmix stimulated β-oxidation of t10c12 in plasma. On the other hand, the higher concentration of CLAmix in the diet enriched in SeVI disturbed the our results revealed a slight preference towards accumulation of c9t11 in comparison with t10c12 in the plasma of rats fed the diets enriched in 1 or 2% CLAmix, irrespective of the presence of Se (as SeVI and Se-yeast). Indeed, the concentration of t10c12 as well as t10t12 isomers tended to be lower than c9t11 and t9t11 isomers in tissues of rats due to more efﬁcient β-oxidation of t10c12 and t10t12 isomers than their 9,11 homologues (Alasnier et al., 2002; Czauderna et al., 2004a,b). Therefore, the value of the concentration ratios of c9t11/t10c12 in the plasma of rats fed the diets enriched in 1 or 2% CLAmix, irrespective of the presence of Se (as SeVI and Se-yeast), was higher compared with the value of the concentration ratios of these isomers in the dietary CLAmix (see data in Tables 2-5 vs Table 1). Addition of SeVI to the diet enriched in 1% CLAmix stimulated β-oxidation of t10c12 in plasma. On the other hand, the higher concentration of CLAmix in the diet enriched in SeVI disturbed the 267 NIEDŹWIEDZKA K.M. ET AL. Effect of CLA isomer(s) and Se on the concentration of non-conjugated fatty acids in plasma The current study was designed to determine whether changes of the concentration of non-conjugated fatty acids depended upon the chemical form of dietary Se and the positional and geometrical structure of CLA isomers. As can be seen from the results summarized in Tables 2-5, the dietary 1% CLAmix showed a tendency towards decreasing the concentration of C16:0 and the sum of all assayed saturated fatty acids (SFA) in plasma. Unexpectedly, the diets enriched in 1 or 2% CLAmix and Se (as SeVI or Se-yeast) tended or statistically signiﬁcantly increased of the concentration of C16:0, SFAC16:0+C18:0 as well as the sum of SFA in plasma. Likewise, adding Se-yeast to the diets containing 1 or 2% CLAmix revealed a similar effect on these saturated fatty acids, although the inﬂuence of dietary high- selenized yeast was considerably stronger (Tables 2 and 3 vs 4 and 5). Dietary Se-yeast more efﬁciently stimulated the accumulation these saturated fatty acids in plasma of rats fed the diets enriched in c9t11 or t10c12 compared with rats fed the diets containing SeVI and c9t11or SeVI and t10c12. On the other hand, the diet enriched in only Se-yeast showed a tendency towards a slight decrease in the 268 FA AND AA PROFILES IN BLOOD PLASMA OF RATS concentration of these saturated fatty acids in plasma, whereas dietary SeVI tended to or statistically signiﬁcantly increased the concentration of these fatty acids. The obtained results documented that inorganic Se as selenate stimulated the accumulation of saturated fatty acids in plasma, while Se-yeast, mainly as seleno- methionine, lowered the yield of saturated fatty acid formation in rat plasma. Dietary CLA isomers exert a variety of inﬂuences on the capacity of Δ9-desaturation of fatty acids (Belury, 2002; Wahle at al., 2004). Indeed, the value of the desaturase index (Δ9-index) as well as the concentration of c9C18:1 tended to decrease in the plasma of rats fed the diet enriched in t10c12 or 1% CLAmix, regardless of the presence of SeVI (Tables 2-5), whereas the addition of Se-yeast to the diet containing these isomers almost eliminated this effect of dietary t10c12 (Table 4). This is in agreement with our previous studies (Czauderna et al., 2004a,b; Korniluk et al., 2006; Niedźwiedzka et al., 2006a) in which dietary t10c12 also reduced the Δ9-desaturation activity and inhibited steaoryl-CoA desaturase mRNA expression and fatty acid synthesis in rats (Terpsta, 2004). Effect of CLA isomer(s) and Se on the concentration of non-conjugated fatty acids in plasma Terpsta (2004) explained that a 12-double bond appears to be a key structure for inhibiting stearoyl-CoA desaturase activity, especially when coupled with a 10-double bond, however not with a 9-double bond (Belury, 2002). On the other hand, the diet containing c9t11 tended to increase the value of the Δ9-index as well as the concentration of c9C18:1 in plasma, whereas addition of this isomer to the diets enriched in Se (as SeVI and Se-yeast) tended to increase the concentration of c9C18:1 in plasma compared with the concentration of this isomer in plasma of rats fed the diets containing only c9t11. So, these results show that simultaneous addition of c9t11 and Se (as SeVI and Se-yeast) to the diets stimulated Δ9-desaturation activity or/ and increased steaoryl-CoA desaturase mRNA expression in the body of rats. y p y We found that feeding Se as SeVI and Se-yeast usually tended to increase, or signiﬁcantly increased, the concentration of linoleic (LA) and linolenic (LNA) acids in plasma of rats fed the diets containing CLA isomer(s) and Se regardless of its chemical form (a positive interaction) (Tables 2-5). A signiﬁcantly higher increase of the concentrations of LA and LNA was observed in plasma of rats fed the diets enriched in CLA isomer(s) and Se-yeast, despite the diet enriched in only Se-yeast tending to decrease the concentrations of LA, LNA as well as MUFA, PUFA and the sum of unsaturated fatty acids (UFA) as well as the sum of all assayed fatty acids (ΣFA) in plasma (Tables 4 and 5). A similar effect was observed in the liver, spleen and pancreas of rats fed the diet enriched in only Se-yeast (Korniluk et al., 2006, 2007). The positive correlation between the concentration of the sum of UFA (i.e. MUFA and PUFA) and simultaneous addition of CLA isomer(s) and Se (as SeVI and Se-yeast) to the diet was observed in plasma of rats. As expected, the addition of Se-yeast together with CLA isomer(s) caused a signiﬁcantly higher increase in the concentration of UFA in plasma than the addition of SeVI and CLA isomer(s). These results are consistent with our recent 269 NIEDŹWIEDZKA K.M. ET AL. Effect of CLA isomer(s) and Se on the concentration of non-conjugated fatty acids in plasma investigation in rats showing that the interaction between Se-yeast and CLA isomer(s) was also responsible for stimulating the accumulation of LA, LNA as well as other PUFA in spleen, pancreas and kidneys of rats fed the diets containing Se-yeast and CLA isomer(s) (Korniluk et al., 2006, 2007). The explanation for the interaction mechanism of the increase in the concentration of unsaturated fatty acids, particularly long-chain PUFA (Tables 4 and 5), is through the increase of the capacity of Δ9-, Δ6-, Δ4-desaturations and elongation of fatty acids. As a consequence of the above observations, feeding CLA isomer(s), particularly with Se (as SeVI or Se-yeast), has been shown to usually increase the sum of all assayed fatty acids in plasma of rats. FA AND AA PROFILES IN BLOOD PLASMA OF RATS 270 Table 6. Mean concentrations1 of the sum of protein essential-(ΣE-AAs)2, non-essential- (ΣNE-AA)2 amino acids and the sum of all protein amino acids (ΣAA)2 in femoral muscles and liver of rats fed the diet enriched in CLA isomer(s) and Se (as SeVI and Se-yeast) Group Tissue Se, µg/g3 ΣCLA, µg/g3 ΣE-AA, mg/g ΣNE-AA, mg/g ΣAA, mg/g SeVI Se-yeast SeVI Se-yeast SeVI Se-yeast SeVI Se-yeast SeVI Se-yeast Control Liver 4.48ABCDEFGHa 3.03ABCD 0.07 - 230 319 228 220 458 775 Muscles -4 0.44ABa - - 190 214 221 168 411 538 Se Liver 5.77AIJb 3.45DEFGH 0.08 - 211 229 230 212 440 646 Muscles - 0.99ACDEb - - 153 181 156 164 310 503 1%CLAmix Liver 4.17a 2.70A 2.60a 1.35a 233 236 238 225 471 686 Muscles - 0.50a 4.92Aa 7.44A 196 258 221 219 417 691 c9t11 Liver 3.91B 2.71B 2.60 1.71b 222 178 227 195 449 561 Muscles - 0.50 6.19 8.92a 144 221 164 139 308 500 t10c12 Liver 4.07C 3.12 2.90 1.71 265 220 275 210 541 635 Muscles - 0.48 5.80b 5.50b 181 227 206 141 388 502 2%CLAmix Liver 3.89D 2.75C 9.62ab 1.93a 226 240 237 227 463 687 Muscles - 0.58B 9.31A 12.5A 145 241 178 143 323 523 1%CLAmix + Se Liver 5.15Ej 3.98E 2.85 1.24 221 203 245 187 466 567 Muscles - 1.16C 7.37a 8.49 188 283 192 213 379 708 c9t11+Se Liver 5.50F 3.95F 2.98 1.31b 222 289 229 268 452 816 Muscles - 1.16D 6.85 11.5a 171 294 164 179 335 647 t10c12+Se Liver 5.23Gb 4.11G 2.71 1.76 212 230 223 207 434 643 Muscles - 1.14b 8.56b 8.30b 170 313 167 229 336 770 2%CLAmix + Se Liver 5.12Hj 4.22H 7.99b 1.84 226 247 234 238 459 722 Muscles - 1.20E 1.74 10.1 177 303 168 229 344 463 1 the concentrations of AAs analysed in pooled samples prepared by combination of all livers or muscles from rats fed the same diet 2 the sum of essential (ΣE-AA), non-essential (ΣNE-AA) and all assayed (ΣAAs) amino acids determined as described by Czauderna et al. (2002) and Niedźwiedzka et al. (2006c) 3 the concentration of Se and the sum of CLA isomers in the liver and femoral muscles of rats fed the diets enriched in CLA isomer(s) and Se (as SeVI (Czauderna et al., 2004a,b) and Se-yeast (Koniluk et al., 2006, 2007)). Concentrations of amino acids in plasma, liver and femoral muscles of rat Our recent investigations (Czauderna et al., 2004a,b; Niedźwiedzka et al., 2006b) as well as other studies (Alasnier et al., 2002; Terpstra et al., 2002; Wahle et al., 2004) indicated that CLA isomers decreased body fat, increased lean body mass and the amount of protein in the body of laboratory animals. In the presented study, the concentrations of amino acids in blood plasma (Tables 2 and 3), liver and femoral muscles (Table 6) were also affected by the diets enriched in CLA isomer(s). The diet containing individual CLA isomers increased the concentration of the sum of essential (ΣE-AA) and non-essential (ΣNE-AA) amino acids in plasma (Table 2). On the other hand, the addition of 1% of the CLA isomer mixture to the diet showed a tendency to decrease the concentration of these fatty acids in plasma. This result suggests that the interaction between dietary c9t11 and t10c12 in rats resulted in reducing the concentration of ΣE-AA and ΣNE-AA in plasma. Increasing the concentration of the CLAmix in the diet diminished this antagonistic effect of the isomer mixture in rats; consequently, the diet containing 2% CLAmix showed a tendency to slightly increase the concentration of these fatty acids (Table 3). Addition of SeVI to the diet enriched in individual isomers decreased the concentration of ΣE-AA and ΣNE-AA in plasma compared with rats fed the diet enriched in only the individual isomer (Table 2). Similarly, the antagonistic interaction between dietary SeVI, cc, tt, c9t11 and t10c12 CLA isomers decreased the effect of individual CLA isomers and SeVI (Tables 2 and 3) on the concentration of these amino acids. Consequently, the concentrations of ΣE-AA and ΣNE-AA in plasma were similar to the concentration of these fatty acids in the plasma of control rats. The antagonism between dietary geometrical conﬁguration of CLA isomer(s) and SeVI was also reﬂected in the concentration of these fatty acids in the liver and femoral muscle (Table 6). The diet enriched in CLA isomer(s) and SeVI resulted in a small numerical decrease in the concentration of ΣE-AA in the liver and muscle and in the FA AND AA PROFILES IN BLOOD PLASMA OF RATS Means in columns with the different letter are signiﬁcantly different at a,bP<0.05 or at ABP<0.01; statistical analyses of the effects of the CLA isomers or Se were conducted using the non-parametric Mann-Whitney U test for comparing independent experimental groups, while statistical analyses of the simultaneous the CLA isomers and Se treatments were performed applying two-factorial analysis for comparison with the independent control group 4 not measured 271 NIEDŹWIEDZKA K.M. ET AL. concentration of ΣNE-AA in muscles of rats fed the diet containing CLA isomer(s) and SeVI. Similarly, a numerical decrease of the concentration of ΣE-AA was also observed in the liver of rats receiving the diets enriched in Se-yeast and CLA isomer(s). A similar effect, although weaker, was found in the liver of rats fed the diets containing pure CLA isomer(s), i.e. possessing smaller concentrations of ttCLA and ccCLA (Table 1). The current results conﬁrm those of our recent investigations and other studies in rodents showing that dietary CLA isomer(s) increased liver weight (up to 50%); this effect on liver weight of CLA isomer(s) was mainly due to the increase the total lipid content per gram of liver in rats and mice (Terpstra et al., 2002; Czauderna et al., 2004a; Korniluk et al., 2006), while the total protein content in the liver decreased. p The data summarized in Table 6 conﬁrm the results of other studies showing that CLA isomer(s) appear to increase the lean body mass and the amount of protein in the body of laboratory animals (Belury, 2002). Indeed, dietary CLA isomer(s), possessing a lower concentration of ttCLA, numerically stimulated the accumulation of ΣE-AA in muscles, while decreasing the sum of all amino acids (ΣAA) (Table 6). This effect on the concentration of ΣE-AA was stronger in muscles of rats fed the diets containing these CLA isomer(s) and Se-yeast. The obtained results suggest that dietary CLA isomers decreased the biosynthesis of NE-AA, so the mean concentration of ΣNE-AA in muscles numerically declined; this effect was stronger in the muscles of rats (Table 6) fed the diets enriched in CLA isomer(s) containing a higher level of ttCLA and ccCLA isomers (Table 1), irrespective of the presence of SeVI. REFERENCES REFERENCES Alasnier C., Berdeaux O., Chardigny J.M., Sébédio J.L., 2002. Fatty acid composition and conjugated linoleic acid content of different tissues in rats fed individual conjugated linoleic acid isomers given as triacylglycerols. J. Nutr. Biochem. 13, 337-345 Arthur J.R., 2003. Selenium supplementation: does soil supplementation help and why? Proc. Nutr. Soc. 62, 393-397 Belury M.A., 2002. Dietary conjugated linoleic acids in health: Physiological effects and mechanisms of action. Annu. Rev. Nutr. 22, 505-531 ombs G.F., 2004. Status of selenium in prostate cancer prevention. Brit. J. Cancer 91, 195-199 Czauderna M., Kowalczyk J., 2001. Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection. J. Chromatogr. B 760, 165-178 Czauderna M., Kowalczyk J., Korniluk K., Wąsowska I., 2005. Improving the analysis of fatty acids using combination of gas chromatography and Ag+-liquid chromatography. J. Anim. Feed Sci. 14, Suppl. 1, 263-266 Czauderna M., Kowalczyk J., Niedźwiedzka K.M., Wasowska I., 2002. Determination of free- and protein primary amino acids in biological materials by high-performance liquid chromatography and photodiode array detection. J. Anim. Feed Sci. 11, 143-167 Czauderna M., Kowalczyk J., Niedźwiedzka K.M., Wąsowska I., Pastuszewska B., 2004a. Conjugated linoleic acid (CLA) content and fatty acids composition of muscle in rats fed isomers of CLA and selenium. J. Anim. Feed Sci. 13, 183-196 Czauderna M., Kowalczyk J., Niedźwiedzka K.M., Wąsowska I., Pastuszewska B., Bulska E., Ruszczyńska A., 2004b. Liver and body mass gain, content of CLA isomers and other fatty acids in the liver of rats fed CLA isomers and selenium. J. Anim. Feed Sci. 13, 353-369 Czauderna M., Kowalczyk J., Wąsowska I., Niedźwiedzka K.M., 2003. Determination of conjugated linoleic acid isomers by liquid chromatography and photodiode array detection. J. Anim. Feed Sci. 12, 269-282 De La Tore A., Debiton E., Juaneda P., Durand D., Chardigny J-M, Barthomeuf C., Bauchart D., Gruffat D., 2006. Beef conjugated linoloic acid isomers reduce human cancer cell growth even when associated with other beef fatty acids. Brit. J. Nutr. 95, 346-352 Korniluk K., Czauderna M., Kowalczyk J., Mieczkowska A., Taciak M., Leng L'., 2006. Inﬂuence of dietary conjugated linoleic acid isomers and selenium on growth, feed efﬁciency, and liver fatty acid proﬁle in rats. J. Anim. Feed Sci. 15, 131-146 Korniluk K., Kowalczyk J., Czauderna M., 2007. CONCLUSIONS As reported in other studies on rodents, the effectiveness of dietary CLA isomers in stimulating the body weight gain of rats and feed conversion efﬁciency was conﬁrmed in our current study. In it, we also unexpectedly found that the strongest positive interactions on these parameters occurred between t10c12 and Se, regardless of the latter’s chemical form. Therefore, we hypothesize that these effects can be explained by the interaction between metabolite(s) of dietary SeVI and Se-yeast (probably the same ones) and t10c12 and/or its metabolite(s). More importantly, we suggest that dietary SeVI and Se-yeast increased the capacity of β-oxidation of t10c12 as well as t10t12; therefore in our current and previous studies, the c9t11 isomer was preferentially accumulated in the body of rats and sheep, especially those fed the diets simultaneously enriched in the mixture of CLA isomers and selenium. Moreover, we hypothesize that the interaction between dietary CLA isomer(s) and Se (as SeVI or Se-yeast) stimulates the capacity of Δ9-, Δ6-, Δ4-desaturation and elongation of fatty acids. FA AND AA PROFILES IN BLOOD PLASMA OF RATS 272 REFERENCES The inﬂuence of dietary conjugated linoleic acid isomers and selenized yeast on the fatty acid proﬁle of spleen, pancreas and kidneys of rats. J. Anim. Feed Sci. 16, 121-139 Lyons T.P., Jacques K.A., 2004. Nutritional biotechnology in the feed and food industries. In: Proceedings of Alltech’s Twentieth Annual Symposium. 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https://openalex.org/W4295759489	https://microbiomejournal.biomedcentral.com/counter/pdf/10.1186/s40168-022-01341-9	English	null	The effect of 3-nitrooxypropanol, a potent methane inhibitor, on ruminal microbial gene expression profiles in dairy cows	Microbiome	2,022	cc-by	16,368	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pitta et al. Microbiome (2022) 10:146 https://doi.org/10.1186/s40168-022-01341-9 Open Access Abstract Keywords: Enteric methane, Hydrogenases, Methane mitigation, Ruminal methanogenesis, Total and metabolically active microbes ric methane, Hydrogenases, Methane mitigation, Ruminal methanogenesis, Total and metabolically Keywords: Enteric methane, Hydrogenases, Methane mitigation, Ruminal methanogenesis, Total and metabolically active microbes anthropogenic GHG emissions originate from the live- stock sector [2] and enteric CH4 from livestock pro- duction is the largest anthropogenic source of global CH4, having contributed approximately 97 Tg CH4/year, which is approximately 29.5% of total global CH4 emis- sions from 2000 to 2009 [3]. In addition to the GHG effect, CH4 emission results in a gross feed energy loss of approximately 2 to 12% to the ruminant host [4]. Glob- ally, research efforts and funding have been invested to develop novel CH4 mitigation strategies including The effect of 3‑nitrooxypropanol, a potent methane inhibitor, on ruminal microbial gene expression profiles in dairy cows Dipti W. Pitta1, Nagaraju Indugu1, Audino Melgar2, Alexander Hristov2, Krishna Challa1, Bonnie Vecchiarelli1, Meagan Hennessy1, Kapil Narayan1, Stephane Duval3, Maik Kindermann3 and Nicola Walker3 Dipti W. Pitta1, Nagaraju Indugu1, Audino Melgar2, Alexander Hristov2, Krishna Challa1, Bonnie Vecchiarelli1, Meagan Hennessy1, Kapil Narayan1, Stephane Duval3, Maik Kindermann3 and Nicola Walker3 Abstract Background: Enteric methane emissions from dairy cows are an environmental problem as well as a gross feed energy loss to the animal. Methane is generated in the rumen by methanogenic archaea from hydrogen (H2) + carbon dioxide and from H2 + methanol or methylamines. The methanogenic substrates are provided by non-meth- anogens during feed fermentation. Methane mitigation approaches have yielded variable results, partially due to an incomplete understanding of the contribution of hydrogenotrophic and methylotrophic archaea to methanogenesis. Research indicates that 3-nitrooxypropanol (3-NOP) reduces enteric methane formation in dairy cows by inhibiting methyl-coenzyme M reductase (MCR), the enzyme responsible for methane formation. The purpose of this study was to utilize metagenomic and metatranscriptomic approaches to investigate the effect of 3-NOP on the rumen microbi- ome and to determine the fate of H2 that accumulates less than expected under inhibited methanogenesis. Results: The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methano- sphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H2 concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H2-producing bacte- ria to regulate the amount of H2 production. No previously reported alternative H2 sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway. Results: The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methano- sphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H2 concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H2-producing bacte- ria to regulate the amount of H2 production. No previously reported alternative H2 sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway. Conclusion: By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H2 metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen. Conclusion: By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H2 metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen. Background g Methane (CH4) accounts for 20% of total global green- house gas (GHG) emissions and is the second largest contributor to global warming [1]. About 14.5% of all Correspondence: dpitta@vet.upenn.edu 1 Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square, PA 19348, USA Full list of author information is available at the end of the article 1 Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square, PA 19348, USA Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pitta et al. Microbiome (2022) 10:146 Pitta et al. Microbiome (2022) 10:146 Page 2 of 21 Page 2 of 21 altering the dietary regimen, feed supplements, and the use of both organic and inorganic molecules such as halomethanes and ionophores [2]. However, these dif- ferent mitigation strategies have had varying degrees of success when implemented, with some strategies accom- panied by adverse effects on animal health and produc- tion and also on the environment [5], indicating their limited practical use on farms. Furthermore, some of these mitigation strategies, including some CH4 inhibi- tors, that were found to be active in in vitro studies were reported to be ineffective in vivo [6]. Although a few CH4 mitigation practices have been found to be effective in inhibiting methanogenesis in vivo [7–9], their impact on archaea and other microbes present in the foregut of ruminants is unknown. decreased CH4 emissions [12, 18–20]. Therefore, the fate of H2 under normal and inhibited methanogenesis remains to be determined, as well as whether there are unidentified alternative sinks that can capture H2 when it is spared from methanogens in the rumen.h The inhibitor 3-NOP was designed to inhibit methyl- coenzyme M reductase (MCR), a highly conserved enzyme family that is essential for methanogenesis and is found in all methanogenic archaea. Notably, a study by Duin et al. [21] revealed that 3-NOP inhibited the growth of methanogenic archaea but had varying effects on individual methanogenic lineages in vitro. However, the effect of 3-NOP on the rumen microbiome remains to be investigated. We hypothesized that 3-NOP would significantly reduce the methanogenic community com- posed of hydrogenotrophic methanogens (4H2 + CO2 → CH4) and methylotrophic methanogens (1H2 + methanol or methylamines → CH4). We also hypothesized that reduction of methanogenesis would induce changes in ruminal H2-forming microbial communities via altering H2 concentrations in the rumen of dairy cows. To test this hypothesis, we employed a combination of metagenomic (metaG) and metatranscriptomic (metaT) approaches to determine changes in microbial diversity and microbial gene expression in the rumen of dairy cows with and without 3-NOP supplementation to the feed. Billions of microbes (bacteria, protozoa, fungi, and archaea) inhabit the rumen and work synergistically to digest what is indigestible feed for the host. Methane is a natural byproduct formed by methanogens (archaea) by utilizing hydrogen (H2), carbon dioxide (CO2), meth- anol, and methylamines that are released during carbo- hydrate and lipid breakdown by other microbes [10, 11]. Therefore, any mitigation strategy developed to reduce methanogenesis can perturb the symbiotic relationship between archaea and other microbial domains, which can negatively impact the rumen fermentation pattern and ultimately reduce feed intake and animal productiv- ity. Strategies that result in depression of feed intakes are not practical for the US dairy and beef production sys- tem. Therefore, a greater understanding of the impact of CH4 mitigation strategies on the symbiotic relationship among microbial domains and their selective inhibition of microbial genes/enzymes that control CH4 produc- tion is critically needed before the inhibitors are recom- mended for on-farm use. However, such information is not available as our knowledge on the functional role of archaea and interactions between bacteria and archaea in the rumen is limited. Changes in total and metabolically active methanogenic communities in response to 3‑NOP supplementation Changes in total and metabolically active methanogenic communities in response to 3‑NOP supplementation Rumen samples from 8 cows (4 control and 4 3-NOP supplemented cows) collected at weeks 4, 8, and 12 of a 15-week experiment were separated into solid and liquid fractions and analyzed for total (DNA) and metabolically active (RNA) methanogenic archaeal and bacterial diversity (solid communities; Fig. 1; liquid communities; SI Additional file 1: Figure S1). For beta diversity, the methanogenic community profiles dif- fered by treatment (DNA solid: P = 0.001; RNA solid: P = 0.002; RNA liquid: P = 0.004) and week (DNA solid: P = 0.001; RNA solid: P = 0.001; DNA liquid: P = 0.052; RNA liquid: P = 0.014), but no interac- tion was observed between treatment and week in either DNA-based or RNA-based archaeal commu- nities across either fraction (Fig. 1A; SI Additional file 1: Table 1). The solid fraction of rumen samples was further used for metagenomic and metatranscrip- tomic analysis to understand mechanisms of methano- genesis and to what extent microbial gene expression was altered when dairy cows were supplemented with 3-NOP. Recently, it was demonstrated that supplementing high- yielding dairy cows with 3-nitrooxypropanol (3-NOP; DSM Nutritional Products, CH-4303, Kaiseraugst, Swit- zerland), a potent CH4 inhibitor, led to a 27% and 57% reduction in enteric CH4 emissions in beef cattle [12, 13] and 23–37% in dairy cattle [14, 15]. Similarly, supplemen- tation of 3-NOP to dairy cows over a 15-week experi- mental period reduced enteric CH4 emissions by 26 to 30% with no observable impact on feed intake and pro- ductivity [7, 16–18]. Further, these authors observed that H2 emissions increased under inhibited methanogenesis for the first 8 weeks and then steadily declined in 3-NOP supplemented cows over the remainder of the 15-week period [7]. In this and other studies, the increased H2 emissions could only partially be accounted for by the Based on sequencing information (Supplementary information text), methanogenic archaea constituted Page 3 of 21 Pitta et al. Microbiome (2022) 10:146 Pitta et al. Microbiome Page 3 of 21 Pitta et al. Microbiome (2022) 10:146 Fig. 1 Rumen archaeal diversity and composition in control and 3-nitrooxypropanol (3-NOP) treated cows at weeks 4, 8, and 12. 16S rRNA archaeal diversity: A species richness, B Shannon diversity, and C comparison of overall community between samples by weighted UniFrac distances in DNA-based and RNA-based 16S rRNA analysis (beta diversity). Changes in total and metabolically active methanogenic communities in response to 3‑NOP supplementation (.) P < 0.1; () P < 0.05; () P < 0.01; () P < 0.001; NS, not significant DNA solid RNA solid R2 P value R2 P value Treatment 0.219 0.001 Treatment 0.231 0.002 Week 0.254 0.001 * Week 0.247 0.001 * Treatment: Week 0.030 0.30 NS Treatment: Week 0.021 0.36 NS DNA liquid RNA liquid Treatment 0.144 0.16 NS Treatment 0.157 0.004 ** Week 0.187 0.05 . NS Week 0.207 0.01 * Treatment: Week 0.010 0.68 NS Treatment: Week 0.010 0.66 NS Liquid vs solid in DNA Liquid vs solid in RNA Phase 0.152 0.004 Phase 0.237 0.001 * DNA vs RNA in solid DNA vs RNA in liquid Nucleic acid 0.202 0.001 * Nucleic acid 0.182 0.001 * the relative abundance of Methanobrevibacter was lower while that of unclassified Methanobacteriaceae and Meth- anosphaera was higher (P < 0.05) at weeks 4 and 8 when compared with control cows (SI Additional file 1: Table 2; Table S3). From metatranscriptomic data (Table 2; SI Additional file 1: Table S4), the relative abundance of Methanosphaera and genera from Thermoplasmata was nearly doubled compared with metagenomics. The rela- tive abundance of Methanobrevibacter was reduced (P < 0.001) with 3-NOP supplementation at weeks 4, 8, and 12 whereas that of Methanosphaera was higher (P < 0.001) at week 4 and week 8 but reduced at week 12. Based on rt-PCR results (Fig. 1H–K), it was evident that 3-NOP significantly reduced Methanobrevibacter ruminantium (P = 0.03) but had no effect on Methanobrevibacter smithii or 16S rRNA gene copy number in either DNA or RNA-based analysis. Methanosphaera stadtmanae was numerically increased with 3-NOP supplementation compared with control samples. all methanogenic pathways. Genes coding for the corre- sponding enzymes involved in steps 1–5 were identified using metagenomics, and gene expression was compared by identifying transcripts using metatranscriptomics (Fig. 2A). In this study, we were not able to identify genes associated with EC: 1.17.1.9, an enzyme required to con- vert formate to CO2 and therefore not shown in Fig. 2A. Only a small number of transcripts were identified for EC: 1.17.1.9, suggesting that in this particular experi- ment, formate was not a major substrate for methano- genesis. The copy number for genes encoding for enzyme EC: 1.2.7.12 (step 1 in Fig. 2A), required for reduction of CO2 to formylmethanofuran, was highest compared with all other enzymes involved in steps 1–5. Impact of 3‑NOP on methanogenesis pathways Using metagenomic and metatranscriptomic data, we quantified the genes and transcripts of enzymes involved in the 3 predominant ruminal methanogenesis pathways (CO2-, methanol-, and methylamine-reducing pathways) in cows with and without 3-NOP supplementation at weeks 4, 8, and 12 of the experimental period (Fig. 2). In addition, taxonomy of the annotated genes and tran- scripts was also tracked to help us understand the role of individual methanogenic lineages in methanogenesis. Changes in total and metabolically active methanogenic communities in response to 3‑NOP supplementation The transcript copy number for EC: 1.2.7.12 was higher compared with transcripts of enzymes involved in steps 2, 3, and 4 but lower than those in step 5. The number of gene copies for enzyme EC: 1.2.7.12 tended to be lower (P = 0.115), but the corresponding transcripts were lower (P = 0.05) in 3-NOP supplemented cows compared with control cows. The transcript copy numbers for EC: 2.3.1.101 (P < 0.001; step 2), 3.5.4.27 (P < 0.001; step 3), 1.5.98.1 (P < 0.001; step 4), and 1.5.98.2 (P < 0.001; step 5) were substantially reduced in 3-NOP supplemented cows compared with control cows. the relative abundance of Methanobrevibacter was lower while that of unclassified Methanobacteriaceae and Meth- anosphaera was higher (P < 0.05) at weeks 4 and 8 when compared with control cows (SI Additional file 1: Table 2; Table S3). From metatranscriptomic data (Table 2; SI Additional file 1: Table S4), the relative abundance of Methanosphaera and genera from Thermoplasmata was nearly doubled compared with metagenomics. The rela- tive abundance of Methanobrevibacter was reduced (P < 0.001) with 3-NOP supplementation at weeks 4, 8, and 12 whereas that of Methanosphaera was higher (P < 0.001) at week 4 and week 8 but reduced at week 12. Based on rt-PCR results (Fig. 1H–K), it was evident that 3-NOP significantly reduced Methanobrevibacter ruminantium (P = 0.03) but had no effect on Methanobrevibacter smithii or 16S rRNA gene copy number in either DNA or RNA-based analysis. Methanosphaera stadtmanae was numerically increased with 3-NOP supplementation compared with control samples. Changes in total and metabolically active methanogenic communities in response to 3‑NOP supplementation Archaeal genera: comparison of archaeal composition at genus level for D DNA-based 16S rRNA, E RNA-based 16S rRNA, F metagenomics (metaG), and G metatranscriptomics (metaT). rt-PCR: quantification of selected methanogens at week 8 by real time PCR (rt-PCR) for HMethanosphaera stadtmanae DSM309 (mtaB), IMethanobrevibacter ruminantium M1 (mcrG), JMethanobrevibacter smithii ATCC35061 (mtaB), and K 16S rRNA copy number. PCoA, principal coordinates analysis. NS, no statistical significance in generalized linear mixed model (glmer); P < 0.05; P < 0.01; *P < 0.001. The black circles appearing on the boxplots are the outlier samples Fig. 1 Rumen archaeal diversity and composition in control and 3-nitrooxypropanol (3-NOP) treated cows at weeks 4, 8, and 12. 16S rRNA archaeal diversity: A species richness, B Shannon diversity, and C comparison of overall community between samples by weighted UniFrac distances in DNA-based and RNA-based 16S rRNA analysis (beta diversity). Archaeal genera: comparison of archaeal composition at genus level for D DNA-based 16S rRNA, E RNA-based 16S rRNA, F metagenomics (metaG), and G metatranscriptomics (metaT). rt-PCR: quantification of selected methanogens at week 8 by real time PCR (rt-PCR) for HMethanosphaera stadtmanae DSM309 (mtaB), IMethanobrevibacter ruminantium M1 (mcrG), JMethanobrevibacter smithii ATCC35061 (mtaB), and K 16S rRNA copy number. PCoA, principal coordinates analysis. NS, no statistical significance in generalized linear mixed model (glmer); P < 0.05; P < 0.01; P < 0.001. The black circles appearing on the boxplots are the outlier samples about 6% and 8% of metagenomes and metatranscrip- tomes, respectively (Supplementary information text, SI Additional file 1: Table S1, Table S2). Across all sam- ples, within methanogenic archaea from metagenomic data, we found that the genus Methanobrevibacter alone contributed to 66–82% of total ruminal archaeal abundance followed by unclassified genera of Metha- nobacteriaceae and Methanosphaera at 3–6 and 3–5%, respectively (Fig. 1F; Table 2; SI Additional file 1: Table S3). In cows that received 3-NOP supplementation, Pitta et al. Microbiome (2022) 10:146 Page 4 of 21 Table 1 PERMANOVA analysis for 16S rRNA archaeal amplicon sequencing data. Treatment: control and 3-nitrooxypropanol (3-NOP); week: W4, W8, and W12. (.) P < 0.1; () P < 0.05; () P < 0.01; () P < 0.001; NS, not significant week: W4, W8, and W12. Methanogens involved in the CO2‑reducing pathway and impact of 3‑NOPh The most abundant archaea that contributed to the CO2-reducing pathway were identified as Methanobrevi- bacter species in metagenomic data (SI Additional file 1: Table S5). Six species of Methanobrevibacter (M. rumi- nantium M1, M. olleyae YLM1, M. millerae SM9, M. sp. YE315, M. sp AbM4, and M. smithii ATCC 35061) were identified. These 6 archaea species contributed to more CO2‑reducing methanogenesis pathwayh The CO2-reducing pathway is presented as the central pathway represented by steps 1–8 (Fig. 2A). Steps 1–5 are unique to this pathway, and steps 6–8 are common to Pitta et al. Microbiome (2022) 10:146 Page 5 of 21 Pitta et al. CO2‑reducing methanogenesis pathwayh Microbiome Table 2 The most abundant (> 1%) archaeal and bacterial genera identified in metagenomics and metatranscriptomics data in cows treated with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12 weeks Genus Control 3-NOP Significance W4 W8 W12 SEM W4 W8 W12 SEM Trt W4 vs W8 W4 vs W12 Trt:W4 vs W8 Trt: Trt:W4 vs W12 Archaea-metagenomics Methanobrevibacter 82.36 75.27 70.38 2.28 73.89 66.96 69.74 2.39 0.02 < 0.001 < 0.001 0.55 < 0.001 Methanobacteriaceae 3.76 5.84 5.52 0.33 4.35 6.61 6.39 0.35 0.02 < 0.001 < 0.001 0.74 0.34 Methanosphaera 3.17 4.81 5.26 0.48 4.41 5.63 5.31 0.46 0.13 < 0.001 < 0.001 0.006 < 0.001 Methanobacterium 0.87 1.39 1.38 0.11 1.21 1.84 1.73 0.13 0.03 < 0.001 < 0.001 0.82 0.27 Methanosarcina 0.9 1.11 1.58 0.16 1.54 1.65 1.44 0.19 0.02 < 0.001 < 0.001 0.65 < 0.001 Thermoplasmata 0.79 1.04 1.3 0.17 1.01 1.59 1.56 0.16 0.01 < 0.001 < 0.001 0.82 < 0.001 Archaea-metatranscriptomics Methanobrevibacter 78.6 72.19 69.46 1.66 74.01 67.18 66.82 1.84 0.12 < 0.001 < 0.001 < 0.001 < 0.001 Methanosphaera 6.14 9.65 11.07 0.88 7.32 10.68 7.92 0.81 0.76 < 0.001 < 0.001 0.002 < 0.001 Methanobacteriaceae 4.24 6.98 6.86 0.46 6.47 7.24 8.07 0.45 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Thermoplasmata 4.83 4.14 4.51 0.39 5.13 6.82 8.03 0.8 0.17 < 0.001 < 0.001 < 0.001 < 0.001 Bacteria- metagenomics Bacteroidetes Prevotella 16.19 16.14 18.56 0.826 17.54 16.52 14.66 0.81 0.24 < 0.001 < 0.001 < 0.001 < 0.001 Bacteria Unclassified 4.29 4.45 4.32 0.094 4.17 4.41 4.57 0.092 0.40 < 0.001 0.004 < 0.001 < 0.001 Fibrobacteres Fibrobacter 3.16 3.07 3.27 0.346 3.32 2.41 3.64 0.309 0.97 < 0.001 0.36 < 0.001 < 0.001 Firmicutes Butyrivibrio 3.6 3.33 2.72 0.332 2.72 2.85 3.18 0.209 0.006 < 0.001 < 0.001 < 0.001 < 0.001 Bacteroidetes Bacteroides 2.55 2.52 2.75 0.092 3 2.75 2.54 0.094 0.002 0.66 < 0.001 < 0.001 < 0.001 Firmicutes Lachnospiraceae 2.06 2.13 1.94 0.068 1.97 2.02 2.07 0.03 0.007 < 0.001 < 0.001 0.42 < 0.001 Firmicutes Clostridium 1.86 1.98 1.87 0.041 1.74 2.03 1.98 0.061 0.02 < 0.001 0.38 < 0.001 < 0.001 Actinobacteria Bifidobacterium 2.24 2.13 1.45 0.471 2.1 1.93 1.4 0.323 0.82 < 0.001 < 0.001 0.001 < 0.001 Firmicutes Lachnoclostridium 1.51 1.64 1.45 0.052 1.49 1.56 1.59 0.026 0.10 < 0.001 < 0.001 0.002 < 0.001 Firmicutes Clostridia 1.38 1.47 1.33 0.045 1.27 1.42 1.5 0.053 0.19 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Ruminococcus 1.38 1.35 1.29 0.071 1.05 1.31 1.27 0.07 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Actinobacteria Olsenella 1.1 1.16 1.4 0.137 1.25 1.15 1.16 0.114 0.38 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Clostridiales 1.14 1.16 1.07 0.036 1.1 1.13 1.2 0.044 0.41 0.09 < 0.001 < 0.001 < 0.001 Bacteroidetes Prevotellaceae 0.89 0.96 1.11 0.069 1.11 1.05 0.88 0.059 0.01 < 0.001 < 0.001 < 0.001 < 0.001 Bacteria-metatranscriptomics Bacteroidetes Prevotella 17.65 16.96 18.74 0.749 16.1 16.42 14.9 0.559 0.26 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Lachnospiraceae 3.81 4.16 3.9 0.144 4.39 4.05 4.62 0.15 0.20 < 0.001 < 0.001 < 0.001 < 0.001 Bacteria 3.74 3.68 3.61 0.07 3.63 3.58 3.77 0.056 0.71 < 0.001 0.56 0.001 < 0.001 Fibrobacteres Fibrobacter 3.77 3.65 3.92 0.425 3.15 2.8 3.22 0.269 0.21 < 0.001 < 0.001 < 0.001 < 0.001 Page 6 of 21 Pitta et al. CO2‑reducing methanogenesis pathwayh Microbiome (2022) 10:146 Genus Control 3-NOP Significance W4 W8 W12 SEM W4 W8 W12 SEM Trt W4 vs W8 W4 vs W12 Trt:W4 vs W8 Trt: Trt:W4 vs W12 Firmicutes Ruminococcus 3.47 3.71 3.05 0.218 2.59 2.83 2.86 0.145 0.16 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Butyrivibrio 3.17 2.98 2.77 0.218 3 2.95 2.9 0.195 0.52 0.002 < 0.001 < 0.001 < 0.001 Firmicutes Lachnoclostridium 1.97 2.13 1.99 0.068 2.28 2.05 2.53 0.1 0.005 < 0.001 < 0.001 < 0.001 < 0.001 Bacteroidetes Bacteroides 2.01 1.87 1.96 0.074 2.04 1.92 1.74 0.069 0.98 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Clostridium 1.24 1.47 1.71 0.199 2.5 1.56 1.96 0.259 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Eubacterium 1.51 1.72 1.6 0.073 2.06 1.68 1.71 0.111 0.10 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Clostridia 1.54 1.54 1.41 0.065 1.61 1.49 1.79 0.075 0.51 < 0.001 0.65 < 0.001 < 0.001 Firmicutes Clostridiales 1.34 1.36 1.28 0.05 1.4 1.36 1.6 0.06 0.42 < 0.001 < 0.001 < 0.001 < 0.001 Actinobacteria Olsenella 1.12 1.15 1.49 0.116 0.94 1.27 1.53 0.129 0.34 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Faecalibacterium 1.24 1.24 1.16 0.036 1.16 1.21 1.38 0.04 0.92 < 0.001 < 0.001 < 0.001 < 0.001 Actinobacteria Bifidobacterium 1.34 1.57 0.77 0.407 1.72 0.78 1.11 0.17 0.45 < 0.001 < 0.001 < 0.001 < 0.001 Proteobacteria Proteobacteria 1.3 1.2 1.19 0.045 1.1 1.21 1.1 0.056 0.003 < 0.001 < 0.001 < 0.001 < 0.001 Bacteroidetes Prevotellaceae 1.12 1.24 1.4 0.11 1.12 1.17 1.04 0.06 0.65 < 0.001 < 0.001 < 0.001 < 0.001 Firmicutes Megasphaera 0.27 0.3 1.08 0.29 0.23 3.83 0.3 1.302 0.92 < 0.001 < 0.001 < 0.001 < 0.001 Pitta et al. Microbiome (2022) 10:146 Page 7 of 21 Fig. 2 Comparisons of genes (metagenomics; metaG) and transcripts (metatranscriptomics; metaG) abundance in copies per million (cpm) for enzymes involved in methanogenesis between control and 3-nitrooxypropanol (3-NOP)-treated cows at weeks 4, 8, and 12. A Carbon dioxide (CO2)/hydrogen (H2) methanogenesis pathway (KEGG pathway entry MD:M00567), B methanol methanogenesis pathway (KEGG pathway entry MD:M00356), and C methylamine methanogenesis pathway (KEGG pathway entry MD:M00563). NS, no statistical significance in generalized linear mixed model (glmer); P < 0.05; P < 0.01; P < 0.001. The black circles appearing on the boxplots are the outlier samples Fig. CO2‑reducing methanogenesis pathwayh 2 Comparisons of genes (metagenomics; metaG) and transcripts (metatranscriptomics; metaG) abundance in copies per million (cpm) for enzymes involved in methanogenesis between control and 3-nitrooxypropanol (3-NOP)-treated cows at weeks 4, 8, and 12. A Carbon dioxide (CO2)/hydrogen (H2) methanogenesis pathway (KEGG pathway entry MD:M00567), B methanol methanogenesis pathway (KEGG pathway entry MD:M00356), and C methylamine methanogenesis pathway (KEGG pathway entry MD:M00563). NS, no statistical significance in generalized linear mixed model (glmer); P < 0.05; P < 0.01; P < 0.001. The black circles appearing on the boxplots are the outlier samples expression was reduced at all sampling times in 3-NOP supplemented dairy cows.h than 93% of the genes as well as transcripts coding for enzymes involved in steps 1–5 of the CO2-reducing path- way. The most abundant was M. ruminantium M1 fol- lowed by the other 5 Methanobrevibacter species with small variations in their contributions towards gene and transcript abundance. The archaea populations that contributed genes/tran- scripts to the methanol-utilizing pathway were identified as Methanosphaera BMS, Methanosphaera stadtmanae, Methanogenic archaeon ISO4-H5, and Methanobrevibac- ter smithii (SI Additional file 1: Table S6). Metagenomic results showed that these 4 archaea species comprised about 97–99% of identified methanol-utilizing archaea with 46–50% of gene copies from M. stadtmanae, 39–44% from M. BMS, 5–9% from M. smithii, and 2–4% from M. archaeon. In metatranscriptomics, M. BMS had the greatest metabolic activity with nearly 56% of contribution followed by M. stadtmanae at 38% and M. archaeon at 5%, indicating their significant role in utiliz- ing methanol. Although M. smithii had considerable gene copies for methanol utilization, there was no expression found for these genes indicating that M. smithii is not capable of utilizing methanol. Impact of 3‑NOP on methanol‑ and methylamine‑utilizing pathways In the methanol-utilizing pathway (Fig. 2B), the genes and transcripts for the enzyme methanol-corrinoid pro- tein co-methyltransferase (mtaB; EC: 2.1.1.90) were found in greater numbers compared with the other 2 enzymes involved in this pathway. Gene copies were numerically reduced with 3-NOP supplementation at weeks 4 and 8 but not at week 12. However, transcripts for this enzyme were reduced (P = 0.001) at weeks 4, 8, and 12 in cows supplemented with 3-NOP with the greatest reduction at week 12. These data indicate that although changes in gene copies were inconsistent, their In the methylamine-utilizing pathway (Fig. 2C), most of the genes coding for enzymes involved in transfer of Pitta et al. Microbiome (2022) 10:146 Pitta et al. Microbiome (2022) 10:146 Page 8 of 21 Table 3 Changes in genes and transcripts (copies per million, CPM) encoding for methyl-coenzyme M reductase (MCR) enzyme (EC: 2.8.4.1) in the rumen of dairy cows supplemented with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12. Based on raw reads and based on metagenome assembled genomes (MAGs). K00399, alpha subunit of MCR; K00401, beta subunit of MCR; K00402, gamma subunit of MCR Table 3 Changes in genes and transcripts (copies per million, CPM) encoding for methyl-coenzyme M reductase (MCR) enzyme (EC: 2.8.4.1) in the rumen of dairy cows supplemented with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12. Based on raw reads and based on metagenome assembled genomes (MAGs). Impact of 3‑NOP on methanol‑ and methylamine‑utilizing pathways K00399, alpha subunit of MCR; K00401, beta subunit of MCR; K00402, gamma subunit of MCR Percent reduction = (Control)−(3−NOP) (Control) X 100 MCR genes identification based on raw reads Metagenomics W4 W8 W12 Control 3-NOP % reduction Control 3-NOP % reduction Control 3-NOP % reduction Treatment P value K00399 265 213 20 306 230 25 227 253 −11 0.18 K00401 245 178 27 239 163 32 169 192 −14 0.18 K00402 253 197 22 293 223 24 210 251 −20 0.22 2.8.4.1 763 588 23 838 616 26 606 697 −15 0.12 Metatranscriptomics K00399 2756 2578 6 3656 2548 30 2667 2333 13 0.15 K00401 2135 1781 17 2529 1606 36 1885 1415 25 0.001 K00402 2679 2908 −9 3946 2905 26 2829 2571 9 0.64 EC:2.8.4.1 7570 7268 4 10131 7059 30 7381 6320 14 0.06 MCR gene identification based on MAGs Metagenomics K00399 32 33 −3 49 47 5 36 51 −42 0.47 K00401 52 43 17 55 37 33 38 42 −11 0.10 K00402 142 92 35 88 41 54 69 54 22 0.06 EC:2.8.4.1 75 56 26 64 42 35 48 49 −3 0.07 Metatranscriptomics K00399 411 507 −24 777 607 22 596 591 1 0.92 K00401 537 565 −5 877 570 35 681 545 20 0.08 K00402 1282 988 23 1276 816 36 891 605 32 0.02 EC:2.8.4.1 743 687 8 977 664 32 723 580 20 0.01 MCR genes identification based on raw reads methylamines were found in greater numbers relative to dimethyl or trimethylamines, although the total gene/ transcript copies were much lower than those involved in other pathways. Across all animals, copies of the gene coding for the enzyme methylamine-corrinoid protein co-methyltransferase (EC: 2.1.1.248) were negligible (<10 copies per million [cpm]) whereas the correspond- ing transcript contribution was 10 times greater than its gene abundance. No differences were noted between treatment groups in either their genes or transcripts. The archaea populations that contributed to the methylamine pathway were Methanogenic archaeon ISO4 and Thermo- plasmatales archaeon BRNA1 with some contribution from Candidatus Methanomethylophilus alvus Mx1201 (SI Additional file 1: Table S7). heterodisulphide and releasing CH4 in the penultimate step [22]. In the current study, the copy number of genes and transcripts for EC: 2.8.4.1 were among the most abundant genes/transcripts involved in methanogenesis pathways. Impact of 3‑NOP on methanol‑ and methylamine‑utilizing pathways Although variable responses were noted for gene copies, transcripts tended to be reduced (P = 0.062) in 3-NOP sup- plemented dairy cows indicating that the reduction in CH4 emissions by 3-NOP as described [18] is accompanied by a reduction in expression of genes coding for MCR (Fig. 2A).h methylamines were found in greater numbers relative to dimethyl or trimethylamines, although the total gene/ transcript copies were much lower than those involved in other pathways. Across all animals, copies of the gene coding for the enzyme methylamine-corrinoid protein co-methyltransferase (EC: 2.1.1.248) were negligible (<10 copies per million [cpm]) whereas the correspond- ing transcript contribution was 10 times greater than its gene abundance. No differences were noted between treatment groups in either their genes or transcripts. The archaea populations that contributed to the methylamine pathway were Methanogenic archaeon ISO4 and Thermo- plasmatales archaeon BRNA1 with some contribution from Candidatus Methanomethylophilus alvus Mx1201 (SI Additional file 1: Table S7). The enzyme MCR has 3 subunits (alpha, beta, and gamma) encoded by K00399, K00401, and K00402, respectively [23]. Using metagenomics and metatran- scriptomics, across all samples, we found that gene and transcript copy numbers for K00399 and K00402 were similar to each other whereas K00401 was numerically lower compared with K00399 and K00402 (Table 3). With 3-NOP supplementation, gene copies for MCR tended to be lower with a 23% and 26% reduction observed at weeks 4 and 8 and a 15% increase at week 12. The transcripts of the MCR enzyme also tended to be lower (P = 0.062) Impact of 3‑NOP on methyl‑coenzyme M reductase, h i i f ll h i h Impact of 3‑NOP on methyl‑coenzyme M reductase, the connecting point for all methanogenesis pathways The enzyme MCR (EC: 2.8.4.1) is responsible for CH4 for- mation by incorporating methyl Co-M and Co-B to form a Fig. 3 Effect of 3-nitrooxypropanol (3-NOP) on rumen bacteria via changes in dissolved hydrogen (H2) concentrations. A Effect of 3-NOP on dissolved H2 in lactating dairy cows; the bacterial genera appearing in the oval shapes are rapid (orange) and slow (blue) fermenters that were increased in 3-NOP treated cows at week 8 and week 12, respectively (see panel B). B Fold change (log 10) between control and 3-NOP-treated cows at weeks 4, 8, and 12 in the relative abundance of selected bacterial genera in DNA-based 16S rRNA analysis. The selection of bacterial genera was based on significant differences (glmer test) between treatment groups (P < 0.05). C Fold change (log 10) between control and 3-NOP treated cows at weeks 4, 8, and 12 in hydrogenase content in metagenomes (metaG) and metatranscriptomes (metaT). Hydrogenase content is shown based on hydrogenase subgroup. These are divided into fermentative hydrogenases (H2-producing; group A1, A2, B [FeFe]-hydrogenases), bifurcating hydrogenases (bidirectional; group A3, A4 [FeFe]-hydrogenases), respiratory hydrogenases (H2-uptake; group 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j [NiFe]-hydrogenases), respiratory hydrogenases (H2-evolving; group 4b, 4d [NiFe]-hydrogenases), alternative and sensory hydrogenases (H2-uptake; 2a, 2b [NiFe]-hydrogenases), cofactor-coupled bidirectional hydrogenases (3b, 3d, [NiFe]-hydrogenases), methanogenic hydrogenases (H2-uptake; group 1k, 3a, 3c, 4h, 4i [NiFe]-hydrogenases, [Fe]-hydrogenases), energy-converting hydrogenases (bidirectional; group 4a, 4c, 4e, 4f, 4g [NiFe]-hydrogenases), and sensory hydrogenases (group C [FeFe]-hydrogenases). Positive and negative log 10-fold change is the increased and decreased relative abundance, respectively, in 3-NOP compared with controls cows. (See figure on next page.) Impact of 3‑NOP on methyl‑coenzyme M reductase, the connecting point for all methanogenesis pathwaysh y The enzyme MCR (EC: 2.8.4.1) is responsible for CH4 for- mation by incorporating methyl Co-M and Co-B to form a Pitta et al. Microbiome (2022) 10:146 Page 9 of 21 with a 4%, 30%, and 14% reduction in 3-NOP supple- mented cows at weeks 4, 8, and 12, respectively (Table 3) compared with control cows. Notably, the reduction in transcript copies was greater for the beta chain (K00401; P < 0.001) in response to 3-NOP, followed by the alpha and then gamma subunits, indicating that the beta sub- unit may be more vulnerable to inhibition by 3-NOP. The effect of 3-NOP was greatest at week 8, which may be attributed to an increase in dry matter intakes during that period [18] which may have increased the allowance of 3-NOP in the rumen compared with weeks 4 and 12 [24]. In addition, we identified the three genes (mcr𝛼, mcrB, mcr𝛾) from the methanogen MAGs across all sam- ples. Although the percent reduction varied between raw reads and methanogen MAGs between treatments at each sampling week, overall differences in MCR enzyme followed the same trend for raw reads as well as MAGs (Table 3). Other genes and transcripts involved in step 8 had very low copy number and are discussed in the Sup- plementary information text and Table S8 (SI Additional file 1). 3-NOP groups were selected to indicate changes in the relative abundance between weeks (Fig. 3A, B). In cows that received 3-NOP, Lachnospiraceae, Ruminococcaceae, and Clostridiales were reduced at weeks 4 and 8 but increased by week 12, whereas Succinivibrionaceae, Suc- ciniclasticum, Veillonellaceae, and Sharpea showed the opposite trend compared with control cows. Hydrogenases regulating H2 production under normal and inhibited methanogenesis Under inhibited methanogenesis, concentrations of both gaseous and dissolved H2 increased from week 4 through week 8 and then declined from week 9 through week 12 as described in Melgar et al. ([18] Fig. 3A; SI Additional file 1: Table S13). The dynamics in H2 concentrations in response to 3-NOP supplementation were also associated with changes in hydrogenases, which are metalloenzymes that interchangeably convert H2 to 2[H] + 2e-. Hydroge- nases are broadly classified as [FeFe], which aid in sensing H2 concentrations and production of H2; [NiFe], which facilitate H2 uptake; and [Fe], of which the function is currently unknown. The [FeFe] are further differentiated into A1-A4, B, and C1-C3 with the first two groups regu- lating H2 production and the latter sensing H2 concentra- tions. Both metaG and metaT data revealed that [FeFe] A1, [FeFe]A3, [FeFe]B, [FeFe]C2, and [FeFe]C3 consti- tuted the majority of the [FeFe] hydrogenases (Fig. 3C). Impact of 3‑NOP on bacterial populations In DNA-based 16S rRNA sequencing and metagenomics, Firmicutes and Bacteroidetes were the most dominant bacterial phyla (Table 2; SI Additional file 1: Table S9; Table S11). However, in RNA-based 16S rRNA sequenc- ing and metatranscriptomics (Table 2; SI Additional file 1: Table S10; Table S12), the contribution of Fir- micutes was much higher and that of Bacteroidetes was much lower than their corresponding gene copies. Fur- ther, the relative abundance of Fibrobacteres and Proteo- bacteria was much higher in RNA compared with DNA in 16S rRNA sequencing. Firmicutes was dominated by Lachnospiraceae, Ruminococcaceae, Clostridiales, and Butyrivibrio, Bacteroidetes was dominated by Prevo- tella, Bacteroidales, and S24-7, and Proteobacteria was dominated by Succinivibrionaceae. Bacterial lineages that showed significant differences (generalized linear mixed model [glmer] test; P < 0.05) between control and Interestingly, the gene copies encoding for hydro- genases A1, A3, B, C1, and C3 were fairly consistent between weeks 4, 8, and 12 in the control group. How- ever, variations were noted in hydrogenases between sampling weeks within 3-NOP supplemented cows, with A3, B, and C3 progressively increasing from week 4 through week 12 (SI Additional file 1: Table S14, Table S15). When compared with control, the gene cop- ies for B (P = 0.007) and C3 (P < 0.001) were higher in 3-NOP supplemented cows at all sampling weeks. Both A1 and A3 had a greater number of transcripts, as cpm, compared with gene copies, whereas transcripts of genes encoding for B, C2, and C3 were only slightly greater than Page 10 of 21 Pitta et al. Microbiome (2022) 10:146 Pitta et al. Microbiome Fig. 3 (See legend on previous page.) Pitta et al. Microbiome (2022) 10:146 Page 11 of 21 Pitta et al. Microbiome Fig. 4 Associations between rumen bacteria and fermentation profiles. Correlation between bacteria (DNA-based [top] and RNA-based 16S rRNA sequencing analysis [bottom]) and fermentation profiles. Different colors of the bacterial genera show the corresponding phylum. dH2, dissolved hydrogen; VFA, volatile fatty acids; mol%, molar proportion Fig. 4 Associations between rumen bacteria and fermentation profiles. Correlation between bacteria (DNA-based [top] and RNA-based 16S rRNA sequencing analysis [bottom]) and fermentation profiles. Different colors of the bacterial genera show the corresponding phylum. dH2, dissolved hydrogen; VFA, volatile fatty acids; mol%, molar proportion fermentation variables including volatile fatty acids (VFA; Table S18), we sought to investigate differences in fer- mentation pathways leading to VFA production (Fig. 4). Impact of 3‑NOP on bacterial populations their corresponding gene copies. The ratio of A1:A3 was consistent at weeks 4 and 8 whereas A3 was numerically reduced and A1 numerically increased by week 12 in the control group. In the 3-NOP supplemented group, tran- scripts of genes coding for A1 were numerically reduced from week 4 through week 12 whereas the opposite pat- tern was observed for genes coding for A3. Transcripts of genes coding for A3 showed a trend (P = 0.11) to be lower at weeks 4 and 8 but increased by week 12 in 3-NOP compared with control samples. First, we performed correlation analysis between molar proportions of individual VFA and bacteria popula- tions identified from DNA- and RNA-based 16S rRNA sequencing across all samples (Fig. 4). Associations between bacteria and individual VFA were more evident with RNA-based analysis than those identified using DNA-based methods. Bacteria that showed differences (P < 0.05) between control and 3-NOP treatments across all sampling weeks were selected to perform correlation analysis with fermentation parameters. We found that most of the lineages from Firmicutes were positively associated with acetate proportions and a few genera, such as Prevotella and Succinivibrionaceae, were posi- tively associated with propionate. Alternative H2 sinks under inhibited methanogenesis 5 Comparisons of metagenomic (metaG) and metatranscriptomic (metaG) abundance for enzymes involved in the butyrate pathway (A) and propanoate pathway (B) between control and 3-nitrooxypropanol (3-NOP) treated cows at weeks 4, 8, and 12. cpm, copies per million; NS, no statistical significance in generalized linear mixed model (glmer); P < 0.05; P < 0.01; P < 0.001. The black circles appearing on the boxplots are the outlier samples Fig. 5 Comparisons of metagenomic (metaG) and metatranscriptomic (metaG) abundance for enzymes involved in the butyrate pathway (A) and propanoate pathway (B) between control and 3-nitrooxypropanol (3-NOP) treated cows at weeks 4, 8, and 12. cpm, copies per million; NS, no statistical significance in generalized linear mixed model (glmer); P < 0.05; P < 0.01; P < 0.001. The black circles appearing on the boxplots are the outlier samples The bacterium that had the predominant role in the propi- onate pathway was identified as Prevotella ruminicola fol- lowed by several other bacteria. The bacterium that had the predominant role in the propi- onate pathway was identified as Prevotella ruminicola fol- lowed by several other bacteria. (malonate semialdehyde pathway), the propanoate path- way, and the acrylate pathway via lactate (Fig. 5B). No differences were detected in genes involved in propion- ate pathways between control and 3-NOP group-treated cows. The M00741 pathway appeared to be the predomi- nant pathway. Although there were small changes in tran- scripts, there was no particular pattern in response to 3-NOP treatment. Similarly, no differences were noted in the molar proportion of ruminal propionate. Bacteria shown to be associated with the propionate pathway are shown in Table S21 and Table S22 (SI Additional file 1). Alternative H2 sinks under inhibited methanogenesis 2 g We attempted to investigate whether H2 spared under inhibited methanogenesis was directed to alternate H2 sinks (SI Additional file 1: Table S16; Table S17) that may directly or indirectly compete with methanogens. We identified that methanogenesis is the largest H2 sink followed by reduction of CO2 to acetate, reduction of fumarate to succinate, reduction of nitrate/nitrite to ammonia, and reduction of sulfate to H2S. Lastly, there is a very small contribution from sulfite reductase, fuma- rate reductase, and ammonia-forming nitrite reductase. Although acetyl-CoA synthetase, a marker gene for ace- togenesis, was not significantly increased in either gene or transcripts copies, we found a significant increase (P = 0.008) in transcript copies of formyl-tetrahydrofolate synthetase, another marker gene for acetogens, in 3-NOP supplemented cows compared to control at all sampling weeks (SI Additional file 1: Table S16, Table S17). Meth- ane inhibition did not result in a sudden increase of either genes or transcripts of other alternative H2 sinks that were investigated in this study (SI Additional file 1: Table S16, Table S17). Because there were differences in Next, we identified genes and transcripts coding for enzymes involved in different pathways leading to butyrate and propionate formation (Fig. 5A, B). There were differences in gene and transcript copies between control and 3-NOP treatment groups for enzymes involved in the butyrate pathway. Notably, genes and transcripts for the enzyme EC: 1.3.8.1, which cata- lyzes conversion of crotonyl-CoA to butyryl-CoA, were increased (genes: P = 0.001; transcripts: P < 0.001) in 3-NOP samples compared with control at all weeks of sampling. These data agree with increased (P < 0.05) molar proportions of butyrate in 3-NOP samples com- pared with control. Bacteria shown to be associated with the butyrate pathway are shown in Table S19 and Table S20 (SI Additional file 1). i The pathways leading to propionate were identified as M00741 (propanoyl-CoA via succinyl-CoA), M00013 Pitta et al. Microbiome (2022) 10:146 Page 12 of 21 Pitta et al. Microbiome Fig. 5 Comparisons of metagenomic (metaG) and metatranscriptomic (metaG) abundance for enzymes involved in the butyrate pathway (A) and propanoate pathway (B) between control and 3-nitrooxypropanol (3-NOP) treated cows at weeks 4, 8, and 12. cpm, copies per million; NS, no statistical significance in generalized linear mixed model (glmer); P < 0.05; P < 0.01; *P < 0.001. The black circles appearing on the boxplots are the outlier samples Fig. Discussion Methanogenesis in the rumen can occur via hydrog- enotrophic, methylotrophic, and aceticlastic pathways [25, 26], although the contribution of these pathways to total CH4 formation has not been clearly described. In the current study, using both metagenomic and metatran- scriptomic approaches, we have identified that the CO2-reducing (hydrogenotrophic) pathway was predomi- nant followed by the methanol-utilizing pathway and the methylamine-utilizing pathway regardless of treatment. However, metatranscriptomics predicts that the methy- lotrophic methanogenic (methanol- and methylamine- reducing) pathways may have greater contributions to total methanogenesis compared with their corresponding gene content, revealing that methylotrophic methano- gens may have a greater contribution to CH4 formation in the rumen than what was originally anticipated. For example, we found that the total number of gene copies for the methanol-utilizing enzyme methanol--corrinoid protein co-methyltransferase (EC: 2.1.1.90) in Methano- sphaera was < 100 cpm in metagenomic data, whereas it was > 600 cpm in metatranscriptomics (Fig. 2B), revealing that Methanosphaera has greater metabolic activity than what we would anticipate from the total number of gene copies alone. We also found that 2 lineages of Methano- massiliicoccales, Methanogenic archaeon ISO4-H5, and Thermoplasmatales archaeon BRNA1, which contributed to the methylamine-reducing pathway, also had a greater contribution from transcripts than their corresponding genes. These findings agree with those of Söllinger et al. [27] who employed metatranscriptomics to report that methylotrophic methanogens may have a greater role in methanogenesis than was originally thought. In the current study, although the overall contribution of hydrogenotrophic methanogens was much greater than methanol-utilizing and methylamine-utilizing methanogens, the genes encoding for the enzyme EC: 1.2.7.12, which facilitates conversion of CO2 to formyl- methanofuran, were approximately 1000 cpm while its transcripts were 1500 cpm. In contrast, the genes encoding for the enzyme EC: 2.1.1.90, which facilitates conversion of methanol, were less than 50 cpm and its transcripts were less than 1000 cpm, suggesting a greater contribution from methanol-utilizing methanogens than what would be anticipated from their gene copies. Fur- ther, experimental dairy cows were transitioned from a fresh diet to a lactation diet by week 4 after calving and continued on the same lactation diet throughout the study. There were minor changes in the ingredient composition of fresh and lactation diets, but the chemi- cal composition remained similar. Discussion Enteric CH4 formation is an intractable problem and is a consequence of complex microbial interactions in the forestomach of ruminant animals. Using 3-NOP, a potent CH4 inhibitor, as a feed supplement to dairy cow diets has been previously shown to reduce CH4 formation by 26 to 30% [7, 18] and by 23–37% [14, 15]. This study Pitta et al. Microbiome (2022) 10:146 Page 13 of 21 Page 13 of 21 Differences in metabolic capabilities in hydrogenotrophic and methylotrophic methanogens have been discussed in our recent paper [31]. Although both Methanobrevi- bacter and Methanosphaera belong to the same order, Methanobacteriales, there are contrasting features between the 2 methanogenic genera that have functional relevance to CH4 formation and rumen fermentation. First, Methanobrevibacter lineages reduce CO2 or for- mate whereas Methanosphaera has acquired the ability to extract the methyl group from methanol and therefore adopts a methanogenic pathway that is a hybrid between the hydrogenotrophic and methylotrophic pathways [32]. Second, affinity and thresholds for H2 are lower for Methanosphaera compared with those of Methanobrevi- bacter [33]. Recently, Feldewert et al. [34] reported that CO2-reducing Methanobrevibacter species have higher H2 thresholds (> 5.0 Pa) compared with methanol-utiliz- ing Methanosphaera (1.0 Pa) and methylamine-utilizing Methanomassiliicoccales (< 0.1 Pa) suggesting that meth- ylamine- and methanol-utilizing methanogens have an advantage over CO2-reducing methanogens. Particularly under conditions when the dissolved H2 concentrations in the rumen are low and there is availability of methyl- amines and methanol substrates, methylotrophic metha- nogens may outcompete hydrogenotrophic methanogens and may serve as the major pathways for methanogene- sis. However, when the concentrations of dissolved H2 in the rumen fluid are higher than the thresholds of hydrog- enotrophic methanogens, the latter methanogens may dominate due to the abundance of CO2 concentrations in the rumen. enabled us to gain a deeper understanding of the com- plex interdependencies between methanogens and bac- teria for H2 under normal and inhibited methanogenesis in the rumen of dairy cows supplemented with 3-NOP. Using a combination of omic approaches, this study pro- vides new information on temporal dynamics in individ- ual methanogenic lineages and their contribute to total methanogenesis. Further, this study has elucidated both temporal changes in bacteria populations in response to fluctuating H2 concentrations in the rumen and some of the possible mechanisms by which the spared H2 under inhibited methanogenesis by 3-NOP was directed to alternate sinks. Discussion Based on DNA- and RNA-based 16S rRNA sequencing analysis, we found that certain bacteria including Prevotella, Succinivibrionaceae, Veil- lonellaceae, Succiniclasticum, and Sharpea, have been associated with rapid fermentation of hexoses, similar to the findings [38], were increased at week 8 when dis- solved H2 concentrations were the highest but then were significantly lower at weeks 4 and 12. In contrast, the slow fermenting bacteria such as Clostridiales, Butyrivi- brio, and Ruminococcus did not fluctuate as did the rapid fermenting bacteria but were increased by week 12. Because methanogens were inhibited, dissolved H2 may be directed towards other hydrogenotrophic bacteria that transiently increased in response to dissolved H2 accu- mulation. However, this increase in dissolved H2 concen- tration in the rumen may have stimulated the expression of H2-sensing hydrogenases that then began the second phase of H2 dynamics in the rumen. These H2-sensing [FeFe] hydrogenases, as described in Zheng et al. [39], then enabled expression of A1 [FeFe] hydrogenases that led to a reduction in H2 production by H2-producing bacteria. Interestingly, this shift was also accompanied by an increase in ethanol production ([26, 40] and 65 mg/ kg of rumen contents at weeks 4, 8, and 12, respectively) suggesting that the amount of H2 released is indeed regu- lated in H2-producing bacteria under inhibited metha- nogenesis as described in Melgar et al. [18]. This process was also accompanied by a significant decrease in the molar proportion of acetate and an increase in molar proportion of butyrate in the rumen. The CH4 inhibitor 3-NOP is an analog of methyl Co-M and therefore inhibits MCR, an enzyme that catalyzes CH4 formation [36]. In the current study, abundance of genes encoding for MCR (EC: 2.8.4.1) did not differ between control and supplemented treatment groups. However, their corresponding transcripts were 3 times higher compared with their gene copies across all sam- ples and showed a tendency to reduce in 3-NOP sup- plemented dairy cows compared with control cows. The effective inhibition of methanogenesis by 3-NOP was clearly observed in reduced gene expression more than gene content which corroborates the findings of Shi et al. [37] that CH4 yield in sheep is tightly correlated with gene expression. Discussion There was a gradual increase in the relative abundance of Methanosphaera and transcripts of the methanol-reducing pathway from week 4 through week 12, but these increases may be attributed to an increase in DMI as dairy cows advanced Several studies have reported negative correlations between Methanobrevibacter and Methanosphaera line- ages in the rumen [28, 29]. Because it was assumed that Methanobrevibacter is the predominant methanogenic genus and the contribution of Methanosphaera lineages is small, the competition among methanogens has been largely ignored until metatranscriptomic approaches were employed [27, 30] to understand methanogenesis. Pitta et al. Microbiome (2022) 10:146 Page 14 of 21 Page 14 of 21 in lactation rather than a carryover effect of the fresh diet at week 4. Recently, we reported a greater contribution of Methanosphaera compared with other methanogens in the rumen of dairy cows during the first 6 h post-feeding owing to the abundance of methylated substrates due to rapid fermentation of carbohydrates; a gradual decrease in Methanosphaera was observed after 6 h post-feeding with an increasing abundance of Methanobrevibacter around 10 h post-feeding [35]. Dietary sources includ- ing pectin, hemicellulose, choline, and glycine betaine are methyl compounds that are ultimately converted by microbial enzymes to methanol and methylamines in the rumen [27] that are then utilized by methylotrophic methanogens. With an increase in DMI, substrates sup- plying methyl groups may support an increase in the methanol-reducing pathway; however, more information on the different types of substrates and what conditions favor the abundance of Methanosphaera and Methano- massiliicoccales representatives and their contribution to total methanogenesis in a temporal manner using RNA- based approaches may help us better understand CH4 formation in the rumen. samples via GreenFeed and dissolved H2 concentra- tions in the rumen increased progressively from week 1 through week 9 and then declined by week 15 (SI Addi- tional file 1: Table S13; Table S18). These changes in H2 emissions, although very difficult to measure and change rapidly with time and location in the rumen, may be explained 2 phases as follows: the first phase was char- acterized by a drop in CH4 formation resulting in a spike in dissolved H2 concentrations that accumulated dur- ing this time period. Discussion In the current study, H2 concentrations were higher at weeks 4 and 8 which may have resulted in a relatively lower expression of bifurcating enzymes [FeFe]A3 in 3-NOP supplemented dairy cows. By week 12, there had been adjustments within fiber-digesting bacteria result- ing in a decrease in H2 concentrations in the rumen which was accompanied by an increase in electron- bifurcating enzymes. Based on these H2 concentrations in the rumen, the ratio of A1:A3 hydrogenase expres- sion was regulated. This may be directly associated with H2 concentrations within the rumen with increasing H2 concentrations inversely related to bifurcating enzymes whereas these hydrogenases increased with a reduced H2 concentrations. In the rumen, methanogens serve as the major H2 sink and this interdependency for H2 between methanogens and other fermenting microbes drives fermentation of feeds [43, 44]. Janssen [41] conceptualized a model in which changes in diet (altering forage to grain ratio), low- ering pH in the rumen, and inhibiting methanogens may lead to an increase in P[H2], thus creating a negative feed- back mechanism on H2-producing bacteria to reduce H2 production. This negative feedback mechanism results in a shift in fermentation pathways in H2-producing bac- teria from higher H2 and acetate production to the for- mation of more reduced products such as succinate or ethanol as described in Greening et al. [42]. Melgar et al. [18] observed that molar concentration as well as propor- tion of acetate was reduced in the rumen contents col- lected from 3-NOP supplemented cows compared with control cows at weeks 4, 8, and 12. However, the H2 that would be spared by an approximately 26% reduction in CH4 has not been completely accounted for in that study. Although these authors reported a significant increase in The steady-state acetate concentration in the rumen was found to be lower after 3-NOP supplementation, which does not exclude sugar-fermenting acetogens being involved as indirect sinks. In the rumen, ethanol is formed from sugars by bacteria such as Ruminococ- cus albus when the H2 concentration is high. The etha- nol reacts with acetate to butyrate and caproate and with propionate to valarate in a fermentation catalyzed by Clostridium kluyveri [46]. Indeed, Melgar et al. [18] found that the steady-state ethanol concentration was significantly higher in 3-NOP supplemented rumen sam- ples compared to control samples (26.68 mg/kg versus 16.51 mg/kg). Discussion Further, as indicated in our previous report, the higher dose of 3-NOP ingested by the cows as a result of greater dry matter intakes may have led to greater inhibition of 3-NOP on the methanol pathway at week 8 [35], because a higher dose (>1 μM) of 3-NOP is needed to inhibit Methanosphaera whereas only 0.25 μM of 3-NOP is sufficient to inhibit Methanobrevibacter ruminantium [21]. Collectively, it can be inferred that 3-NOP inhibits MCR but has differential effects on indi- vidual methanogenic linages. It is important to under- stand the factors governing the distribution of individual methanogenic lineages and at what doses these lineages are inhibited to more effectively reduce CH4 formation in the rumen. It has been reported that inhibited methanogenesis and increased H2 concentrations may result in an increase in partial pressure of hydrogen (P[H2]) [41] which may have happened in the rumen of cows that received 3-NOP supplementation. Greening et al. [42] reported differ- ences in stoichiometries of Ruminococcus albus 7 in response to high and low H2 concentrations in the rumen which have been attributed to the presence of putative sensory group C [FeFe] hydrogenases that can sense H2 concentrations. Accordingly, we have identified that group C hydrogenases were increased in 3-NOP supple- mented cows compared with control cows at weeks 4, 8, and 12, thus indicating that increasing H2 concentrations in the rumen under inhibited methanogenesis induced Previous reports [7, 18] showed that during 3-NOP supplementation to dairy cows for prolonged periods (15 weeks), while CH4 emissions were persistently reduced by 26 to 30%, both gaseous H2 measured in breath Pitta et al. Microbiome (2022) 10:146 Page 15 of 21 Pitta et al. Microbiome (2022) 10:146 Page 15 of 21 pH, formic acid, ethanol, butyrate, gaseous H2 emission, and dissolved H2 in ruminal contents of cows supple- mented with 3-NOP compared with control cows, these increases were not able to account for H2 spared under inhibited methanogenesis by 3-NOP. It is interesting to note that the genes or transcripts that code for some of the alternative sinks [42] such as nitrate and nitrite reduc- tase, CO-dehydrogenase/acetyl CoA synthase, fumarate reductase, and sulfite reductase showed only marginal increases, and their overall contribution to the total gene or transcript abundance was insignificant. Discussion However, tran- scripts of formyl-tetrahydrofolate synthetase, a marker enzyme of acetogenesis or the Wood-Ljungdahl pathway, were increased in 3-NOP samples indicating that aceto- gens may have increased under inhibited methanogen- esis. It has been reported that acetogens may serve as one of the alternative H2 sinks under reduced methano- genesis in sheep with low CH4-yield phenotype [42] and they are the main H2 sink in the intestinal tract of marsu- pials and termites. In a monoculture of Acetobacterium woodii, an acetogen, 1 mole of fructose is fermented to 3 moles of acetate, but this bacterium was shown to shift its fermentation pathway to produce 2 moles of acetate, 2 CO2, and 4 H2 when co-cultured with Methanobacterium strain AZ, which kept the H2 concentration in the media low [45]. Other than hydrogenotrophic methanogens, all known acetogens can grow on sugars and reduce CO2 with H2 to acetate only when sugars are not available. It is thus very likely that the acetogens in the rumen normally ferment sugars to 2 moles acetate, 2 CO2, and 4 H2 when the H2 concentration is very low and that they switch to forming 3 moles acetate when the H2 concentration increases after 3-NOP inhibition. Rather than reducing CO2 with 4 H2 to acetate, 4 H2 are spared by not being produced. by 3-NOP were sensed by group C [FeFe] hydrogenases, whereas these remained fairly stable in control cows. Although we did not see a consistent increase in expres- sion of A1, we found that B group [FeFe] hydrogenases were consistently increased in 3-NOP supplemented cows to regulate the amount of H2 produced. In pure cul- tures of R. albus, H2 production is regulated by either A1 [FeFe] hydrogenases, which are ferredoxin-only hydro- genases, or the A3 group which are electron-bifurcating hydrogenases. Under low P[H2], such as when grown in the presence of methanogens, R. albus favors the energy- efficient pathway via production of acetate and H2 which is regulated by the electron-bifurcating A3 group of [FeFe] hydrogenases. However, under high P[H2], i.e., in the absence of methanogens, H2 production is regulated by the ferredoxin-only hydrogenase (group A1 [FeFe]- hydrogenase), a bifunctional alcohol and aldehyde dehy- drogenase, and regulatory elements including a putative sensory hydrogenase (group C [FeFe]-hydrogenase) [42]. Discussion Further, both butyrate and caproate were significantly higher in 3-NOP rumen samples compared to control samples. Butyrate and caproate formation from ethanol and acetate in C. kluyveri involves butyryl-CoA dehydrogenase, which catalyzes the reduction of ferre- doxin and crotonyl-CoA with 2 NADH to butyryl-CoA Pitta et al. Microbiome (2022) 10:146 Page 16 of 21 Page 16 of 21 methanogenic lineages, which may be driven by several factors including dietary composition, dry matter intake, host genetics, and ruminal conditions including pH, VFA molar proportions, and partial pressure of H2 in the rumen. Further, increases in concentrations of spared H2 under inhibited methanogenesis may lead to a shift in fermentation pathways in H2-producing and H2-utilizing bacteria such as acetogens. More information is needed to determine how much H2 is spared under inhibited methanogenesis by 3-NOP, as the amount of H2 spared varied in a temporal manner. Fluctuations in H2 were accompanied by changes in hydrogenases, possibly indi- cating regulation of H2 concentrations by hydrogenases in H2-producing bacteria. While there appears to be no alternative sinks that can compete with methanogens for H2 under inhibited methanogenesis by 3-NOP, butyrate synthesis seems to be the compensatory H2 sink. The increase in butyrate concentrations may only be the con- sequence of a shift in acetogen fermentation pathways that lead to higher acetate production; however, certain bacteria such as C. Kluyveri may increase with 3-NOP supplementation and can ferment acetate and ethanol to form butyrate. and reduced ferredoxin [46]. In the current study, we noticed a significant increase in the genes and transcripts (overall across all sampling weeks) for butyryl-CoA dehy- drogenase (EC: 1.3.8.1), by 10% and 15%, respectively. Furthermore, we also noted that C. kluyveri’s contribu- tion for this enzyme was increased in 3-NOP samples compared to control samples. There are 2 major pathways for butyrate synthe- sis in the rumen [27]: one mechanism mediated via the butyrate kinase pathway (BP1), which is mostly predomi- nant in Clostridia, and the other mechanism mediated via the butyryl-CoA: acetate-CoA-transferase pathway (BP2) which is mostly predominant in Negativicutes (Selenomondales, Veillonellaceae, and Acidaminococ- caceae) but also in C. kluyveri. It has been shown that BP2 and C. kluyveri are dependent on acetate for butyrate formation [47] which indicates a synergy between ace- tate-producing and butyrate-producing bacteria [48]. Materials and methods Animals and experimental designh The current study was an accompaniment to the ani- mal study described in Melgar et al. [18]. The study and all procedures involving animals were approved by The Pennsylvania State University Institutional Animal Care and Use Committee. The experiment lasted for 15 weeks. As part of the larger experiment (see more details in supplementary information text), 8 ruminally cannu- lated Holstein cows were enrolled in the experiment as they entered into lactation and were randomly assigned to either control (n = 4) or 3-NOP supplementation at 60 mg/kg of feed dry matter (n = 4). The 3-NOP sup- plement was incorporated into the total mixed ration as described in Melgar et al. [18]. It is normal to provide additional Net Energy Lactation (NEL) to meet higher energy requirements in the form of a fresh diet during the transition period. The cows were on a fresh diet for the first 3 weeks after calving and then were transitioned to a lactation diet by week 4 after calving. As the goal of the study was to investigate the effects of 3-NOP in early lactation, cows were enrolled as they entered lactation and remained in the study for 15 weeks. Ingredient and chemical composition of the fresh and lactation diets were described in Melgar et al. [18]. Feed analysis, meas- urement of enteric CH4 and dissolved H2 concentrations in ruminal fluid, and VFA analysis were described in Mel- gar et al. [18] and also included in supplementary infor- mation text. Methane (g/d), CH4 yield (g/kg DMI) and H2 Discussion In the current study, we found that 3-NOP numerically increased the gene expression of the enzyme acetate- CoA transferase (EC: 2.8.3.8) by approximately 25%, sug- gesting that the BP2 pathway of butyrate synthesis may be an alternative pathway under inhibited methanogen- esis by 3-NOP. However, it remains to be determined how much of spared H2 is diverted to butyrate and other reduced products such as ethanol and formic acid. Before we determine the fate of spared H2, it is also essential to determine how much H2 is spared. The amount of H2 spared under inhibited methanogenesis is dependent on methanogen diversity and to what extent individual methanogenic lineages are inhibited. Methanogens that reduce CO2 require 4 moles of H2 whereas methylo- trophic methanogens require only 1 mole of H2; thus, the amount of H2 spared when the latter methanogens are inhibited is much lower than when the former metha- nogens are inhibited. Further studies on methanogen diversity and to what extent different CH4 inhibitors may inhibit individual methanogenic lineages may help to bet- ter quantify the amount of H2 spared and then to deter- mine the diversion of H2 to alternate sinks to understand energy conservation in the rumen. Such information may help design strategies to formulate diets to reduce enteric CH4 formation without perturbing rumen microbiota and to safely divert H2 to more reduced fermentation products that are then available for the host metabolism. Conclusionshi The main findings of this study indicate that methylo- trophic methanogens may have a greater contribution to a total methanogenesis than what was originally thought and that metatranscriptomic approaches provide deeper insights on methanogenesis in the rumen. The inhibi- tor 3-NOP may have a differential effect on individual Pitta et al. Microbiome (2022) 10:146 Page 17 of 21 Page 17 of 21 Pitta et al. Microbiome (2022) 10:146 amplification of the bacterial 16S rRNA gene involved an initial denaturing step at 95°C for 5 min followed by 20 cycles (denaturing at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s) and a final extension step at 72°C for 8 min. The thermal cycling conditions for PCR amplification of the archaeal 16S rRNA gene involved an initial denaturing step at 94°C for 2 min followed by 30 cycles (denaturing at 94°C for 30 s, annealing at 56°C for 1 min 30 s, extension at 72°C for 30 s) and a final exten- sion step at 72°C for 8 min. The triplicate amplicon prod- ucts from each sample were pooled and then quantified using a Spectramax M2e microplate reader (Molecular Devices; San Jose, CA). The quantified amplicons were combined by adding each sample to a pool in equimo- lar concentration, and pools were bead purified using Agencourt AMPure XP Beads (Beckman-Coulter; Indi- anapolis, IN, USA). Sequencing was performed at the PennCHOP Microbiome Core, University of Pennsylva- nia, using the Illumina MiSeq platform. The rt-PCR assay on rumen samples was performed as described in Pitta et al. [35]. measurements from animals in the study at experimental weeks 2, 6, 9, and 15 were found to be significantly differ- ent between the two treatment groups (P <0.01; Melgar et al. [18]) and are presented in Table S13. The 8 can- nulated dairy cows were sampled for rumen contents at 2 h after feeding in experimental weeks 4, 8, and 12. Rumen samples were collected from 4 different (the ven- tral sac, the atrium or reticulum, and two samples from the feed mat) locations in the rumen, combined to rep- resent a homogenous sample, filtered through 4 layers of cheesecloth to separate solid and liquid fractions, snap- frozen at cow side using liquid nitrogen, and then stored at −80°C until analysis. Metagenomic and metatranscriptomic analysis Metagenomic and metatranscriptomic analysis Metagenomic and metatranscriptomic analysis For metagenomics, DNA was prepared for whole- genome shotgun sequencing using the Nextera DNA Library Prep Kit (Illumina; San Diego, CA, USA). The library (tight insert size of 250 bp for high-through- put sequencing from both ends by 2 × 150 bp) was sequenced on an Illumina HiSeq 2500 at the Center for Host-Microbe Interactions at the University of Penn- sylvania School of Veterinary Medicine. For metatran- scriptomics, ribosomal RNA was depleted from total RNA using the Ribo-Zero Plus rRNA Removal Kit (Illu- mina). Double-stranded cDNA was synthesized from the mRNA-enriched RNA using the TruSeq Stranded mRNA Kit (Illumina), and a library was constructed. The library was sequenced on an Illumina NextSeq 500 at the Pen- nCHOP Microbiome Core, University of Pennsylvania. DNA and RNA extraction, PCR amplification, and sequencingh The genomic DNA from both the solid and liquid ruminal samples was extracted using the repeated bead beating and column (RBB + C) method followed by extraction with the QIAmp Fast DNA Stool Mini Kit (Qiagen Sci- ences; Germantown, MD, USA) as described in Yu and Morrison [49]. The RNA extraction from rumen sam- ples was performed using the Trizol method. The steps involve bead beating in Trizol followed by chloroform and isopropanol extractions and finally by ethanol pre- cipitation. RNA was reverse transcribed to cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s pro- tocol. For each extracted genomic DNA and cDNA sam- ple, both the V1–V2 regions of the bacterial 16S rRNA gene and the V6–V8 regions of the archaeal 16S rRNA gene were PCR-amplified in triplicate. The bacterial-spe- cific primers used were F27 (5′-AGAGTTTGATCCTGG CTCAG-3′) and R338 (5′-TGCTGCCTCCCGTAGGAG T-3′); the archaeal-specific primers used were i958aF (5’- AATTGGAKTCAACGCCKGR-3’) and i1378aR (5’-TGT GTGCAAGGAGCAGGGAC-3’). Both sets of primers were barcoded with a unique 12-base error-correcting Golay code for multiplexing as described in Song et al. [50]. Polymerase chain reaction was performed in tripli- cate using the Accuprime Taq DNA Polymerase System (Invitrogen). The thermal cycling conditions for PCR Conclusionshi Both solid and liquid samples were processed for bacterial and archaeal diversity analy- sis, whereas only solid samples were used for metagen- omic, metatranscriptomic, and rt-PCR analysis. Because the microbiome associated with the liquid fractions was found to vary with several factors including dilution with water intake, time of the day and accumulation of fermentation variables, and that the microbiome associ- ated with the solid fraction remained stable throughout the day for individual cows [29], we selected only the solid samples for metagenomic and metatranscriptomic analysis. Statistical analysis To facilitate comparisons between samples with differ- ent sequencing depths, the gene orthologs were normal- ized to cpm. Absolute abundance of taxonomy values was compositionally normalized (relative abundance) such that each sample summed to 1. All of the statisti- cal analysis was performed in R [58]. To test for differ- ences in 16S-based amplicons and metagenomic and metatranscriptomic taxonomy analysis, a generalized lin- ear mixed model was constructed with the lme4 package for R [59]. The model used treatment and week as fixed effects with a binomial family object and cow included as a random effect term. For the KEGG gene orthologs, we used treatment as fixed effect with Poisson family object and included a random effect term of week: treatment. The P values for multiple tests were corrected using the Benjamini-Hochberg approach. To determine whether the individual methanogens derived from rt-PCR were significantly different between treatment groups, we conducted the Wilcoxon test. The Spearman correla- tion coefficients were calculated to evaluate correlations between relative abundance of bacterial genera (DNA- and RNA-based 16S rRNA) and fermentation profiles. We considered the relationships with the criteria of abso- lute correlation coefficients greater than 0.5 and P values less than 0.05 as significant. Bioinformatic analysish In addition, taxonomy of the annotated genes and transcripts was also tracked to help us understand the role of individ- ual methanogenic lineages in methanogenesis. Further, hydrogenases were confirmed and classified by aligning the quality filtered reads to a hydrogenase database [42] using the DIAMOND [56] search tool. for selected enzymes such as MCR which is encoded by mcra, mcrb, and mcry. The methodology used MAG con- struction, and other bioinformatics details are described in supplementary note (SI Additional file 1). they were shorter than 36 bases in length. Reads align- ing to the host genome (ARS-UCD1.2/bosTau9) were identified and removed using Bowtie2 (v2.2.7) [52] with parameters set by the flag --very sensitive local --un- conc. For metatranscriptomic sequences, an additional quality filtering step was applied in which the rRNA and tRNA sequences were removed using SortmeRNA (v2.1) [53]. Taxonomic labels were assigned to quality-con- trolled reads by mapping sequences to a low-complexity masked database of bacterial, archaeal, viral, fungal, and protozoal sequences from NCBI complete genomes. The relative abundance of the bacterial and archaeal taxon- omy was estimated using Kraken2, version 2.1.1 [54]. The reads were mapped to the KEGG [55] protein database to estimate abundance of microbial gene orthologs using DIAMOND [56], and the functional profiles were per- formed by HUMAnN2 [57]. The abundance of orthologs was then annotated to Enzyme Commission (EC) num- bers already present in the KEGG database. The genes/ transcripts that encoded for enzymes at each step of the methanogenesis pathway were identified based on a lit- erature search [27, 30, 37, 40] and the Kyoto Encyclope- dia of Genes and Genomes (KEGG) database, and the different methanogenesis pathways were constructed and described by our group in Pitta et al. ([23] Figure S2). Using metagenomic and metatranscriptomic data, we quantified the genes and transcripts of enzymes involved in the 3 predominant ruminal methanogenesis pathways (CO2-, methanol-, and methylamine-reducing pathways) along with the butyrate and propanoate pathways in cows with and without 3-NOP supplementation at weeks 4, 8, and 12 of the experimental period (Fig. 2). In addition, taxonomy of the annotated genes and transcripts was also tracked to help us understand the role of individ- ual methanogenic lineages in methanogenesis. Further, hydrogenases were confirmed and classified by aligning the quality filtered reads to a hydrogenase database [42] using the DIAMOND [56] search tool. Bioinformatic analysish y The DNA- and RNA-based amplicon 16S rRNA sequences for archaeal and bacterial diversity were ana- lyzed according to the method previously described [35]. The metagenomic and metatranscriptomic sequences were demultiplexed, and the adapter was trimmed at the sequencing facility. These raw sequences were sub- jected to quality trimming using Trimmomatic (0.36) [51] according to the following parameters: starting from either end of the sequence, bases were trimmed off if their Phred quality score was < 3 or if they appeared as N; bases were trimmed off if their average Phred qual- ity score was < 15 when the sequence was analyzed on a 4-base sliding window; and sequences were removed if Pitta et al. Microbiome (2022) 10:146 Pitta et al. Microbiome (2022) 10:146 Page 18 of 21 they were shorter than 36 bases in length. Reads align- ing to the host genome (ARS-UCD1.2/bosTau9) were identified and removed using Bowtie2 (v2.2.7) [52] with parameters set by the flag --very sensitive local --un- conc. For metatranscriptomic sequences, an additional quality filtering step was applied in which the rRNA and tRNA sequences were removed using SortmeRNA (v2.1) [53]. Taxonomic labels were assigned to quality-con- trolled reads by mapping sequences to a low-complexity masked database of bacterial, archaeal, viral, fungal, and protozoal sequences from NCBI complete genomes. The relative abundance of the bacterial and archaeal taxon- omy was estimated using Kraken2, version 2.1.1 [54]. The reads were mapped to the KEGG [55] protein database to estimate abundance of microbial gene orthologs using DIAMOND [56], and the functional profiles were per- formed by HUMAnN2 [57]. The abundance of orthologs was then annotated to Enzyme Commission (EC) num- bers already present in the KEGG database. The genes/ transcripts that encoded for enzymes at each step of the methanogenesis pathway were identified based on a lit- erature search [27, 30, 37, 40] and the Kyoto Encyclope- dia of Genes and Genomes (KEGG) database, and the different methanogenesis pathways were constructed and described by our group in Pitta et al. ([23] Figure S2). Using metagenomic and metatranscriptomic data, we quantified the genes and transcripts of enzymes involved in the 3 predominant ruminal methanogenesis pathways (CO2-, methanol-, and methylamine-reducing pathways) along with the butyrate and propanoate pathways in cows with and without 3-NOP supplementation at weeks 4, 8, and 12 of the experimental period (Fig. 2). Availability of data and materials The raw sequences from DNA- and RNA-based 16S rRNA archaeal and bacte- rial sequencing, metagenomics, and metatranscriptomics have been depos- ited in the NCBI Sequence Read Archive (SRA) database under Bio Project accession number PRJNA666417. error of mean; Trt: treatment group; W: week; 3-NOP: 3-nitrooxypropanol. Table S10: Effect of 3-nitrooxypropanol (3-NOP) on bacterial taxonomical composition (relative abundance %) in RNA-based 16S rRNA sequencing. SEM: Standard error of mean; Trt: treatment group; W: week. Table S11: Effect of 3-nitrooxypropanol (3-NOP) on bacterial taxonomical composi- tion (relative abundance %) in metagenomics. SEM: Standard error of mean; Trt: treatment group; W: week. Table S12: Effect of 3-nitrooxypro- panol (3-NOP) on bacterial taxonomical composition (relative abundance %) in metatranscriptomics. SEM: Standard error of mean; Trt: treatment group; W: week. Table S13: Average methane (CH4) and hydrogen (H2) gas data in control and 3-nitrooxypropanol (3-NOP) treated cows at weeks 2, 6, 9, and 15. Table S14: Effect of 3-nitrooxpropanol (3-NOP) on hydroge- nase subgroups (cpm; copies per million) including [FeFe], [Fe], and [NiFe] hydrogenases in metagenomics. Table S15: Effect of 3-nitrooxpropanol (3-NOP) on hydrogenase subgroups (cpm; copies per million) including [FeFe], [Fe], and [NiFe] hydrogenases in metatranscriptomics. Table S16: Effect of 3-NOP on hydrogenases (cpm; copies per million) classified into H2 production and H2 consumption in metagenomics. Table S17: Effect of 3-NOP on hydrogenases (cpm; copies per million) classified into H2 production and H2 consumption in metatranscriptomics. Table S18: Effect of 3-nitrooxypropanol (3-NOP) on rumen fermentation variables in early- lactation dairy cows at weeks 4, 8, and 12. Table S19: Taxonomy (cpm; copies per million) associated with the butyrate pathway in metagenom- ics in cows supplemented with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12. Table S20: Taxonomy (cpm; copies per million) associated with the butyrate pathway in metatranscriptom- ics in cows supplemented with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12. Table S21: Taxonomy (cpm; copies per million) associated with the propionate pathway in metagenomics in cows supplemented with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12. Table S22: Taxonomy (cpm; copies per million) associated with the propionate pathway in metatranscriptom- ics in cows supplemented with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12. Table S23: List of metagenomes assembled genomes (MAGs) identified in early-lactation dairy cows. Availability of data and materials Table S24: Transcripts (cpm; copies per million) coding for EC: 1.8.98.1 in cows supplemented with 3-nitrooxypropanol (3-NOP) compared to con- trol cows at weeks 4, 8, and 12. ND = not detected. Table S25: Transcripts (cpm; copies per million) coding for EC: 1.8.98.5 in cows supplemented with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12. ND = not detected. Table S26: Transcripts (cpm; copies per million) coding for EC: 1.8.98.6 in cows supplemented with 3-nitrooxypro- panol (3-NOP) compared to control cows at weeks 4, 8, and 12. ND = not detected. Acknowledgements The authors would like to thank DSM Nutritional Products, Kaiseraugst, Switzerland, for providing 3-nitrooxypropanol. The authors also thank Prof. Rolf Thauer for providing assistance with data interpretation and editing the manuscript. The authors acknowledge the assistance and resources provided by Center for Host-Microbe Interactions, School of Veterinary Medicine, University of Pennsylvania, and the PennCHOP Sequencing Core, Perelman School of Medicine, University of Pennsylvania. 5. Hristov AN, Oh J, Firkins JL, Dijkstra J, Kebreab E, Waghorn G, et al. Special topics—mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options. J Anim Sci. 2013;91(11):5045–69. https://doi.org/10.2527/jas.2013-6583. 6. Klevenhusen F, Zeitz JO, Duval S, Kreuzer M, Soliva CR. Garlic oil and its principal component diallyl disulfide fail to mitigate methane, but improve digestibility in sheep. Anim Feed Sci Technol. 2011;166:356–63. https://doi.org/10.1016/j.anifeedsci.2011.04.071. 6. Klevenhusen F, Zeitz JO, Duval S, Kreuzer M, Soliva CR. Garlic oil and its principal component diallyl disulfide fail to mitigate methane, but improve digestibility in sheep. Anim Feed Sci Technol. 2011;166:356–63. https://doi.org/10.1016/j.anifeedsci.2011.04.071. Funding 3-NOP compared to control at weeks 4, 8, and 12. Table S8: Transcripts (cpm; copies per million) coding for EC: 1.8.7.3 (HdrA, HdrB and HdrC) in cows supplemented with 3-nitrooxypropanol (3-NOP) compared to This study was sponsored by DSM Nutritional Products, Kaiseraugst, Swit- zerland, and was also supported by the USDA National Institute of Food and Agriculture Federal Appropriations under project PEN 04538 and accession number 1000803 and USDA-NIFA-AFRI-006351 number 2017-05832. control cows at weeks 4, 8, and 12. ND = not detected. Table S9: Effect of 3-nitrooxypropanol (3-NOP) on bacterial taxonomical composition (rela- tive abundance %) in DNA-based 16S rRNA sequencing. SEM: Standard error of mean; Trt: treatment group; W: week; 3-NOP: 3-nitrooxypropanol Competing interests d SD, MK, and NW are employees of DSM Nutritional Products. The other authors declare no competing interests. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s40168-022-01341-9. Additional file 1: Supplementary Information Text. Figure S1: Alpha diversity based on 16S rRNA amplicon archaeal sequencing data in cows supplemented with 3-nitrooxypropanol (3-NOP) at weeks 4, 8, and 12. (A) observed species in DNA liquid; (B) Shannon diversity in DNA liquid; (C) observed species in RNA liquid; (D) Shannon diversity in RNA liquid. NS = not significant. Figure S2: Schematic diagram of possible methanogenesis pathways. Table S1: Sequencing information for metagenomics. Trt: treat- ment group; W: week; 3-NOP: 3-nitrooxypropanol. Table S2: Sequencing information for metatranscriptomics. Trt: treatment group; W: week 3-NOP: 3-nitrooxypropanol. Table S3: Relative abundance (%) of archaeal taxon- omy in metagenomics. SEM: Standard error of mean; Trt: treatment group; W: week; 3-NOP: 3-nitrooxypropanol. Table S4: Relative abundance (%) of archaeal taxonomy in metatranscriptomics, SEM: Standard error of mean; Trt: treatment group; W: week; 3-NOP: 3-nitrooxypropanol. Table S5: The 6 most abundant archaea (cpm; copies per million) contributing to steps 1-5 in the carbon dioxide (CO2)-hydrogen (H2) methanogenic pathway in the rumen of dairy cows supplemented with 3-nitrooxypropanol (3-NOP) compared to control cows at weeks 4, 8, and 12. Table S6: The most abun- dant archaea (cpm; copies per million) utilizing methanol as a substrate for methanogenesis in the rumen of dairy cows supplemented with 3-NOP compared to control at weeks 4, 8, and 12. Table S7: The most abundant archaea (cpm; copies per million) utilizing methylamines as a substrate for methanogenesis in the rumen of dairy cows supplemented with We have also derived MCR gene profiles for metagen- omics and metatranscriptomic data using metagenome assembled genomes (MAGs) approach. Only 61 MAGs were assembled (Table S25); of these, 54 are bacteria and 7 are methanogens. Among the bacteria, MAG were identified only to class and order level except for Metha- nobrevibacter, Methanosphaera, Prevotella, Butyrivibrio, Succiniclasticum ruminis, Lachnospiraceae bacterium NE2001, Sarcina, Butyrivibrio, Bifidobacterium, and Pseudobutyrivibrio, which showed taxonomy only to the genus or species level. Among the 7 methanogen MAG, 6 were identified as Methanobrevibacter genus (species not identified) and only one MAG was identified as Methano- sphaera. 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